**Meet the editors**

Dr. Bassam H. Mahboub is currently heading the department of Pulmonary Medicine and Allergy and Sleep Medicine, Rashid Hospital, Dubai & Assistant professor of Medicine and Chest disease, University of Sharjah, UAE. His main area of research and clinical interest includes asthma, respiratory allergy and COPD. Dr Bassam Completed his American Board of Internal Medicine, Pulmonology, Ameri-

can Board Allergy Clinical Immunology. Dr bassam got the fellowship of Royal college of physicians and Royal college Allergy & Clinical Immunology, Canada. Before that He was assistant medical director and Consultant and Head of division of Allergy/Immunology and Respiratory Medicine, Al Baraha Hospital, Ministry of Health, Dubai. Dr Bassam, is holding the position of Head and vice chairman of various medical professional societies in UAE & GCC countries. Dr Bassam has the credit of supervising graduates, postgraduates and doctoral students focusing on research in the field of respiratory medicine and allergy and immunolongy. He has authored many peer reviewed articles including original research, chapters published in international books and journals. He is presently also involved in numerous research projects undergoing in Gulf countries and the academic activities for the specialist doctors in UAE.

Dr. Mayank G. Vats is Senior Specialist in the department of Pulmonary Medicine and Allergy and Sleep Medicine, Rashid Hospital, Dubai. His main area of research and clinical interest includes asthma, respiratory allergy and COPD & Sleep Medicine. Dr Vats Completed his postgraduation (MD) in Pulmonary Medicine, then he did DNB in Respiratory Medicine & DNB in Critical Care medicine from the

prestigious Escorts Heart Institute in new delhi, India and then served as Consultant Pulmonologist, Intensivist & Sleep Physician at Escorts Heart Institute & Apollo Hospital, New Delhi , India for More than 3 years. Dr Vats also got extensive Training in Sleep Medicine by American Academy of Sleep Medicine at LDS Hospital, Salt Lake City, UTAH, USA. Dr Vats got the Membership of Royal college of physicians and Surgeons of Glasgow, UK & was elected as fellow of American College of Physicians (FACP) & Fellow of American College of Chest Physicians (FCCP) & Holds the European Diplomat in Respiratory Medicine. Before that he provided his services as Specialist (A) in the department of Medicine and Chest disease, Al Qassimi Hspital, Sharjah, UAE. Dr Vats is the member in good standing of various medical professional societies viz. ERS, ACCP, AASM, APSR, ISCCM to name a few. He has authored many peer reviewed articles including original research, Case reports, Review articles & chapters published in international books and journals. He is presently also involved in many research projects in Gulf countries and the academic activities for the specialist doctors in UAE.

Contents

**Preface VII**

Cabello-Gutiérrez

**Virus (RSV) 33**

**Coronaviruses 47**

**Section 2 Bacterial Infections 77**

**Biofilms 79**

**Pneumonia 99**

Chapter 6 **Pneumonia in Children 137**

Fuenzalida

Bartley

Chapter 1 **Pathogenesis of Viral Respiratory Infection 3**

Sameera Al Johani and Javed Akhter

Ma. Eugenia Manjarrez-Zavala, Dora Patricia Rosete-Olvera, Luis Horacio Gutiérrez-González, Rodolfo Ocadiz-Delgado and Carlos

Chapter 2 **Virology and Molecular Epidemiology of Respiratory Syncytial**

Chapter 3 **Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and**

Wahyu Surya, Montserrat Samsó and Jaume Torres

Mohammed Al-Haddad, Ahmed Al-Jumaily, John Brooks and Jim

**Immunocompetent Adult Patients with Community-Acquired**

Fernando Peñafiel Saldías, Orlando Díaz Patiño and Pablo Aguilera

Chapter 4 **Biophysical Effects on Chronic Rhinosinusitis Bacterial**

Irena Wojsyk-Banaszak and Anna Bręborowicz

Chapter 5 **Clinical Diagnosis and Severity Assessment in**

**Section 1 Viral Infections 1**

### Contents

#### **Preface XI**




#### **Section 3 Helminthic Infections of Lung 205**

Chapter 8 **Helminthic Infections and Asthma: Still a Challenge for Developing Countries 207** Isabel Hagel, Maira Cabrera and Maria Cristina Di Prisco

Preface

Medicine is an ever-changing science. Every day we are encountered with the new develop‐ ments and knowledge in the pathogenesis, mechanism of disease, newer diagnostic modali‐ ties, treatment options and new challenges in the management of the various diseases. The same holds true for respiratory diseases with the emergence of new respiratory pathogens

Respiratory Diseases are an important contributor to the morbidity and mortality of man‐ kind since antiquity and its prevalence is on rise in with new disease are being recognized, however little importance has been given to the respiratory disease due to low level of

This book has been designed to deliver the detailed knowledge about the various respirato‐

The first section covers the updated pathogenesis of the respiratory viral infections. The chap‐ ters covers the comprehensive view of the virology and molecular epidemiology of RSV, the commonest respiratory pathogen in children and in adults as well. In the same section detailed

The section on bacteriology primarily emphasize upon the very important but often over‐ looked cause of bacterial infection of the lung, the biofilm which acts as a persistent reser‐ voir for the bacterial load and gives rise to frequent exacerbation in all population. Appropriate weightage has been given to the Clinical diagnosis and severity assessment of community acquired pneumonia which is a very common cause of morbidity and mortality in all age groups especially at extremes of age including the latest guidelines and recom‐

Various challenges associated with the diagnosis and management of Helminthic infections

As the prevalence of smoking has increased remarkably worldwide hence a dedicated chap‐ ter has been included on Smoking Cessation focusing on the Psychological approach to increase Smoking abstinence, which is very important component in any smoking cessa‐

The authors and the publishers of this book have made sure that the contents and the knowledge delivered by the book is evidence based, updated and comprehensive and taken

The experience and knowledge of each of the editors have been directed to ensure that all specialized aspects of respiratory diseases and infection have been expertly covered and

discussion has been done about the structural and functional aspects of viroporins.

and lung especially patients with asthma has been dealt in a very concise way.

having significant impact on the respiratory system.

ry infections including viral, bacterial, and helminthic infections.

awareness in physicians and general public.

mendation from various professional societies.

tion programme.

from the reliable sources.

**Section 4 Smoking Cessation and Lung 229**

Chapter 9 **Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary Disease: A Review 231** Jennifer Lira-Mandujano, M. Carmen Míguez-Varela and Sara E. Cruz-Morales

### Preface

Chapter 7 **Cystic Fibrosis Pulmonary Exacerbation – Natural History, Causative Factors and Management 173** Iara Maria Sequeiros and Nabil Jarad

Chapter 8 **Helminthic Infections and Asthma: Still a Challenge for**

Isabel Hagel, Maira Cabrera and Maria Cristina Di Prisco

Chapter 9 **Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary Disease:**

Jennifer Lira-Mandujano, M. Carmen Míguez-Varela and Sara E.

**Section 3 Helminthic Infections of Lung 205**

**VI** Contents

**Developing Countries 207**

**Section 4 Smoking Cessation and Lung 229**

**A Review 231**

Cruz-Morales

Medicine is an ever-changing science. Every day we are encountered with the new develop‐ ments and knowledge in the pathogenesis, mechanism of disease, newer diagnostic modali‐ ties, treatment options and new challenges in the management of the various diseases. The same holds true for respiratory diseases with the emergence of new respiratory pathogens having significant impact on the respiratory system.

Respiratory Diseases are an important contributor to the morbidity and mortality of man‐ kind since antiquity and its prevalence is on rise in with new disease are being recognized, however little importance has been given to the respiratory disease due to low level of awareness in physicians and general public.

This book has been designed to deliver the detailed knowledge about the various respirato‐ ry infections including viral, bacterial, and helminthic infections.

The first section covers the updated pathogenesis of the respiratory viral infections. The chap‐ ters covers the comprehensive view of the virology and molecular epidemiology of RSV, the commonest respiratory pathogen in children and in adults as well. In the same section detailed discussion has been done about the structural and functional aspects of viroporins.

The section on bacteriology primarily emphasize upon the very important but often over‐ looked cause of bacterial infection of the lung, the biofilm which acts as a persistent reser‐ voir for the bacterial load and gives rise to frequent exacerbation in all population. Appropriate weightage has been given to the Clinical diagnosis and severity assessment of community acquired pneumonia which is a very common cause of morbidity and mortality in all age groups especially at extremes of age including the latest guidelines and recom‐ mendation from various professional societies.

Various challenges associated with the diagnosis and management of Helminthic infections and lung especially patients with asthma has been dealt in a very concise way.

As the prevalence of smoking has increased remarkably worldwide hence a dedicated chap‐ ter has been included on Smoking Cessation focusing on the Psychological approach to increase Smoking abstinence, which is very important component in any smoking cessa‐ tion programme.

The authors and the publishers of this book have made sure that the contents and the knowledge delivered by the book is evidence based, updated and comprehensive and taken from the reliable sources.

The experience and knowledge of each of the editors have been directed to ensure that all specialized aspects of respiratory diseases and infection have been expertly covered and

well presented in view of scientific content. After undergoing peer review, the book aspires to provide a readable and updated coverage of all the latest updates in respiratory diseases and infection.

The book Respiratory Disease and Infections- A New Insight has been intended for the in‐ ternists, general practitioners and the respiratory physicians in order to broaden the horizon of knowledge about the respiratory diseases and infection.

We owe a great deal to all authors who worked hard to contribute the chapters in the book. We are greatly indebted to all and especially InTech publisher for their dedicated efforts and close collaboration with all the authors to publish the book for the advancement of knowl‐ edge and new insight in the field of Respiratory Disease and Infections.

Lastly, we owe a great deal to our family, who provided constant aspiration, encouragement, peace of mind and unwavering support to us to complete the editorial work of this book.

> Editor: **Dr Bassam H. Mahboub** Director, Department of Pulmonary Medicine and Allergy, Rashid Hospital & Dubai Hospital,

**Section 1**

**Viral Infections**

Assistant Professor, Dept of Medicine & Respiratory Disease & Allergy, University of Sharjah, UAE

Co-editor: **Dr Mayank Vats** Specialist - A, Pulmonary Medicine, Intensive Care Medicine & sleep Medicine, AL Qassimi Hospital, Sharjah, UAE

**Section 1**

## **Viral Infections**

well presented in view of scientific content. After undergoing peer review, the book aspires to provide a readable and updated coverage of all the latest updates in respiratory diseases

The book Respiratory Disease and Infections- A New Insight has been intended for the in‐ ternists, general practitioners and the respiratory physicians in order to broaden the horizon

We owe a great deal to all authors who worked hard to contribute the chapters in the book. We are greatly indebted to all and especially InTech publisher for their dedicated efforts and close collaboration with all the authors to publish the book for the advancement of knowl‐

Lastly, we owe a great deal to our family, who provided constant aspiration, encouragement, peace of mind and unwavering support to us to complete the editorial work of this book.

Editor:

Director,

Co-editor: **Dr Mayank Vats** Specialist - A,

**Dr Bassam H. Mahboub**

Assistant Professor,

AL Qassimi Hospital,

Sharjah, UAE

University of Sharjah, UAE

Rashid Hospital & Dubai Hospital,

Department of Pulmonary Medicine and Allergy,

Dept of Medicine & Respiratory Disease & Allergy,

Pulmonary Medicine, Intensive Care Medicine & sleep Medicine,

of knowledge about the respiratory diseases and infection.

edge and new insight in the field of Respiratory Disease and Infections.

and infection.

VIII Preface

**Chapter 1**

**Pathogenesis of Viral Respiratory Infection**

Ma. Eugenia Manjarrez-Zavala, Dora Patricia Rosete-Olvera, Luis Horacio Gutiérrez-González, Rodolfo Ocadiz-Delgado and

Speaking of viral pathogenesis, it must describe the features and factors of viral pathogen, hosts and environment. Over its lifetime, an individual is exposed to many infectious agents, however, in most situations does not develop a disease thanks to factors such as physical and chemical host barriers. In other cases, pathogens circumvent these barriers and cause infection; however, a "biological war" will start between the determinants of pathoge‐ nicity and early host defenses. If the virus is able to overcome these first lines of defense, a type of highly specialized and specific protection will be activated. This defense will ach‐ ieve, in most situations, the infection control and subsequent eradication of the disease. Fur‐ thermore, this process will initiate the generation of the immunological memory, enabling the individual with a more quickly and effectively response at the next contact with the same agent. On the contrary, if the foreign agent can overcome both defenses, the result is disease. In certain cases, the line of defense, when triggered, can also cooperate with the damage instead of healing, making the disease more severe. Thus, the immunopathology vi‐ ral respiratory infection is a frequent consequence of the immune response against many of respiratory pathogens. Furthermore, if the infection is established, the factors or viral viru‐ lence determinants and physiological conditions of the host cell will determine which direc‐ tion the infection will take. A virus is pathogenic when it is able to infect and cause disease in a host, while it is virulent when it causes more severe disease than another virus of a dif‐ ferent strain, although both remain pathogens. Each virus can cause different cytopathic ef‐ fects in the host cell, which may lead to several symptoms and disease. In addition, developing a disease reflects the existence of an abnormality of the host, either structural or

> © 2013 Manjarrez-Zavala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Carlos Cabello-Gutiérrez

http://dx.doi.org/10.5772/54287

functional, induced by the invading virus.

**1. Introduction**

### **Pathogenesis of Viral Respiratory Infection**

Ma. Eugenia Manjarrez-Zavala, Dora Patricia Rosete-Olvera, Luis Horacio Gutiérrez-González, Rodolfo Ocadiz-Delgado and Carlos Cabello-Gutiérrez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54287

#### **1. Introduction**

Speaking of viral pathogenesis, it must describe the features and factors of viral pathogen, hosts and environment. Over its lifetime, an individual is exposed to many infectious agents, however, in most situations does not develop a disease thanks to factors such as physical and chemical host barriers. In other cases, pathogens circumvent these barriers and cause infection; however, a "biological war" will start between the determinants of pathoge‐ nicity and early host defenses. If the virus is able to overcome these first lines of defense, a type of highly specialized and specific protection will be activated. This defense will ach‐ ieve, in most situations, the infection control and subsequent eradication of the disease. Fur‐ thermore, this process will initiate the generation of the immunological memory, enabling the individual with a more quickly and effectively response at the next contact with the same agent. On the contrary, if the foreign agent can overcome both defenses, the result is disease. In certain cases, the line of defense, when triggered, can also cooperate with the damage instead of healing, making the disease more severe. Thus, the immunopathology vi‐ ral respiratory infection is a frequent consequence of the immune response against many of respiratory pathogens. Furthermore, if the infection is established, the factors or viral viru‐ lence determinants and physiological conditions of the host cell will determine which direc‐ tion the infection will take. A virus is pathogenic when it is able to infect and cause disease in a host, while it is virulent when it causes more severe disease than another virus of a dif‐ ferent strain, although both remain pathogens. Each virus can cause different cytopathic ef‐ fects in the host cell, which may lead to several symptoms and disease. In addition, developing a disease reflects the existence of an abnormality of the host, either structural or functional, induced by the invading virus.

© 2013 Manjarrez-Zavala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Viral pathogenesis**

The term "pathogenesis" refers to the processes or mechanisms to generate an injury or ill‐ ness, in this case induced by a viral infection. The results of a viral infection depend on fac‐ tors related to the nature of the virus, the host and the environment. They include: number of infectious particles, the way to reach the target tissue, the rate of multiplication, the effect of virus on cell functions and the host's immune response. Three requirements must be sat‐ isfied to ensure the infection of an individual host [1]:

utes to the kinetics of viral spread [2]. The amount of virus inoculated may influence or

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 5

**d. Speed of replication.** Some viruses replicate so rapidly that they often cause acute in‐ fections, others are slow virus replication, or some have to travel greater distances,

**e. Viral Spread.** Following replication at the site of entry, virus particles can remain local‐ ized, or can spread to other tissues. Local spread of the infection in the epithelium oc‐ curs when newly released virus infects adjacent cells. These infections are usually contained by the physical constraints of the tissue and brought under control by the in‐ trinsic and immune defenses. Respiratory infections are the typical example of local spread. An infection that spreads beyond the primary site of infection is called dissemi‐ nated (for example: measles virus). If many organs become infected, the infection is de‐ scribed as systemic. For an infection to spread beyond the primary site, physical and immune barriers must be breached. After crossing the epithelium, virus particles reach the basement membrane. The integrity of that structure may be compromised by epi‐ thelial cell destruction and inflammation. Below the basement membrane are sub-epi‐ thelial tissues, where the virus encounters tissue fluids, the lymphatic system and phagocytes. All three biological environments play significant roles in clearing viruses, but also may disseminate infectious virus from the primary site of infection. One impor‐ tant mechanism for avoiding local host defenses and facilitating spread within the body is the directional release of virus particles from polarized cells at the mucosal surface. Virions can be released from the apical surface, from the basolateral surface, or from both. After replication, virus released from the apical surface is outside the host. Such directional release facilitates the dispersal of many newly replicated enteric viruses in the feces (e.g., poliovirus). In contrast, virus particles released from the basolateral sur‐ faces of polarized epithelial cells have been moved away from the defenses of the lume‐ nal surface. Directional release is therefore a major determinant of the infection pattern. In general, viruses released at apical membranes establish a localized or limited infec‐ tion. Release of viruses at the basal membrane provides access to the underlying tissues

**f. Virulence genes.** Despite modern technology, identification and analysis of virulence genes is not easy. Part of the problem is that many of the effects of viral pathogenesis are the result of the action of the immune response mechanisms, including both innate and adaptive, and can not reproduce these effects in tissue culture assays. Another problem limiting the studies is that no one knows precisely what is being observed and what for. So, to address this field, most studies begin with the premise that if a virus has a defective vir‐ ulence gene, it may not cause disease or, if at all, can only cause a weak disease, such that this reasoning can cause confusion. Molecular directed mutations has been a tool that, al‐ though difficult to control, has greatly contributed to the characterization of virulence genes. Thus, the reversion of mutations (mutations repair), the mixture of mutant and wild viruses, among others, have identified genetic defects in virulence. Some mutations lead to eliminated, reduced or increased protein function, whereas other proteins affect

determine if it causes a mild or severe infection.

and may facilitate systemic spread [1].

which slows replication.


To infect its host, a virus must first enter cells at a body surface. Common sites of entry in‐ clude the mucosal linings of the respiratory, alimentary and urogenital tracts, the outer sur‐ face of the eye (conjunctival membranes or cornea), and the skin.

Among the factors that affect the infection process are:


utes to the kinetics of viral spread [2]. The amount of virus inoculated may influence or determine if it causes a mild or severe infection.

**d. Speed of replication.** Some viruses replicate so rapidly that they often cause acute in‐ fections, others are slow virus replication, or some have to travel greater distances, which slows replication.

**2. Viral pathogenesis**

4 Respiratory Disease and Infection - A New Insight

isfied to ensure the infection of an individual host [1]:

**•** Sufficient virus must be available to initiate infection,

Among the factors that affect the infection process are:

result of virus-induced liver damage [1].

disease, respectively.

The term "pathogenesis" refers to the processes or mechanisms to generate an injury or ill‐ ness, in this case induced by a viral infection. The results of a viral infection depend on fac‐ tors related to the nature of the virus, the host and the environment. They include: number of infectious particles, the way to reach the target tissue, the rate of multiplication, the effect of virus on cell functions and the host's immune response. Three requirements must be sat‐

**•** Cells at the site of infection must be accessible, susceptible, and permissive for the virus

To infect its host, a virus must first enter cells at a body surface. Common sites of entry in‐ clude the mucosal linings of the respiratory, alimentary and urogenital tracts, the outer sur‐

**a. Virulence.** Virulence is under polygenic control and is not assignable to any isolated property of the virus, but is often associated to characteristics that favor viral repli‐ cation and cellular injury. For example, virulent viruses multiply themselves readily at high temperatures prevailing during the disease, block the synthesis of interferon and macromolecules related to immune system. Viral virulence is a quantitative statement of the degree or extent of pathogenesis. In general, a virulent virus causes significant disease, whereas an avirulent or attenuated virus causes no or reduced

**b. Measuring Viral Virulence.** Virulence can be quantified in a number of different ways. One approach is to determine the concentration of virus that causes death or disease in 50% of the infected organisms. This parameter is called the 50% lethal dose (LD50), the 50% paralytic dose (PD50), or the 50% infectious dose (ID50), depending on the parame‐ ter that is measured. Other measurements of virulence include mean time to death or appearance of symptoms, as well as the measurement of fever or weight loss. Virus-in‐ duced tissue damage can be measured directly by examining histological sections or blood samples. For example, safety of live attenuated poliovirus vaccine is determined by assessing the extent of pathological lesions in the central nervous system in experi‐ mentally inoculated monkeys. Indirect measures of virulence include assays for liver enzymes (alanine or aspartate amino-transferases) that are released into the blood as a

**c. The amount of inoculum.** The impact of virus dose on the outcome of infection is poor‐ ly understood. It has been shown that, for rhinovirus, the size of the inoculum contrib‐

**•** Local host anti-viral defense systems must be absent or initially ineffective.

**1. Virus-dependent factors.** They usually are dependent on the virus structure.

face of the eye (conjunctival membranes or cornea), and the skin.


the level of transcription, translation or replication of the genetic information [1]. The viral genes that affect virulence status can be classified into four groups or classes: 1. those af‐ fecting the ability of the virus to replicate; 2. genes that modify the host's defense mecha‐ nisms; 3. genes that allow the virus to spread in the host, and 4. genes that codify proteins having toxic effects [1].

Usually, viruses that cause respiratory infections penetrate through the epithelium replicat‐ ing at the site and causing localized infections. Sometimes, as in the case of herpes infection, virions bind to nerve endings in the nasopharyngeal cavity until they find the trigeminal ganglion and even spread to the brain, causing encephalitis; other viruses, such as measles, rubella, mumps etc., may enter through airways not being this site its target organ, so viral

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 7

**Tropism.** It is the ability of a virus to infect or damage specific cells, tissues, organs or spe‐ cific cells. In some virus is strictly limited, other are pantropic and are able to infect and rep‐ licate in different types of cells and tissues. The tropism contributes significantly to the virulence and pathogenesis of viral infections, and is determined by several factors that in‐ tervere in the virus-host relationship such as the gateway and route of viral spread, the per‐ missibility of the cell (receptors, cell differentiation), the nature of the innate and adaptive

**Cell membrane receptors.** A cell may be susceptible to viral infection if viral receptors are present and functional. In other words, if the viral receptor is not expressed, the tis‐ sue can not be infected. In epithelial cells from human respiratory tract, some receptors habe been identified such as N-acetyl neuraminic acid, glycosaminoglycans and glycoli‐ pids, ICAM integrin and molecules of the Major Histocompatibility Complex. In airways the sialic acid receptor that binds to the influenza virus has been identified. This recep‐ tor is found in several tissues of the body, although the infection in humans is restricted to the respiratory tract. Influenza A viruses infect a variety of animals. While viruses that infect humans bind to sialic acid type α-2,6, in birds they bind to α-2,3 type that is localized in the gastrointestinal epithelium where the virus replicates. In pigs, the virus can recognize both types, which facilitates the generation of gene arrangements between

**Virus-cell interaction.** The interaction of a virus with its cellular receptor is mediated by one or more surface proteins. In enveloped viruses, the envelope glycoproteins (e.g. the influen‐ za virus hemagglutinin); in naked viruses, the capsid proteins (e.g. exon protein of the ade‐ novirus). Enveloped viruses have the ability to fuse directly to the cell membrane allowing the entry of the nucleocapside into the cytoplasm. Naked viruses and some enveloped virus‐ es have the capacity to fuse to che cell membrane by means of endocytosis. Some viruses

Some viruses require cellular proteases that cut viral proteins to form an infectious viral par‐ ticle. During an influenza virus infection, a cellular protease cuts an HA precursor generat‐ ing two subunits in order to activate and allow the fusion between the viral envelope and the cell membrane. It has been described that alterations in the cleavage site of the HA of influenza virus causes changes in the pathogenicity of the virus, in fact, highly pathogenic strains of birds contain multiple basic amino acids at the cleavage site of the HA that is rec‐ ognized by different proteases. As a result these strains are capable of infecting various or‐ gans such as spleen, liver, lung, kidney and brain. This same cutting activation procedure is

require co-receptor molecules to penetrate the cell as happens with Adenovirus [7].

performed with the F protein of the virus of the *Paramyxoviridae* family.

particles will be spread through various mechanisms.

immune response of the host and specific tisular features.

strains of different origin [1, 5, 6].

**2. Host-dependent factors.** There are factors that are innate to host such as: race and ge‐ netic load, sex, age, immunological and nutritional status, weight, etc.. These factors and the presence of specific cellular receptors for a given virus can determine resistance or susceptibility to viral infection. Subsequently, adaptive immune defense will enter into action and influence the success or the elimination of the infection.

**Cellular virulence genes.** Numerous studies have shown that certain cellular genes can be considered as virulence determinants [1]. Among the candidate genes are genes encoding com‐ ponents of the host immune response such as proteins required for T- and B-cell function, as well as cytokines. When these genes are altered, proteins do not perform correctly their func‐ tion, which can have adverse effects during viral infection; thus, the disease may be more or less severe. Other candidate genes are cellular genes that encode proteins required for replica‐ tion, translation, transcription and mRNA synthesis and are considered cellular virulence de‐ terminants; however, there are few studies that demonstrate this condition [1]. This field is still poor studied, but with the current tools and knowledge on the pathogenesis mechanisms, re‐ sults are being achieved that in a near future will help us to learn more about the subject.

**3. Enviromental factors.** Environmental conditions such as temperature, moisture, pH, aeration, etc., can influence the viability of the virus before reaching their target organ and affect or facilitate its infectivity. A well-known example is the winter predominance of respiratory viral viruses and the summer propagation of enteric viruses.

#### **3. Cellular level pathogenesis**

Molecular interactions between the virus and the cell result in a phenomenon called patho‐ genesis. It can be analyzed at different levels ranging from the early interactions (cellular re‐ ceptors) to the expression and suppression of cellular and viral genes, resulting in the production of inflammatory, pro-apoptotic or anti-apoptotic proteins, whose presence or ab‐ sence induce the activation of complex networks of proteins that interact in cellular signal‐ ing pathways [3]. The sensitivity or resistance of a cell to viral infection is determined by early interactions with the virus, such as the adhesion and release of nucleic acids in the cell, and is strongly related to the characteristics of the cell, such as physiological maturation, ge‐ netic characteristics and specific receptors for a given virus [4].

**Molecular gateway and viral spread.** The site of entry of a virus is defined by the presence of specific receptors for a virus. Also, the gateway sets the path of its spread and conse‐ quently the disease process, which in some viral infections are not always predictable.

Usually, viruses that cause respiratory infections penetrate through the epithelium replicat‐ ing at the site and causing localized infections. Sometimes, as in the case of herpes infection, virions bind to nerve endings in the nasopharyngeal cavity until they find the trigeminal ganglion and even spread to the brain, causing encephalitis; other viruses, such as measles, rubella, mumps etc., may enter through airways not being this site its target organ, so viral particles will be spread through various mechanisms.

the level of transcription, translation or replication of the genetic information [1]. The viral genes that affect virulence status can be classified into four groups or classes: 1. those af‐ fecting the ability of the virus to replicate; 2. genes that modify the host's defense mecha‐ nisms; 3. genes that allow the virus to spread in the host, and 4. genes that codify proteins

**2. Host-dependent factors.** There are factors that are innate to host such as: race and ge‐ netic load, sex, age, immunological and nutritional status, weight, etc.. These factors and the presence of specific cellular receptors for a given virus can determine resistance or susceptibility to viral infection. Subsequently, adaptive immune defense will enter

**Cellular virulence genes.** Numerous studies have shown that certain cellular genes can be considered as virulence determinants [1]. Among the candidate genes are genes encoding com‐ ponents of the host immune response such as proteins required for T- and B-cell function, as well as cytokines. When these genes are altered, proteins do not perform correctly their func‐ tion, which can have adverse effects during viral infection; thus, the disease may be more or less severe. Other candidate genes are cellular genes that encode proteins required for replica‐ tion, translation, transcription and mRNA synthesis and are considered cellular virulence de‐ terminants; however, there are few studies that demonstrate this condition [1]. This field is still poor studied, but with the current tools and knowledge on the pathogenesis mechanisms, re‐ sults are being achieved that in a near future will help us to learn more about the subject.

**3. Enviromental factors.** Environmental conditions such as temperature, moisture, pH, aeration, etc., can influence the viability of the virus before reaching their target organ and affect or facilitate its infectivity. A well-known example is the winter predominance

Molecular interactions between the virus and the cell result in a phenomenon called patho‐ genesis. It can be analyzed at different levels ranging from the early interactions (cellular re‐ ceptors) to the expression and suppression of cellular and viral genes, resulting in the production of inflammatory, pro-apoptotic or anti-apoptotic proteins, whose presence or ab‐ sence induce the activation of complex networks of proteins that interact in cellular signal‐ ing pathways [3]. The sensitivity or resistance of a cell to viral infection is determined by early interactions with the virus, such as the adhesion and release of nucleic acids in the cell, and is strongly related to the characteristics of the cell, such as physiological maturation, ge‐

**Molecular gateway and viral spread.** The site of entry of a virus is defined by the presence of specific receptors for a virus. Also, the gateway sets the path of its spread and conse‐ quently the disease process, which in some viral infections are not always predictable.

of respiratory viral viruses and the summer propagation of enteric viruses.

into action and influence the success or the elimination of the infection.

having toxic effects [1].

6 Respiratory Disease and Infection - A New Insight

**3. Cellular level pathogenesis**

netic characteristics and specific receptors for a given virus [4].

**Tropism.** It is the ability of a virus to infect or damage specific cells, tissues, organs or spe‐ cific cells. In some virus is strictly limited, other are pantropic and are able to infect and rep‐ licate in different types of cells and tissues. The tropism contributes significantly to the virulence and pathogenesis of viral infections, and is determined by several factors that in‐ tervere in the virus-host relationship such as the gateway and route of viral spread, the per‐ missibility of the cell (receptors, cell differentiation), the nature of the innate and adaptive immune response of the host and specific tisular features.

**Cell membrane receptors.** A cell may be susceptible to viral infection if viral receptors are present and functional. In other words, if the viral receptor is not expressed, the tis‐ sue can not be infected. In epithelial cells from human respiratory tract, some receptors habe been identified such as N-acetyl neuraminic acid, glycosaminoglycans and glycoli‐ pids, ICAM integrin and molecules of the Major Histocompatibility Complex. In airways the sialic acid receptor that binds to the influenza virus has been identified. This recep‐ tor is found in several tissues of the body, although the infection in humans is restricted to the respiratory tract. Influenza A viruses infect a variety of animals. While viruses that infect humans bind to sialic acid type α-2,6, in birds they bind to α-2,3 type that is localized in the gastrointestinal epithelium where the virus replicates. In pigs, the virus can recognize both types, which facilitates the generation of gene arrangements between strains of different origin [1, 5, 6].

**Virus-cell interaction.** The interaction of a virus with its cellular receptor is mediated by one or more surface proteins. In enveloped viruses, the envelope glycoproteins (e.g. the influen‐ za virus hemagglutinin); in naked viruses, the capsid proteins (e.g. exon protein of the ade‐ novirus). Enveloped viruses have the ability to fuse directly to the cell membrane allowing the entry of the nucleocapside into the cytoplasm. Naked viruses and some enveloped virus‐ es have the capacity to fuse to che cell membrane by means of endocytosis. Some viruses require co-receptor molecules to penetrate the cell as happens with Adenovirus [7].

Some viruses require cellular proteases that cut viral proteins to form an infectious viral par‐ ticle. During an influenza virus infection, a cellular protease cuts an HA precursor generat‐ ing two subunits in order to activate and allow the fusion between the viral envelope and the cell membrane. It has been described that alterations in the cleavage site of the HA of influenza virus causes changes in the pathogenicity of the virus, in fact, highly pathogenic strains of birds contain multiple basic amino acids at the cleavage site of the HA that is rec‐ ognized by different proteases. As a result these strains are capable of infecting various or‐ gans such as spleen, liver, lung, kidney and brain. This same cutting activation procedure is performed with the F protein of the virus of the *Paramyxoviridae* family.

**Sensitive cells.** These cells have specific receptors on the cell membrane, capable of interact‐ ing with the virus antigenic proteins and to allow the infectious process. According to whether the cell allows or not the virus replication, it can differentiate them into permissive and non-permissive [8].

During the viral infection, cells may respond in different way, such that the ECP is dif‐ ferent for each type of virus which might allow us to identify the virus. However, there are cases in which the cells show no apparent change. The ECP is a manifestation of the infectious process, and is defined as "morphological and functional changes of cells caused by a virus and is visible under the microscope, resulting in cell death". In cul‐ tures infected with influenza virus, cells were rounded and clustered like a bunch of grapes (Figure 1a) Adenovirus also rounded the cells but retract into a sphere. Respirato‐ ry syncytial virus (Figure 1b) and herpes simplex type I and II induce fusion of cell

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 9

Alteration of membranes. The plasmatic membrane is the first part of the cell with a vi‐ rus contacts, this interaction occurs at the junction between the individual components of the cell surface proteins and the virus surface. After entry of the intact viral particle, and if penetration was by endocytosis, the genome is released into the cytoplasm after dis‐ ruption of the membrane endocytic. In the case of paramyxovirus, a family of enveloped viruses and RNA genome, viruses contain two glycoproteins on its surface, one is the F protein that is able to initiate membrane fusion at acidic pH, the viral genome is intro‐ duced directly into the cell as a result of the fusion between the viral envelope and the cell plasma membrane. During the acute infection by cytolytic virus, especially the nonenveloped in the infected cell which finally releases large amounts of virus, the plasma membrane is damaged until to rupture. At this time, cytoplasmic proteins that are fil‐ tered, and ions such as Na + and Κ+ allow the entry of water and the development of

**Cell lysis.** Besides membrane damage by the entry of viral particles there are differene cell membrane alterations, including the nucleous and organelles that lead to cell lysis. Cell lysis is mainly due to the inhibition of cellular macromolecular synthesis by some viral proteins. DNA viruses inhibit early the cellular DNA synthesis and during late pe‐ riods cellular RNA and proteins (e.g. adenovirus). RNA viruses inhibit the synthesis of RNA and proteins from earlier periods. The accumulation of viral products causes cell

**Effect on the cytoskeleton.** Some viral and cellular proteins synthesized during infection act on the cell cytoskeleton. This alteration induces that cell is made round; this occurs mainly in cells infected with adenovirus. Other changes in the cytoskeleton are caused by oncogenic viruses that cause a cell morphology change (e.g. human papilloma virus in laryngeal papil‐ lomatosis). Cells that possess cilia, such as respiratory tract, lack their ciliary functionality

**Cellular fusion.** Some viruses have structural proteins (e.g. F protein) which have the prop‐ erty of fusing cell membranes. In infected cells, same viral protein allows the fusion between neighboring cells, giving rise to multinucleated cells that are called polykaryocytes or syncy‐ tia. Among the viruses that show syncytia formation are RSV, measles, parainfluenza, her‐ pes simplex, as they have fusion proteins and are able to move from one cell to another

membranes forming syncytia or multinucleated giant cells (figure 1c).

cellular inflammation (cell swelling), which leads to cell lysis.

lysis and release of virions.

during influenza virus infection [9].

without having to leave cell.

**Permissive cells.** Are those that allow the virus enter and allowing the complete viral life cycle, dividing, and producing offspring. So, the virus enters to the cell cytoplasm or nu‐ cleus, depending on the type of virus. In what is called early phase, several viral compo‐ nents are synthesized such as viral proteins. In the subsequent phase, these components are assembled and, in the final or lytic phase, cell death occurs, then freeing new generation vi‐ rus. The infection becomes productive.

**No permissive cells.** These cells have viral receptors, but not allow productive infection. The infection is aborted at any step of the viral replication cycle. Upon access of the virus to these cells there is no synthesis of viral components. In some cases, if the virus is lysogenic, or it is an oncogenic virus, it can be observed the phenomenon of integration of the viral ge‐ nome into the host ´genome.

**Resistant cells.** In all types of infection, the initial event is the interaction between the virus and the corresponding receptor present on the cell surface. If a cell lacks the appropriate re‐ ceptor for a particular virus, is then automatically resistant to infection by that virus [8 ].

#### **4. Cell damage caused by virus and cytopathic effect**

**Virus-induced cell damage.** This damage may be a direct result of viral replication as well as the innate or adaptive immune response of the host; here we mention only those caused by viruses.

**Direct effects on cells mediated by cytopathic viruses.** Viruses cause morphological altera‐ tions known as cytopathic effect (CPE) and occur in both the cells of living organisms and *in vitro* culture cells. The alterations produced in virus infected cells ranging from those that do not immediately lead to cell death and those that destroy rapidly and kill the infected cell.

**Figure 1.** Diferent cytopathic effects in cell cultures. A) MDCK cells infected with influenza A H1N1 virus; B) A549 cells infected with respiratory syncytial virus, the virus includes syncytia formation; C) Vero cells infected with herpes sim‐ plex virus 1, the cytopathic effect of the virus is also the syncytia formation.

During the viral infection, cells may respond in different way, such that the ECP is dif‐ ferent for each type of virus which might allow us to identify the virus. However, there are cases in which the cells show no apparent change. The ECP is a manifestation of the infectious process, and is defined as "morphological and functional changes of cells caused by a virus and is visible under the microscope, resulting in cell death". In cul‐ tures infected with influenza virus, cells were rounded and clustered like a bunch of grapes (Figure 1a) Adenovirus also rounded the cells but retract into a sphere. Respirato‐ ry syncytial virus (Figure 1b) and herpes simplex type I and II induce fusion of cell membranes forming syncytia or multinucleated giant cells (figure 1c).

**Sensitive cells.** These cells have specific receptors on the cell membrane, capable of interact‐ ing with the virus antigenic proteins and to allow the infectious process. According to whether the cell allows or not the virus replication, it can differentiate them into permissive

**Permissive cells.** Are those that allow the virus enter and allowing the complete viral life cycle, dividing, and producing offspring. So, the virus enters to the cell cytoplasm or nu‐ cleus, depending on the type of virus. In what is called early phase, several viral compo‐ nents are synthesized such as viral proteins. In the subsequent phase, these components are assembled and, in the final or lytic phase, cell death occurs, then freeing new generation vi‐

**No permissive cells.** These cells have viral receptors, but not allow productive infection. The infection is aborted at any step of the viral replication cycle. Upon access of the virus to these cells there is no synthesis of viral components. In some cases, if the virus is lysogenic, or it is an oncogenic virus, it can be observed the phenomenon of integration of the viral ge‐

**Resistant cells.** In all types of infection, the initial event is the interaction between the virus and the corresponding receptor present on the cell surface. If a cell lacks the appropriate re‐ ceptor for a particular virus, is then automatically resistant to infection by that virus [8 ].

**Virus-induced cell damage.** This damage may be a direct result of viral replication as well as the innate or adaptive immune response of the host; here we mention only those caused

**Direct effects on cells mediated by cytopathic viruses.** Viruses cause morphological altera‐ tions known as cytopathic effect (CPE) and occur in both the cells of living organisms and *in vitro* culture cells. The alterations produced in virus infected cells ranging from those that do not immediately lead to cell death and those that destroy rapidly and kill the infected cell.

**Figure 1.** Diferent cytopathic effects in cell cultures. A) MDCK cells infected with influenza A H1N1 virus; B) A549 cells infected with respiratory syncytial virus, the virus includes syncytia formation; C) Vero cells infected with herpes sim‐

**4. Cell damage caused by virus and cytopathic effect**

plex virus 1, the cytopathic effect of the virus is also the syncytia formation.

and non-permissive [8].

rus. The infection becomes productive.

8 Respiratory Disease and Infection - A New Insight

nome into the host ´genome.

by viruses.

Alteration of membranes. The plasmatic membrane is the first part of the cell with a vi‐ rus contacts, this interaction occurs at the junction between the individual components of the cell surface proteins and the virus surface. After entry of the intact viral particle, and if penetration was by endocytosis, the genome is released into the cytoplasm after dis‐ ruption of the membrane endocytic. In the case of paramyxovirus, a family of enveloped viruses and RNA genome, viruses contain two glycoproteins on its surface, one is the F protein that is able to initiate membrane fusion at acidic pH, the viral genome is intro‐ duced directly into the cell as a result of the fusion between the viral envelope and the cell plasma membrane. During the acute infection by cytolytic virus, especially the nonenveloped in the infected cell which finally releases large amounts of virus, the plasma membrane is damaged until to rupture. At this time, cytoplasmic proteins that are fil‐ tered, and ions such as Na + and Κ+ allow the entry of water and the development of cellular inflammation (cell swelling), which leads to cell lysis.

**Cell lysis.** Besides membrane damage by the entry of viral particles there are differene cell membrane alterations, including the nucleous and organelles that lead to cell lysis. Cell lysis is mainly due to the inhibition of cellular macromolecular synthesis by some viral proteins. DNA viruses inhibit early the cellular DNA synthesis and during late pe‐ riods cellular RNA and proteins (e.g. adenovirus). RNA viruses inhibit the synthesis of RNA and proteins from earlier periods. The accumulation of viral products causes cell lysis and release of virions.

**Effect on the cytoskeleton.** Some viral and cellular proteins synthesized during infection act on the cell cytoskeleton. This alteration induces that cell is made round; this occurs mainly in cells infected with adenovirus. Other changes in the cytoskeleton are caused by oncogenic viruses that cause a cell morphology change (e.g. human papilloma virus in laryngeal papil‐ lomatosis). Cells that possess cilia, such as respiratory tract, lack their ciliary functionality during influenza virus infection [9].

**Cellular fusion.** Some viruses have structural proteins (e.g. F protein) which have the prop‐ erty of fusing cell membranes. In infected cells, same viral protein allows the fusion between neighboring cells, giving rise to multinucleated cells that are called polykaryocytes or syncy‐ tia. Among the viruses that show syncytia formation are RSV, measles, parainfluenza, her‐ pes simplex, as they have fusion proteins and are able to move from one cell to another without having to leave cell.

**Inclusion bodies.** The inclusion bodies are intracellular granules consisting by virions or vi‐ ral subunits. Its location is variable, can be intracytoplasmic as those induced by rabies vi‐ rus, nuclear such as adenovirus or those caused by the virus of measles which are both nuclear and intracytoplasmic. Another example is the eosinophil corpuscles observed in cells infected by herpes simplex. Inclusion bodies break or change the cellular structure and function inducting cell death [1].

**Viremia.** Generally viruses causing the ARI do not cause viremia.

decrease of apoptosis levels [10-12].

level of the virus.

**6. Types of infection**

four basic patterns of infection:

disease becomes severe.

infections in early stages.

Hepatitis B virus.

usually the immune response shows short duration or it is incomplete.

because they represent an important source of transmission.

**Immunity of short duration.** As a result of the alteration of immunity mentioned above,

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 11

**Evasion of the immune response.** The strategies used by viruses to evade the immune re‐ sponse are varied, from antigenic variation to the blocking of on inflammation process, and

**Association with other microorganisms.** Not much is known about this, but there have been some events that suggest it, for example, the bacterium *Staphylococcus aureus* produces a protease that can activate the influenza virus hemagglutinin, thus increasing the virulence

The interactions that occur between the virus and the host can take many forms, there are

**1. Subclinical infections.** Refers to infections that do not show clnical simptoms of dis‐ ease in a host. They are very common in airways and are epidemiologically important

**2. Clinical infections.** These infections show symptoms and signs, the most common are acute respiratory infections which are characterized by quickly presentation with short incubation period as well as the duration of the disease. Usually, the virus is eliminated by the immune system and the physiological condition of the organism. Sometimes the

**3. Abortive infections.** Infection is interrupted in any step of the virus replication cycle. A clear example is the infection with poliomyelitis virus, which causes frequent abortive

**4. Persistent infections.** After an acute infection, the virus is not eliminated and it can still replicate for long periods. The course of the infection can take one of three ways:

**a. Latent infections.** The virus remains most of the time hidden without replication, how‐ ever, it can reactivate resulting in clinical manifestations. The organs or tissues where the virus remains dormant during respiratory tract infections are: The Herpes simplex virus in the trigeminal ganglion; varicella in sensory ganglia; Epstein Barr virus in B lymphocytes; Cytomegalovirus in renal and salivary cells; adenovirus in adenoids. **b. Chronic infections.** After clinical or subclinical infection, the virus continues to mul‐ tiply very slowly but continuously. Some viruses can integrate their genome into the cell, some not. Clinical manifestations may take years to develop but once manifest progress very fast. A typical example, although not a respiratory infection, is the

**Induction of chromosomal aberrations.** Viruses can cause changes at nuclear level that lead to the disintegration of the chromatin of infected cells as occurs in the herpes simplex virus infections. However, nuclear or chromosomal abnormalities can be as subtle to be detected by molecular methodologies, as example, as in the integration of viral genomes into the cel‐ lular genome during transformation mediated by certain viruses, in which the cell is alive, but altered in its properties. Other viruses that cause aberrations are mumps virus, measles, rubella, parainfluenza and adenovirus [10].

**Cellular Transformation and cell proliferation.** DNA and RNA viruses may integrate its genome into the cell, generating transformed cells that behave similarly *in vitro* to cancer cells. Cellular transformation corresponds to a phenomenon that occurs both *in vivo* and *in vitro* and has yielded valuable information regarding the etiology of certain cancers. Some viral proteins inactivate cell proteins which control the cell cycle and hyperplastic processes occur, inducing proliferation or cell growth, for instance, papilloma virus causing laryngeal papillomatosis that can lead to cancer [11]

#### **5. Description and characteristics of virus**

Viruses are microscopic infectious agents that are composed of genetic material (DNA or RNA), surrounded by a protein coat called capsid (naked virus), other viruses have a lipid membrane (enveloped viruses) showing glycoprotein spikes. The entire infectious unit is called virion. The proteins of the capsid of both, naked and enveloped viruses and the glyco‐ proteins of enveloped viruses are the major antigens for inducing immune response of the host. The viruses replicate only in living cells, its genome contains the information needed to program the host cell to synthesize the virus specific molecules required for production of viral progeny [11, 12].

The pathogenicity of a virus is the ability to cause disease and is measured by the degree of virulence which in turn provides for determinants such as: ability to infect, replicate, invade cells, evasion of the host immune system and cause cellular damage. These virulence deter‐ minants are encoded by viral genes.

During the pathogenesis of an acute respiratory infection (ARI) are aspects that are shared by all the viruses that cause them:

**Adherence capacity.** Viruses must evade host innate immunity and defense mechanisms, such as mucociliary barriers, phagocytic cells and NK cells, and to adhere to achieve target.

**Incubation period.** Most ARI causing virus, have short incubation periods.

**Viremia.** Generally viruses causing the ARI do not cause viremia.

**Immunity of short duration.** As a result of the alteration of immunity mentioned above, usually the immune response shows short duration or it is incomplete.

**Evasion of the immune response.** The strategies used by viruses to evade the immune re‐ sponse are varied, from antigenic variation to the blocking of on inflammation process, and decrease of apoptosis levels [10-12].

**Association with other microorganisms.** Not much is known about this, but there have been some events that suggest it, for example, the bacterium *Staphylococcus aureus* produces a protease that can activate the influenza virus hemagglutinin, thus increasing the virulence level of the virus.

#### **6. Types of infection**

**Inclusion bodies.** The inclusion bodies are intracellular granules consisting by virions or vi‐ ral subunits. Its location is variable, can be intracytoplasmic as those induced by rabies vi‐ rus, nuclear such as adenovirus or those caused by the virus of measles which are both nuclear and intracytoplasmic. Another example is the eosinophil corpuscles observed in cells infected by herpes simplex. Inclusion bodies break or change the cellular structure and

**Induction of chromosomal aberrations.** Viruses can cause changes at nuclear level that lead to the disintegration of the chromatin of infected cells as occurs in the herpes simplex virus infections. However, nuclear or chromosomal abnormalities can be as subtle to be detected by molecular methodologies, as example, as in the integration of viral genomes into the cel‐ lular genome during transformation mediated by certain viruses, in which the cell is alive, but altered in its properties. Other viruses that cause aberrations are mumps virus, measles,

**Cellular Transformation and cell proliferation.** DNA and RNA viruses may integrate its genome into the cell, generating transformed cells that behave similarly *in vitro* to cancer cells. Cellular transformation corresponds to a phenomenon that occurs both *in vivo* and *in vitro* and has yielded valuable information regarding the etiology of certain cancers. Some viral proteins inactivate cell proteins which control the cell cycle and hyperplastic processes occur, inducing proliferation or cell growth, for instance, papilloma virus causing laryngeal

Viruses are microscopic infectious agents that are composed of genetic material (DNA or RNA), surrounded by a protein coat called capsid (naked virus), other viruses have a lipid membrane (enveloped viruses) showing glycoprotein spikes. The entire infectious unit is called virion. The proteins of the capsid of both, naked and enveloped viruses and the glyco‐ proteins of enveloped viruses are the major antigens for inducing immune response of the host. The viruses replicate only in living cells, its genome contains the information needed to program the host cell to synthesize the virus specific molecules required for production of

The pathogenicity of a virus is the ability to cause disease and is measured by the degree of virulence which in turn provides for determinants such as: ability to infect, replicate, invade cells, evasion of the host immune system and cause cellular damage. These virulence deter‐

During the pathogenesis of an acute respiratory infection (ARI) are aspects that are shared

**Adherence capacity.** Viruses must evade host innate immunity and defense mechanisms, such as mucociliary barriers, phagocytic cells and NK cells, and to adhere to achieve target.

**Incubation period.** Most ARI causing virus, have short incubation periods.

function inducting cell death [1].

10 Respiratory Disease and Infection - A New Insight

rubella, parainfluenza and adenovirus [10].

papillomatosis that can lead to cancer [11]

viral progeny [11, 12].

minants are encoded by viral genes.

by all the viruses that cause them:

**5. Description and characteristics of virus**

The interactions that occur between the virus and the host can take many forms, there are four basic patterns of infection:


**c. Slow Infections.** This kind of infections have a long incubation period that lasts for months or years, symptoms usually do not occur during the incubation period. A well known example is the persistent infection showed by measles in the nervous system causing SSPE, usually conducting to death.

senteeism is frequent. The virus originated 70-90% of these episodes and viruses that are associated with infections of the upper respiratory tract are: respiratory syncytial vi‐ rus (RSV), rhinovirus (RV), parainfluenza (PIV), influenza A (IA), adenovirus (AD), hu‐ man metapneumovirus (hMPV), human bocavirus (HBoV) and coronavirus (CoV). A virus can cause several syndromes, also too a syndrome may be caused by different vi‐ ruses such that the clinical manifestations are variable. All individuals can be infected by these viruses, however, it has been observed that children are the most affected. The most common syndromes in upper airway are: nasopharyngitis, adenoiditis, pharyngi‐

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 13

**c. Viral infection in lower airways.** Viral infections in lower respiratory airways occupy a smaller percentage, but with high mortality rates. The groups most at risk are young children and older adults. The disease is increased by several factors including anatomi‐ cal disorders, immunological, metabolic or other diseases such as AIDS, asthma or

In the next series of X-ray images are examples of lung damage caused by viral infections,

**Figure 2.** Radiographic images of airways infection by viruses A) pneumonia caused by respiratory syncytial virus; B) bronchiolitis in children caused by respiratory syncytial virus; Croup parainfluenza virus; pneumonia caused by influen‐

tis, sinusitis, laryngitis and croup [14].

upper and lower respiratory tract.

za virus A (H1N1).

chronic obstructive pulmonary disease (COPD).

**5. Transforming infections.** Few respiratory viruses induce transforming infections, usu‐ ally, the viral genome integrates into cellular DNA or remain as an episome. Some of the expressed proteins interact with genes and other cellular proteins, causing changes in cell growth rates. One example is found in laryngeal papillomatosis [10, 11].

#### **7. Respiratory system**


senteeism is frequent. The virus originated 70-90% of these episodes and viruses that are associated with infections of the upper respiratory tract are: respiratory syncytial vi‐ rus (RSV), rhinovirus (RV), parainfluenza (PIV), influenza A (IA), adenovirus (AD), hu‐ man metapneumovirus (hMPV), human bocavirus (HBoV) and coronavirus (CoV). A virus can cause several syndromes, also too a syndrome may be caused by different vi‐ ruses such that the clinical manifestations are variable. All individuals can be infected by these viruses, however, it has been observed that children are the most affected. The most common syndromes in upper airway are: nasopharyngitis, adenoiditis, pharyngi‐ tis, sinusitis, laryngitis and croup [14].

**c. Slow Infections.** This kind of infections have a long incubation period that lasts for months or years, symptoms usually do not occur during the incubation period. A well known example is the persistent infection showed by measles in the nervous system

**5. Transforming infections.** Few respiratory viruses induce transforming infections, usu‐ ally, the viral genome integrates into cellular DNA or remain as an episome. Some of the expressed proteins interact with genes and other cellular proteins, causing changes

**a. Description of the respiratory system and functions.** The respiratory system consists of a set of organs that are grouped into upper respiratory tract (nasal cavity, pharynx, larynx, trachea) and lower airways (bronchi, bronchioles and lungs). The inner part of these organs is covered by epithelial cells which constitute an active physical barrier against pathogens being an important part of the innate immunity. Another structure of the respiratory amembrane is a mucociliary structure found from the nasal cavity to the distal areas of the lungs, consisting of a layer of mucus produced by goblet cells that maintain a continuous flow through the ciliary movement in the luminal surface respi‐ ratory epithelium. The lungs have not these structures, alveolar macrophages are the cells that are responsible for eliminating pathogens. These structures providing protec‐ tion against respiratory viral infections. However, despite these protection mechanisms, respiratory system of a host may be infected by a virus by binding to specific receptors present in epithelial cells of the mucosa, thereby avoiding its removal by the mucocili‐ ary system or by phagocytic cells. Most viruses that infect humans enter into the body through the respiratory tract as in aerosols produced by coughing or sneezing of other infected hosts. Large particles are usually trapped in the turbinates and sinuses and could cause upper respiratory infections. Smaller particles can reach the alveolar spaces and cause infections in the lower respiratory tract [1, 13]. The viruses that cause respira‐ tory infections in both upper and lower airways are distributed in different families: *Orthomyxoviridae, Paramyxoviridae, Picornaviridae, Reoviridae, Adenoviridae, Herpeviridae* and *Coronaviridae*. After penetration of the virus, they can cause local respiratory infec‐ tions as with most respiratory viruses such as influenza, rhinovirus, respiratory syncy‐ tial virus, parainfluenza virus, coronavirus, bocavirus and metapneumovirus occasionally causing lower respiratory infections. Other viruses such as herpes, mea‐ sles, rubella, mumps and varicella among others enter through airways but move to

**b. Viral infection in upper respiratory tract.** Infections of the upper respiratory tract usu‐ ally present acutely and are the most common infections in humans, arise throughout the year but the incidence is higher in winter, are generally of low severity, however, are the main cause of medical consultation and, in consequence, school and work ab‐

in cell growth rates. One example is found in laryngeal papillomatosis [10, 11].

causing SSPE, usually conducting to death.

**7. Respiratory system**

12 Respiratory Disease and Infection - A New Insight

other organs.

**c. Viral infection in lower airways.** Viral infections in lower respiratory airways occupy a smaller percentage, but with high mortality rates. The groups most at risk are young children and older adults. The disease is increased by several factors including anatomi‐ cal disorders, immunological, metabolic or other diseases such as AIDS, asthma or chronic obstructive pulmonary disease (COPD).

In the next series of X-ray images are examples of lung damage caused by viral infections, upper and lower respiratory tract.

**Figure 2.** Radiographic images of airways infection by viruses A) pneumonia caused by respiratory syncytial virus; B) bronchiolitis in children caused by respiratory syncytial virus; Croup parainfluenza virus; pneumonia caused by influen‐ za virus A (H1N1).

The main syndromes caused by viral infections at the lower respiratory tract are bronchioli‐ tis and pneumonia. Bronchiolitis occurs primarily in young infants and preschool children, the most related virus to this syndrome is the RSV (50-75% of the cases). Pneumonia occurs most often in children younger than 3 years of age, as in bronchiolitis, the RSV virus are in‐ volved (50%), as well as the parainfluenza 1 and 3 virus (25%), other viruses participate with lower percentages. In elderly influenza A virus is the most important agent in causing se‐ vere pneumonia with high mortality rates [14], Figure 3.

**Nonspecific or innate response.** Airway use several mechanisms to recognize a virus and to mount a protective response. Cells of the innate immune system use a pattern recognition re‐ ceptors (PRR) that are expressed on their surface and bind to pathogen-associated molecular patterns (PAMs), which are present in microorganisms. Viral PAMs can be: double stranded RNA or RNA produced during replication, surface proteins or glycoproteins. Toll-like recep‐ tors (TLR) represent a PRR family expressed in most cells of the organism, it have been identi‐ fied 10 human types. The TLRs are formed by a binding domain ligand consisting of leucine repeats that interacts directly with viral antigens; a transmembrane domain and a cytoplasmat‐ ic domain responsible for initiating the extracellular signaling. Viral infections activate differ‐ ent TLR receptors (TLRs 3, 7, 8 and 9) that generally induce a protective immune response, however, also can be a part of pathogenic mechanisms. Recently, it has been shown that activa‐ tion of TLRs in epithelial cells by viral infections participate in the regulation of expression of several genes encoding for cytokines, such as: tumor necrosis factor-alpha (TNF-α), Interleu‐ kin-1 (IL-1), IL-6, IL-8, IL-18, interferon alpha and beta (IFN-α and -β), chemokines (leuko‐ trienes, prostaglandins) and antimicrobial peptides (α and β defensins), which are of great

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 15

importance in the organization of the innate and adaptive immune response.

and antimicrobial peptides) [15- 19].

alteration of inflammatory mediators.

The components of the innate immunity are the physical and chemical barriers (epithelia and mucosae), the phagocytic process includes the participation of phagocytic cells (mono‐ cytes, macrophages and neutrophils), dendritic cells (DC) and natural killer cells (NK), also includes the production of soluble molecules (interferons, complement, acute phase proteins

**Epithelial cells.** These cells are actively involved in the production of proteins (lactoferrin), enzymes (lysozyme) and antimicrobial peptides (defensins) which together eliminate or neutralize the virus. When the epithelium loses its integrity by the effect of viral infection, it can be observed the following consequences: exposure of sensory nerve endings, receptors found in the basal membrane is increased, the substances that modulate muscle tone and sensitivity are not working properly, finally, the active inflammatory response results in the

**Natural killer cells (NK).** They are large lymphocytes with intracellular granules. An anti‐ body binds to the surface of a cell infected by a virus, interacts with the Fc receptors and NK cells release proteins (perforins and granzymes) causing cell death. NK cells can be activated by the stimulation of IFN-β and α and other cytokines such as IL-12, IL15 and IL18 pro‐

**Dendritic cells (DC).** DC are present in various tissues as skin, epithelium and mucosal. DC express MHC molecules on their surface localize the virus and migrate to the closest lymph

**Soluble molecules.** Among the soluble molecules involved in innate immunity are: comple‐

**Complement.** Complement is a system consisting of over 30 proteins that are activated by proteolysis in sequence. The complement is found in the human plasma as an inactive form and can be activated by three different pathways: the classical pathway, the alternative path‐

duced by infected cells, dendritic cells (DC) or macrophage (fig.3), [16, 17, 18, 19].

node traveling through the lymphatic vessels eliminating the microorganism [17, 18].

ment, interferons, antimicrobial peptides and acute phase proteins.

**Figure 3.** The Respiratory tract and the main syndromes caused by viral infections. The viruses can infect the respirato‐ ry tract upper and occasionally, some of them can cause infections in the lower respiratory tracts. Others enter trought the respiratory tracts but they move to other organs.

#### **8. Immune response in the respiratory system (innate and adaptive), cells and mechanisms**

The human immune system is divided in two defense mechanisms or responses: a) Innate or nonspecific response that lacks specificity and memory, is the first line of defense of the or‐ ganism, its components are always present to act immediately and b) Specific or adaptive response. This response is more complex, has a memory and identifies the viral specific pep‐ tides processed by antigen presenting cells, which activate the humoral immune response mediated by B cells or a T cell mediated cellular response. An efficient immune response de‐ pends on a correct interaction between the innate and adaptive immune system.

**Nonspecific or innate response.** Airway use several mechanisms to recognize a virus and to mount a protective response. Cells of the innate immune system use a pattern recognition re‐ ceptors (PRR) that are expressed on their surface and bind to pathogen-associated molecular patterns (PAMs), which are present in microorganisms. Viral PAMs can be: double stranded RNA or RNA produced during replication, surface proteins or glycoproteins. Toll-like recep‐ tors (TLR) represent a PRR family expressed in most cells of the organism, it have been identi‐ fied 10 human types. The TLRs are formed by a binding domain ligand consisting of leucine repeats that interacts directly with viral antigens; a transmembrane domain and a cytoplasmat‐ ic domain responsible for initiating the extracellular signaling. Viral infections activate differ‐ ent TLR receptors (TLRs 3, 7, 8 and 9) that generally induce a protective immune response, however, also can be a part of pathogenic mechanisms. Recently, it has been shown that activa‐ tion of TLRs in epithelial cells by viral infections participate in the regulation of expression of several genes encoding for cytokines, such as: tumor necrosis factor-alpha (TNF-α), Interleu‐ kin-1 (IL-1), IL-6, IL-8, IL-18, interferon alpha and beta (IFN-α and -β), chemokines (leuko‐ trienes, prostaglandins) and antimicrobial peptides (α and β defensins), which are of great importance in the organization of the innate and adaptive immune response.

The main syndromes caused by viral infections at the lower respiratory tract are bronchioli‐ tis and pneumonia. Bronchiolitis occurs primarily in young infants and preschool children, the most related virus to this syndrome is the RSV (50-75% of the cases). Pneumonia occurs most often in children younger than 3 years of age, as in bronchiolitis, the RSV virus are in‐ volved (50%), as well as the parainfluenza 1 and 3 virus (25%), other viruses participate with lower percentages. In elderly influenza A virus is the most important agent in causing se‐

**Figure 3.** The Respiratory tract and the main syndromes caused by viral infections. The viruses can infect the respirato‐ ry tract upper and occasionally, some of them can cause infections in the lower respiratory tracts. Others enter trought

**8. Immune response in the respiratory system (innate and adaptive), cells**

The human immune system is divided in two defense mechanisms or responses: a) Innate or nonspecific response that lacks specificity and memory, is the first line of defense of the or‐ ganism, its components are always present to act immediately and b) Specific or adaptive response. This response is more complex, has a memory and identifies the viral specific pep‐ tides processed by antigen presenting cells, which activate the humoral immune response mediated by B cells or a T cell mediated cellular response. An efficient immune response de‐

pends on a correct interaction between the innate and adaptive immune system.

vere pneumonia with high mortality rates [14], Figure 3.

14 Respiratory Disease and Infection - A New Insight

the respiratory tracts but they move to other organs.

**and mechanisms**

The components of the innate immunity are the physical and chemical barriers (epithelia and mucosae), the phagocytic process includes the participation of phagocytic cells (mono‐ cytes, macrophages and neutrophils), dendritic cells (DC) and natural killer cells (NK), also includes the production of soluble molecules (interferons, complement, acute phase proteins and antimicrobial peptides) [15- 19].

**Epithelial cells.** These cells are actively involved in the production of proteins (lactoferrin), enzymes (lysozyme) and antimicrobial peptides (defensins) which together eliminate or neutralize the virus. When the epithelium loses its integrity by the effect of viral infection, it can be observed the following consequences: exposure of sensory nerve endings, receptors found in the basal membrane is increased, the substances that modulate muscle tone and sensitivity are not working properly, finally, the active inflammatory response results in the alteration of inflammatory mediators.

**Natural killer cells (NK).** They are large lymphocytes with intracellular granules. An anti‐ body binds to the surface of a cell infected by a virus, interacts with the Fc receptors and NK cells release proteins (perforins and granzymes) causing cell death. NK cells can be activated by the stimulation of IFN-β and α and other cytokines such as IL-12, IL15 and IL18 pro‐ duced by infected cells, dendritic cells (DC) or macrophage (fig.3), [16, 17, 18, 19].

**Dendritic cells (DC).** DC are present in various tissues as skin, epithelium and mucosal. DC express MHC molecules on their surface localize the virus and migrate to the closest lymph node traveling through the lymphatic vessels eliminating the microorganism [17, 18].

**Soluble molecules.** Among the soluble molecules involved in innate immunity are: comple‐ ment, interferons, antimicrobial peptides and acute phase proteins.

**Complement.** Complement is a system consisting of over 30 proteins that are activated by proteolysis in sequence. The complement is found in the human plasma as an inactive form and can be activated by three different pathways: the classical pathway, the alternative path‐

way and the lectin pathway; viral infections can trigger the three pathways. Complement is more efficient during the attack to enveloped viruses, because complement activation finish‐ ed with the formation of a attack complex which is inserted in to viral membrane, causing the lysis of the virus [16, 19]. Complement anaphylatoxins (C3a and C5a) induce histamine, prostaglandins and leukotrienes release, promoting bronchoconstriction. C5a is a chemotac‐ tic factor for a variety of inflammatory cells. C3a and C5a have been found in high concen‐ trations in the upper airways during infection by influenza virus. It has also been shown that RSV-infected cells activate complement [13, 15].

**Figure 5.** Influenza virus mechanisms to evade interferon action. The, NS1 protein encoded by the virus genome sup‐ presses induction of IFNs-α/β. P58IPK is a cellular inhibitor of PKR that is activated by influenza – virus infection. Modi‐

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 17

**Defensins.** They are cationic small peptides rich in arginine. They are syntethized in leuko‐ cytes, macrophages and epithelial cells constitutively, in response to infection or during in‐ flammation. In humans, there are two types: α and β-defensins. Viral infections induce the production of defensins, which regulate the innate and adaptive immune response (positive and negative). The mechanisms of action and induction of defensins are multiple, generally depend on the type of infecting virus (enveloped and non-enveloped), defensin type and the

The respiratory epithelium induces production of β-defensins, mainly of β-defensin-2. The mechanisms of action include: a) direct, when the peptide binds to the viral membrane by electrostatic attraction, form pores and cause lysis of the viral membrane. This mechanism occurs in the majority of enveloped viruses (influenzavirus, herpes, HIV); and b) indirect, when the defensin inactivate any signaling pathway in any step of the virus replication cycle (herpes, adenovirus). There are few studies on the mechanisms of induction of β-defensins

fied from: Katze 2002.

target cell infected [18, 24].

**Figure 4.** Activation of NK, dendritic cells and soluble molecules during virus infection. The viruses trigger the produc‐ tion of type – 1 interferons (IFNs) by plasmacytoid dendritic cells and other cytokines as interleukin – 12 (IL – 12). IL – 12 and IFN induce the production of IL – 15 by dendritic cells DC). IL – 15 is presented to NK cells, so that NK cells are activated. IL – 15 trigger other pro – inflammatory cytokines, including either the secretion of IFN – γ by NK cells or the release of perforin and grazymes, which leads to citotoxicity. Modified from: Lanier 2008.

**Interferons (α and β).** Interferons are cytokines that are produced in small amounts by cells infected with virus. Are efficiently induced by the presence of double-stranded viral RNA (viral replication intermediary), the process involves three antiviral proteins: protein kinase (PKR), Oas1 and RnasaL, which block the translation and degradation of viral and cellular RNAs. Interesntingly, influenza virus induces high levels of interferon with protective prop‐ erties [20, 21, 22, 23] figure 5.

way and the lectin pathway; viral infections can trigger the three pathways. Complement is more efficient during the attack to enveloped viruses, because complement activation finish‐ ed with the formation of a attack complex which is inserted in to viral membrane, causing the lysis of the virus [16, 19]. Complement anaphylatoxins (C3a and C5a) induce histamine, prostaglandins and leukotrienes release, promoting bronchoconstriction. C5a is a chemotac‐ tic factor for a variety of inflammatory cells. C3a and C5a have been found in high concen‐ trations in the upper airways during infection by influenza virus. It has also been shown

**Figure 4.** Activation of NK, dendritic cells and soluble molecules during virus infection. The viruses trigger the produc‐ tion of type – 1 interferons (IFNs) by plasmacytoid dendritic cells and other cytokines as interleukin – 12 (IL – 12). IL – 12 and IFN induce the production of IL – 15 by dendritic cells DC). IL – 15 is presented to NK cells, so that NK cells are activated. IL – 15 trigger other pro – inflammatory cytokines, including either the secretion of IFN – γ by NK cells or the

**Interferons (α and β).** Interferons are cytokines that are produced in small amounts by cells infected with virus. Are efficiently induced by the presence of double-stranded viral RNA (viral replication intermediary), the process involves three antiviral proteins: protein kinase (PKR), Oas1 and RnasaL, which block the translation and degradation of viral and cellular RNAs. Interesntingly, influenza virus induces high levels of interferon with protective prop‐

release of perforin and grazymes, which leads to citotoxicity. Modified from: Lanier 2008.

erties [20, 21, 22, 23] figure 5.

that RSV-infected cells activate complement [13, 15].

16 Respiratory Disease and Infection - A New Insight

**Figure 5.** Influenza virus mechanisms to evade interferon action. The, NS1 protein encoded by the virus genome sup‐ presses induction of IFNs-α/β. P58IPK is a cellular inhibitor of PKR that is activated by influenza – virus infection. Modi‐ fied from: Katze 2002.

**Defensins.** They are cationic small peptides rich in arginine. They are syntethized in leuko‐ cytes, macrophages and epithelial cells constitutively, in response to infection or during in‐ flammation. In humans, there are two types: α and β-defensins. Viral infections induce the production of defensins, which regulate the innate and adaptive immune response (positive and negative). The mechanisms of action and induction of defensins are multiple, generally depend on the type of infecting virus (enveloped and non-enveloped), defensin type and the target cell infected [18, 24].

The respiratory epithelium induces production of β-defensins, mainly of β-defensin-2. The mechanisms of action include: a) direct, when the peptide binds to the viral membrane by electrostatic attraction, form pores and cause lysis of the viral membrane. This mechanism occurs in the majority of enveloped viruses (influenzavirus, herpes, HIV); and b) indirect, when the defensin inactivate any signaling pathway in any step of the virus replication cycle (herpes, adenovirus). There are few studies on the mechanisms of induction of β-defensins in the respiratory epithelium. When rhinovirus infects the respiratory epithelium, the β-de‐ fensin-2 is induced by virus replication, mRNA activates the transcription factor NF-kB, and therefore, the gene that codifies for defensins; however, defensin has no direct effect on the virus. Influenza virus induces the expression of β-defensin-3, which inhibits the binding of the viral hemagglutinin with epithelium membrane. This same defensin inhibits viral fusion with the cell membrane during a RSV infection [25, 26].

In viral infections, CD8+ T cell response is essential for viral clearance. Lymphocytes are able to recognize through its receptor (TCR) by antigen processing performed by antigen pre‐ senting cells (dendritic cells or macrophages) associated with MHC molecules. For the dif‐ ferentiation and activation of T lymphocyte are required two signals are requare: the first is the specific recognition of the antigen on the target MHC class I-associated cel, and the sec‐ ond produced the cytokines produced by CD4+ T cells which recognize MHC-associated vi‐ ral antigens class II. Cytotoxic T lymphocytes exert their antiviral effects by three mechanisms: producing lysis of infected cells, stimulate the production of enzymes that de‐ grade viral genomes, and secrete cytokines. In severe viral infections including pneumonia caused by RSV, influenza and metapneumovirus, this type of response is important for the resolution of the disease; however, it has been observed that in severe cases this response has no effect and the pattern that develops is through cytokines produced by CD4 Th2 spe‐ cialized cells that induce an inflammatory response increasing damage and thereby aggra‐

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 19

Despite effective defenses, some virus can to evade it by using different mechanisms, for ex‐ ample, in respiratory tract infections influenza virus inhibits the production of interferon by the protein NS1, a non-structural protein that is abundantly expressed in the nucleus of in‐ fected cells. NS1 binds to double-stranded RNA by preventing activation of the dsRNA-de‐ pendent protein kinase (PKR) [28], commonly synthesized during induction of interferon (Figure 5). Another mechanism is the antigenic variation that occur mainly in the HA and NA proteins. PI and RSV respiratory viruses have a surface protein (F), which mediates the fusion of the viral membrane with the cell membrane. This mechanism enables the virus to spread from one cell to another without exposure and avoiding the effect of circulating anti‐ bodies. Another strategy is to make a latent infection. Viruses such as adenovirus and her‐ pes employ transcription and replication strategies to maintain the viral genome in any cell type where the immune response is not efficient and viral particles are not produced for long periods. Other viruses interfere with antigen processing or complement. In summary,

**10. Other mechanisms used by respiratory viruses in the pathogenesis**

In most infections, the viruses cause upper respiratory infections, while others reach the lower airways, may even cause necrosis and cell death, also induce inflammatory processes such as wheezing and hyperreactivity, both important in the development of chronic diseas‐ es such as asthma and chronic bronchitis for which some mechanisms have been proposed. In summary, the strategies used by viruses that cause respiratory infections can be very var‐ ied; however, there are always some strategies are shared between different types of viruses.

vating the condition.

**9. Evasion of the immune response**

viruses use several strategies to evade the immune response [11].

**Acute phase proteins (APP).** APP are serum proteins whose its concentration increases or decreases when there is an infection. APP are induced by pro-inflammatory cytokines like TNF-α, IL-6 and IL-1 that are synthesized in the liver. Examples of APP are the lec‐ tin that binds to mannose, C reactive protein and surfactant proteins A and D. Acute phase proteins recognize PAMs viruses, activate complement and enhance the phagocyt‐ ic capacity of immune cells [24].

#### **8.1. Specific or acquired immunity**

When the adaptive immune system contacts with a antigen it is developed a primary re‐ sponse that generates immunological memory, which at a second contact (secondary re‐ sponse) the response is more rapid and intense. There are two specific types of responses: humoral and cellular.

**Humoral response.** The humoral response has as a major component the B lymphocytes that differentiate into antibody-producing plasmatic cells. The antibodies are displaced by the body fluid to bind to antigens. When antibodies interact with phagocytic cells and com‐ plement, the viruses are neutralized. In humans, there are five classes of immunoglobulins (IgA, IgM, IgG, IgE and IgD). In viral respiratory infections, IgA is of great importance as it is secreted by mucous epithelia, preventing the establishment of virus. IgM is the first anti‐ body that is synthesized and prevailing in a primary response, also fix complement. The IgG is found in greater concentration in serum and is the only one that can cross the placenta in humans. Presents an Fc fragment that binds to complement receptors on phagocytic cells. Most respiratory viruses induce such antibodies that persist, and when a second exposure to the same antigen, the disease is less severe; unfortunately, virus may have mutations that are not recognized by the antibodies, therfore, reinfections are common. IgE is the antibody with the lowest concentration in the serum, but is the most important in allergic disorders. It has been reported that some viruses such as RSV, influenza, bocavirus, parainfluenza and metapneumovirus produce bronchial hyperreactivity or asthma increasing concentrations of this immunoglobulin in blood and secretions. Basophils and mast cells have receptors for these antibodies, when the antigen-antibody binding occurs and consequently activate, these cells release inflammatory mediators that cause many manifestations of these respira‐ tory diseases [27].

**Cellular response.** The cellular response has as its principal components the T lymphocytes, which are divided into two populations according to their surface markers and the pattern of cytokines produced: CD4+, also called helper T cells (Th) and CD8 +, the cytotoxic lym‐ phocytes (CTL). These specialized cells can proliferate and differentiate into effector and memory cells.

In viral infections, CD8+ T cell response is essential for viral clearance. Lymphocytes are able to recognize through its receptor (TCR) by antigen processing performed by antigen pre‐ senting cells (dendritic cells or macrophages) associated with MHC molecules. For the dif‐ ferentiation and activation of T lymphocyte are required two signals are requare: the first is the specific recognition of the antigen on the target MHC class I-associated cel, and the sec‐ ond produced the cytokines produced by CD4+ T cells which recognize MHC-associated vi‐ ral antigens class II. Cytotoxic T lymphocytes exert their antiviral effects by three mechanisms: producing lysis of infected cells, stimulate the production of enzymes that de‐ grade viral genomes, and secrete cytokines. In severe viral infections including pneumonia caused by RSV, influenza and metapneumovirus, this type of response is important for the resolution of the disease; however, it has been observed that in severe cases this response has no effect and the pattern that develops is through cytokines produced by CD4 Th2 spe‐ cialized cells that induce an inflammatory response increasing damage and thereby aggra‐ vating the condition.

#### **9. Evasion of the immune response**

in the respiratory epithelium. When rhinovirus infects the respiratory epithelium, the β-de‐ fensin-2 is induced by virus replication, mRNA activates the transcription factor NF-kB, and therefore, the gene that codifies for defensins; however, defensin has no direct effect on the virus. Influenza virus induces the expression of β-defensin-3, which inhibits the binding of the viral hemagglutinin with epithelium membrane. This same defensin inhibits viral fusion

**Acute phase proteins (APP).** APP are serum proteins whose its concentration increases or decreases when there is an infection. APP are induced by pro-inflammatory cytokines like TNF-α, IL-6 and IL-1 that are synthesized in the liver. Examples of APP are the lec‐ tin that binds to mannose, C reactive protein and surfactant proteins A and D. Acute phase proteins recognize PAMs viruses, activate complement and enhance the phagocyt‐

When the adaptive immune system contacts with a antigen it is developed a primary re‐ sponse that generates immunological memory, which at a second contact (secondary re‐ sponse) the response is more rapid and intense. There are two specific types of responses:

**Humoral response.** The humoral response has as a major component the B lymphocytes that differentiate into antibody-producing plasmatic cells. The antibodies are displaced by the body fluid to bind to antigens. When antibodies interact with phagocytic cells and com‐ plement, the viruses are neutralized. In humans, there are five classes of immunoglobulins (IgA, IgM, IgG, IgE and IgD). In viral respiratory infections, IgA is of great importance as it is secreted by mucous epithelia, preventing the establishment of virus. IgM is the first anti‐ body that is synthesized and prevailing in a primary response, also fix complement. The IgG is found in greater concentration in serum and is the only one that can cross the placenta in humans. Presents an Fc fragment that binds to complement receptors on phagocytic cells. Most respiratory viruses induce such antibodies that persist, and when a second exposure to the same antigen, the disease is less severe; unfortunately, virus may have mutations that are not recognized by the antibodies, therfore, reinfections are common. IgE is the antibody with the lowest concentration in the serum, but is the most important in allergic disorders. It has been reported that some viruses such as RSV, influenza, bocavirus, parainfluenza and metapneumovirus produce bronchial hyperreactivity or asthma increasing concentrations of this immunoglobulin in blood and secretions. Basophils and mast cells have receptors for these antibodies, when the antigen-antibody binding occurs and consequently activate, these cells release inflammatory mediators that cause many manifestations of these respira‐

**Cellular response.** The cellular response has as its principal components the T lymphocytes, which are divided into two populations according to their surface markers and the pattern of cytokines produced: CD4+, also called helper T cells (Th) and CD8 +, the cytotoxic lym‐ phocytes (CTL). These specialized cells can proliferate and differentiate into effector and

with the cell membrane during a RSV infection [25, 26].

ic capacity of immune cells [24].

18 Respiratory Disease and Infection - A New Insight

**8.1. Specific or acquired immunity**

humoral and cellular.

tory diseases [27].

memory cells.

Despite effective defenses, some virus can to evade it by using different mechanisms, for ex‐ ample, in respiratory tract infections influenza virus inhibits the production of interferon by the protein NS1, a non-structural protein that is abundantly expressed in the nucleus of in‐ fected cells. NS1 binds to double-stranded RNA by preventing activation of the dsRNA-de‐ pendent protein kinase (PKR) [28], commonly synthesized during induction of interferon (Figure 5). Another mechanism is the antigenic variation that occur mainly in the HA and NA proteins. PI and RSV respiratory viruses have a surface protein (F), which mediates the fusion of the viral membrane with the cell membrane. This mechanism enables the virus to spread from one cell to another without exposure and avoiding the effect of circulating anti‐ bodies. Another strategy is to make a latent infection. Viruses such as adenovirus and her‐ pes employ transcription and replication strategies to maintain the viral genome in any cell type where the immune response is not efficient and viral particles are not produced for long periods. Other viruses interfere with antigen processing or complement. In summary, viruses use several strategies to evade the immune response [11].

#### **10. Other mechanisms used by respiratory viruses in the pathogenesis**

In most infections, the viruses cause upper respiratory infections, while others reach the lower airways, may even cause necrosis and cell death, also induce inflammatory processes such as wheezing and hyperreactivity, both important in the development of chronic diseas‐ es such as asthma and chronic bronchitis for which some mechanisms have been proposed. In summary, the strategies used by viruses that cause respiratory infections can be very var‐ ied; however, there are always some strategies are shared between different types of viruses.

**Inflammatory cells.** In viral respiratory infections has been observed recruitment of inflam‐ matory cells such as eosinophils, neutrophils, basophils, monocytes, macrophages, mast cells and T lymphocytes. Thus, when activated, these cells release mediators, cytokines or other compounds that increase inflammatory response [29, 30]. **Macrophages.** Alveolar mac‐ rophages are one of the first lines of cellular defense against virus infections. During viral replication in macrophages antiviral mechanisms are activated by stimulating, by stimulat‐ ing the release of interferons or other cytokines, for example, studies have shown that alveo‐ lar macrophage infection by RSV, causes increased secretion of tumor necrosis factor alpha (TNF-α), as well as interleukins IL-8 and IL-6. It has also been observed that the macrophag‐ es express high levels of intercellular adhesion molecule-1 (ICAM-1), receptor molecule spe‐ cific for some virus [17, 31, 32].

struction induces exacerbation of asthma. Several mediators have been reported, which sug‐ gests that during infection can exist the interaction of more than one. Among the most

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 21

**Histamine.** Histamine is released from various cells as basophils, leukocytes, mast cells, among others. The secretion of this mediator is inflammation and airway is inflammation and airway obstruction. *In vitro* and *in vivo* studies with respiratory virus have demonstrat‐ ed high concentrations of histamine in nasopharyngeal secretions and in the plasma of in‐ fected individuals. However, therapeutic success with antihistamines in asthma has not

**Leukotrienes.** Leukotrienes are inflammatory lipid mediators derived from arachidonic acid. Leukotrienes are released by primary inflammatory cells involved in inflammation, as well as endothelial and epithelial cells of the airways. They are very potent bronchoconstric‐ tors that affect both the upper and lower airways. It has also been shown to increase vascu‐ lar permeability and production of mucus, in addition, some evidence suggests that leukotrienes play an important role in the origin of wheezings. Respiratory viruses such as RSV, parainfluenza 3 and influenza A, induce the release of leukotrienes which are detecta‐ ble in nasopharyngeal secretions. High concentrations of Leulotrienes have been found in

**Products of cyclooxygenase, arachidonic acid, prostaglandins and thromboxane.** They are potent bronchoconstrictors and have shown an increase in the concentrations of the primary metabolite of prostaglandin type 2a in plasmatic cells from infants with RSV bronchiolitis and especially in those with recurrent wheezing. It is also reported that the prostaglandin E2 type has an inhibitory effect which may protect the airways of a bronchoconstrictor effect. It is suggested that viral epithelial damage may result in the loss of these protective prosta‐ glandins. It was also found that complexes of RSV-antibody cause an increase in the release

**Platelet activating factor (PAF).** Induces an inflammatory response and stimulates the pro‐ duction of mucus in the airways, alters mucociliary clearance and enhances pulmonary mi‐ crovascular permeability. PAF is released by macrophages, eosinophils and neutrophils. *In vitro* studies have shown that mononuclear phagocytes upon RSV replication, stimulates the synthesis of platelet activating factor. From these results it has been suggested that this fac‐

**Kinins.** These molecules are potent vasoactive peptides that are produced in tissues or flu‐ ids. Kinins may be involved in the pathogenesis of diseases such as asthma by its inflamma‐ tory and bronchoconstrictor action. Kinins are potent stimulus for C fibers, and therefore, improves axon reflex [34, 36]. In unmyelinated sensory nerves in the airways is found the P substance, potent neuropeptide belonging to tachykinin group which when released by lo‐

**Nitric Oxide (NO).** Nitric oxide has a mediator function with different effects such as: anti‐ viral agent, increase bronchial blood flow, eosinophilic infiltration, epithelial damage, po‐ tent vasodilator, inhibit the proliferation of Th1 cells due to a Th2 phenotype change, and, in

tor may play an important role in the inflammatory response caused by RSV [45, 46].

cal axon reflex, potentiate the cholinergic neurotransmission [47].

been confirmed so several authors have questioned the effect of histamine [36, 38].

mentioned are:

infants with RSV infection [40-44].

of thromboxane by neutrophils [36].

**Monocytes.** Also express high levels of ICAM-1. When human monocytes are infected by viruses, monocytes are activated, producing and releasing IFN-α, IFN-β, IL-1β, IL-6 and TNF-α. The production of these cytokines (with the exception of IFN-β) potentiates the pro‐ duction of granulocyte macrophage-colony stimulating factor (GM-CSF) [33].

**T Lymphocytes.** T lymphocytes act as immunomodulators and as producers of cytokines. According to the pattern synthesis of cytokines, helper T cells (Th) are classified into two types: Th1 cells secreting IL-2, IF-γ and lymphotoxin, while Th2 cells secrete IL-4, IL-5, IL -6 and IL-10. The Th1 response is associated with the antiviral immunity. The RSV G protein stimulates Th2 type response and this would explain the symptoms of lower respiratory tract caused by this virus [34].

**Neutrophils.** In viral respiratory infections, neutrophils are activated and recruited into the airways and probably generate oxygen metabolites or other metabolites or inflammatory cy‐ tokines that cause damage and late hyperreactivity 36. They are found in high concentra‐ tions in bronchial secretions of children infected with RSV, with parainfluenza virus and in nasal biopsies of subjects with rhinovirus infection [34].

**Eosinophils.** Eosinophils release mediators such as leukotrienes (LTC4), platelet activat‐ ing factor (PAF), major basic protein and cationic eosinophilic protein. When eosinophils are activated by virus are recruited into the airways causing damage and causing a late hyperreactivity reaction [35]. *In vitro* studies have shown that RSV in humans activates eosinophils [36].

**Basophils.***In vitro* assays using basophils obtained from patients infected with RSV, adeno‐ virus, influenza A, parainfluenza and rhinoviruses have observed an increase in the release of histamine [37, 38, 39].

**Mast cells.** These cells have a high affinity receptor for IgE and participate in hypersensitivi‐ ty reactions, the release of histamine and leukotrienes, molecules that are increased in in‐ fants with respiratory wheezing [38, 39].

**Stimulation of chemical mediators.** It has been proposed that in the respiratory infections, viruses are able to originate the relase of inflammatory mediators, either directly or through viruses-activated cells. Whatever leads to a vigorous inflammatory response, airway ob‐ struction induces exacerbation of asthma. Several mediators have been reported, which sug‐ gests that during infection can exist the interaction of more than one. Among the most mentioned are:

**Inflammatory cells.** In viral respiratory infections has been observed recruitment of inflam‐ matory cells such as eosinophils, neutrophils, basophils, monocytes, macrophages, mast cells and T lymphocytes. Thus, when activated, these cells release mediators, cytokines or other compounds that increase inflammatory response [29, 30]. **Macrophages.** Alveolar mac‐ rophages are one of the first lines of cellular defense against virus infections. During viral replication in macrophages antiviral mechanisms are activated by stimulating, by stimulat‐ ing the release of interferons or other cytokines, for example, studies have shown that alveo‐ lar macrophage infection by RSV, causes increased secretion of tumor necrosis factor alpha (TNF-α), as well as interleukins IL-8 and IL-6. It has also been observed that the macrophag‐ es express high levels of intercellular adhesion molecule-1 (ICAM-1), receptor molecule spe‐

**Monocytes.** Also express high levels of ICAM-1. When human monocytes are infected by viruses, monocytes are activated, producing and releasing IFN-α, IFN-β, IL-1β, IL-6 and TNF-α. The production of these cytokines (with the exception of IFN-β) potentiates the pro‐

**T Lymphocytes.** T lymphocytes act as immunomodulators and as producers of cytokines. According to the pattern synthesis of cytokines, helper T cells (Th) are classified into two types: Th1 cells secreting IL-2, IF-γ and lymphotoxin, while Th2 cells secrete IL-4, IL-5, IL -6 and IL-10. The Th1 response is associated with the antiviral immunity. The RSV G protein stimulates Th2 type response and this would explain the symptoms of lower respiratory

**Neutrophils.** In viral respiratory infections, neutrophils are activated and recruited into the airways and probably generate oxygen metabolites or other metabolites or inflammatory cy‐ tokines that cause damage and late hyperreactivity 36. They are found in high concentra‐ tions in bronchial secretions of children infected with RSV, with parainfluenza virus and in

**Eosinophils.** Eosinophils release mediators such as leukotrienes (LTC4), platelet activat‐ ing factor (PAF), major basic protein and cationic eosinophilic protein. When eosinophils are activated by virus are recruited into the airways causing damage and causing a late hyperreactivity reaction [35]. *In vitro* studies have shown that RSV in humans activates

**Basophils.***In vitro* assays using basophils obtained from patients infected with RSV, adeno‐ virus, influenza A, parainfluenza and rhinoviruses have observed an increase in the release

**Mast cells.** These cells have a high affinity receptor for IgE and participate in hypersensitivi‐ ty reactions, the release of histamine and leukotrienes, molecules that are increased in in‐

**Stimulation of chemical mediators.** It has been proposed that in the respiratory infections, viruses are able to originate the relase of inflammatory mediators, either directly or through viruses-activated cells. Whatever leads to a vigorous inflammatory response, airway ob‐

duction of granulocyte macrophage-colony stimulating factor (GM-CSF) [33].

cific for some virus [17, 31, 32].

20 Respiratory Disease and Infection - A New Insight

tract caused by this virus [34].

eosinophils [36].

of histamine [37, 38, 39].

fants with respiratory wheezing [38, 39].

nasal biopsies of subjects with rhinovirus infection [34].

**Histamine.** Histamine is released from various cells as basophils, leukocytes, mast cells, among others. The secretion of this mediator is inflammation and airway is inflammation and airway obstruction. *In vitro* and *in vivo* studies with respiratory virus have demonstrat‐ ed high concentrations of histamine in nasopharyngeal secretions and in the plasma of in‐ fected individuals. However, therapeutic success with antihistamines in asthma has not been confirmed so several authors have questioned the effect of histamine [36, 38].

**Leukotrienes.** Leukotrienes are inflammatory lipid mediators derived from arachidonic acid. Leukotrienes are released by primary inflammatory cells involved in inflammation, as well as endothelial and epithelial cells of the airways. They are very potent bronchoconstric‐ tors that affect both the upper and lower airways. It has also been shown to increase vascu‐ lar permeability and production of mucus, in addition, some evidence suggests that leukotrienes play an important role in the origin of wheezings. Respiratory viruses such as RSV, parainfluenza 3 and influenza A, induce the release of leukotrienes which are detecta‐ ble in nasopharyngeal secretions. High concentrations of Leulotrienes have been found in infants with RSV infection [40-44].

**Products of cyclooxygenase, arachidonic acid, prostaglandins and thromboxane.** They are potent bronchoconstrictors and have shown an increase in the concentrations of the primary metabolite of prostaglandin type 2a in plasmatic cells from infants with RSV bronchiolitis and especially in those with recurrent wheezing. It is also reported that the prostaglandin E2 type has an inhibitory effect which may protect the airways of a bronchoconstrictor effect. It is suggested that viral epithelial damage may result in the loss of these protective prosta‐ glandins. It was also found that complexes of RSV-antibody cause an increase in the release of thromboxane by neutrophils [36].

**Platelet activating factor (PAF).** Induces an inflammatory response and stimulates the pro‐ duction of mucus in the airways, alters mucociliary clearance and enhances pulmonary mi‐ crovascular permeability. PAF is released by macrophages, eosinophils and neutrophils. *In vitro* studies have shown that mononuclear phagocytes upon RSV replication, stimulates the synthesis of platelet activating factor. From these results it has been suggested that this fac‐ tor may play an important role in the inflammatory response caused by RSV [45, 46].

**Kinins.** These molecules are potent vasoactive peptides that are produced in tissues or flu‐ ids. Kinins may be involved in the pathogenesis of diseases such as asthma by its inflamma‐ tory and bronchoconstrictor action. Kinins are potent stimulus for C fibers, and therefore, improves axon reflex [34, 36]. In unmyelinated sensory nerves in the airways is found the P substance, potent neuropeptide belonging to tachykinin group which when released by lo‐ cal axon reflex, potentiate the cholinergic neurotransmission [47].

**Nitric Oxide (NO).** Nitric oxide has a mediator function with different effects such as: anti‐ viral agent, increase bronchial blood flow, eosinophilic infiltration, epithelial damage, po‐ tent vasodilator, inhibit the proliferation of Th1 cells due to a Th2 phenotype change, and, in asthma patients, it has been observed that after a experimental rhinovirus infection, no in‐ crease in exhaled NO levels [48].

ment of eosinophils and neutrophils, thereby increasing and causing inflammatory activi‐

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 23

**11. Pathogenicity and description of some viral respiratory infections**

The influenza A virus belongs to the *Orthomixoviridae* family, causes high morbidity and mortality. One feature of the virus is the frequent occurrence of new antigenic variants gen‐ erated by both genetic mutations and recombination leading to epidemics and pandemics. Influenza viruses have a fragmented RNA genome (8 fragments). Among others, influenza virus has two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), that are impor‐ tant in their biological activity and pathogenesis. The cellular receptor for this virus is sialic acid, which forms part of mucopolysaccharides found in glycoproteins and cell membranes. HA viral protein binds to the cellular receptor by endocytosis to enter the cell, allowing the virus to remain as an endosome, and is required to be activated so that the fusion peptide is exposed. This step is critical for virus infectivity and depends on both the virus and the cell. Once given membrane fusion, RNA migrates to the nucleus for replication, which is re‐ quired for the cellular RNA polymerase II, as its polymerase is inefficient to generate mRNA, so viral replication depends on the help of the cell. Neuraminidase (NA) is the sec‐ ond virus glycoprotein. Its function is enzymatic and is important because once the new vi‐ rions are synthesized, the glycoprotein is responsible for removing sialic acid residues from the infected cell membrane, which allows the newly synthesized virions can be released without auto aggregation. The viral M2 protein functions as an ion channel that allows the passage of protons into the virion and is the target for the action of the amantadine, its mu‐

The virus enters through the nasopharyngeal region, the target cells are mucus-secreting epi‐ thelial cells and ciliated cells, can be transmitted by droplets expelled by speaking, sneezing or coughing, by contact with contaminated material or hands. The cell binding is via the HA that binds to sialic acid receptor. The incubation time is 1 to 3 days. The virus multiplies rapidly and spreads to neighboring cells. It causes cellular necrosis and apoptosis, altering the ciliar activi‐ ty and increasing mucus secretion. To exit and infect other cells, NA reduces the viscosity of mucus film breaking sialic acid residues. The damage to the epithelium causes respiratory symptoms and signs, stimulates the natural response of the tract and promotes bacterial incor‐ poration. The inflammation process can damage bronchi, bronchioles and alveolar regions. All these events cause initial symptoms of infection like fever, chills, muscle aches, headache, ano‐ rexia and prostration. Local monocytes, lymphocytes and interferon are the main response to the virus. The virus induces an effective humoral response which is important in recovery, but it must be considered that the antibody response is specific for each variant of the virus, where‐ as the T lymphocytes and macrophage response is general and depends on the injury and the condition of the host to perform efficiently the epithelial repair (that can take up to one month).

ty and wheezing [58, 59, 60].

**11.1. Infection with influenza virus A**

tation or changes can lead to viral resistance to this compound.

**Cytokines.** They are small proteins that act generally in cellular processes such as differ‐ entiation, activation and immune defense. All cytokines are secreted by cells due to the interaction with infectious agents and mechanical actions (e.g. cell stress). They interact through a complex network during the immune and inflammatory responses. There are a variety of cytokines and others are continually identified. There are cytokines showing chemotactic properties, therefore, these cytokines are called chemokines. In viral infec‐ tious processes it has been described the participation of various chemokines as a patho‐ logical characteristic of the infection process, so that it has been established that chemokines are directly responsible for the inflammatory processes that occur in respira‐ tory viral infections [49]. Among the viruses that induce the release of chemokines may be mentioned: RSV, rhinovirus, influenza and parainfluenza viruses 3 [50]. It has been observed that, in cell lines, RSV increases the production of IL-6, IL-8, RANTES, macro‐ phage inflammatory protein (MIP-1a), GM-CSF and IL-11.

**Interleukin 8 (IL-8).** IL-8 is a chemokine which promotes the recruitment of neutrophils and eosinophils that are responsible in part for the inflammatory process [49, 50].

**RANTES.** This eosinophil chemokine that induces exocytosis of the eosinophil cationic protein. It is also chemotactic for basophils and CD44 T cells [51, 52].

**MIP-1α.** Less potent than RANTES as eosinophil chemotactic, but it is important mediator in the inflammatory response during virus infection because it stimulates the release of his‐ tamine by basophils and mast cells. Its properties suggest that may be important mediators of asthma exacerbations induced by viral infections. In children, have been found in high concentrations in nasal secretions during asthma exacerbations associated with infection caused by RSV and rhinovirus [50,51].

**Eotaxin.** It is another chemokine with chemotactic activity for eosinophils. Eotaxin has addi‐ tional functions such as endothelial migration, release of reactive oxygen, Ca+ ions mobiliza‐ tion, actin polymerization and is also chemotactic for basophils and Th2 lymphocytes. It is soluble in serum and has been found in high concentrations in patients with asthma and is associated with the severity of the disease [52, 53].

**Intercellular adhesion molecule 1 (ICAM-1).** This a receptor is located in the vascular endothelium, epithelium of the airways and in antigen presenting cells. Its ligands are found in circulating leukocytes [54, 55]. Several studies have shown that, *in vitro*, epithe‐ lial cells of human airways produce increased levels of ICAM-1. This ICAM-1 expression is observed also during the adhesion of eosinophils and neutrophils in response to in‐ flammatory cytokines and in infection processes of various respiratory viruses such as RSV, rhinovirus and parainfluenza [53, 56, 57]. Rhinoviruses attach to the surface of cells via ICAM-1 receptor, suggesting that infection with rhinovirus leads to an increase in the expression of ICAM-1 in the upper airways. In this way, ICAM-I and induces the recruit‐ ment of eosinophils and neutrophils, thereby increasing and causing inflammatory activi‐ ty and wheezing [58, 59, 60].

#### **11. Pathogenicity and description of some viral respiratory infections**

#### **11.1. Infection with influenza virus A**

asthma patients, it has been observed that after a experimental rhinovirus infection, no in‐

**Cytokines.** They are small proteins that act generally in cellular processes such as differ‐ entiation, activation and immune defense. All cytokines are secreted by cells due to the interaction with infectious agents and mechanical actions (e.g. cell stress). They interact through a complex network during the immune and inflammatory responses. There are a variety of cytokines and others are continually identified. There are cytokines showing chemotactic properties, therefore, these cytokines are called chemokines. In viral infec‐ tious processes it has been described the participation of various chemokines as a patho‐ logical characteristic of the infection process, so that it has been established that chemokines are directly responsible for the inflammatory processes that occur in respira‐ tory viral infections [49]. Among the viruses that induce the release of chemokines may be mentioned: RSV, rhinovirus, influenza and parainfluenza viruses 3 [50]. It has been observed that, in cell lines, RSV increases the production of IL-6, IL-8, RANTES, macro‐

**Interleukin 8 (IL-8).** IL-8 is a chemokine which promotes the recruitment of neutrophils

**RANTES.** This eosinophil chemokine that induces exocytosis of the eosinophil cationic

**MIP-1α.** Less potent than RANTES as eosinophil chemotactic, but it is important mediator in the inflammatory response during virus infection because it stimulates the release of his‐ tamine by basophils and mast cells. Its properties suggest that may be important mediators of asthma exacerbations induced by viral infections. In children, have been found in high concentrations in nasal secretions during asthma exacerbations associated with infection

**Eotaxin.** It is another chemokine with chemotactic activity for eosinophils. Eotaxin has addi‐ tional functions such as endothelial migration, release of reactive oxygen, Ca+ ions mobiliza‐ tion, actin polymerization and is also chemotactic for basophils and Th2 lymphocytes. It is soluble in serum and has been found in high concentrations in patients with asthma and is

**Intercellular adhesion molecule 1 (ICAM-1).** This a receptor is located in the vascular endothelium, epithelium of the airways and in antigen presenting cells. Its ligands are found in circulating leukocytes [54, 55]. Several studies have shown that, *in vitro*, epithe‐ lial cells of human airways produce increased levels of ICAM-1. This ICAM-1 expression is observed also during the adhesion of eosinophils and neutrophils in response to in‐ flammatory cytokines and in infection processes of various respiratory viruses such as RSV, rhinovirus and parainfluenza [53, 56, 57]. Rhinoviruses attach to the surface of cells via ICAM-1 receptor, suggesting that infection with rhinovirus leads to an increase in the expression of ICAM-1 in the upper airways. In this way, ICAM-I and induces the recruit‐

and eosinophils that are responsible in part for the inflammatory process [49, 50].

protein. It is also chemotactic for basophils and CD44 T cells [51, 52].

crease in exhaled NO levels [48].

22 Respiratory Disease and Infection - A New Insight

phage inflammatory protein (MIP-1a), GM-CSF and IL-11.

caused by RSV and rhinovirus [50,51].

associated with the severity of the disease [52, 53].

The influenza A virus belongs to the *Orthomixoviridae* family, causes high morbidity and mortality. One feature of the virus is the frequent occurrence of new antigenic variants gen‐ erated by both genetic mutations and recombination leading to epidemics and pandemics. Influenza viruses have a fragmented RNA genome (8 fragments). Among others, influenza virus has two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), that are impor‐ tant in their biological activity and pathogenesis. The cellular receptor for this virus is sialic acid, which forms part of mucopolysaccharides found in glycoproteins and cell membranes. HA viral protein binds to the cellular receptor by endocytosis to enter the cell, allowing the virus to remain as an endosome, and is required to be activated so that the fusion peptide is exposed. This step is critical for virus infectivity and depends on both the virus and the cell. Once given membrane fusion, RNA migrates to the nucleus for replication, which is re‐ quired for the cellular RNA polymerase II, as its polymerase is inefficient to generate mRNA, so viral replication depends on the help of the cell. Neuraminidase (NA) is the sec‐ ond virus glycoprotein. Its function is enzymatic and is important because once the new vi‐ rions are synthesized, the glycoprotein is responsible for removing sialic acid residues from the infected cell membrane, which allows the newly synthesized virions can be released without auto aggregation. The viral M2 protein functions as an ion channel that allows the passage of protons into the virion and is the target for the action of the amantadine, its mu‐ tation or changes can lead to viral resistance to this compound.

The virus enters through the nasopharyngeal region, the target cells are mucus-secreting epi‐ thelial cells and ciliated cells, can be transmitted by droplets expelled by speaking, sneezing or coughing, by contact with contaminated material or hands. The cell binding is via the HA that binds to sialic acid receptor. The incubation time is 1 to 3 days. The virus multiplies rapidly and spreads to neighboring cells. It causes cellular necrosis and apoptosis, altering the ciliar activi‐ ty and increasing mucus secretion. To exit and infect other cells, NA reduces the viscosity of mucus film breaking sialic acid residues. The damage to the epithelium causes respiratory symptoms and signs, stimulates the natural response of the tract and promotes bacterial incor‐ poration. The inflammation process can damage bronchi, bronchioles and alveolar regions. All these events cause initial symptoms of infection like fever, chills, muscle aches, headache, ano‐ rexia and prostration. Local monocytes, lymphocytes and interferon are the main response to the virus. The virus induces an effective humoral response which is important in recovery, but it must be considered that the antibody response is specific for each variant of the virus, where‐ as the T lymphocytes and macrophage response is general and depends on the injury and the condition of the host to perform efficiently the epithelial repair (that can take up to one month). In pandemics and severe cases, it has been observed that the immune response is exacerbated and may cause a greater damage [61].

immune system responds to the infective agent, that is, after approximately two weeks. Up

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 25

to that moment the antibodies could not stop the damage the virus had caused.

**Figure 6.** Hemagglutination inhibition test for the detection of antibodies against influenza A (H1N1) virus.

RSV belongs to the *Paramyxoviridae* family is not segmented and is capable of performing polymerase mediated mRNA synthesis and is characterized by two glycoproteins, the F pro‐

In the case of RSV, this virus infects children under 2 years of age causing high rates of mor‐ bidity and mortality [61]. Usually, children between one and two years of age have had a virus infection [62]; however, reinfections are frequent, as the immune response is incom‐ plete or immature. As all the respiratory viruses, RSV enters through respiratory airways, taking place the first replication in the nasopharyngeal region. For the ability to make syncy‐ tia, RSV virus can move from cell to cell without leaving, which prevents antibody attack. Thus, secretions or by dragging can reach lower airways, causing bronchiolitis and pneumo‐ nia with mucus production. As a child tracks are narrow, respiratory obstruction can be ob‐ served, which can be very serious. The most important mechanism of pathogenesis of the virus is its capacity to infect their terminal paths of the lung during childhood, when the di‐ ameter is quite small. The RSV virus has a propensity to infect the bronchiolar epithelium in comparison with the infection of the rest of the airways; even if the cause is unknown, pathological studies indicate that the inflammatory response is much greater in the area of the terminal airways that in the upper portions of the respiratory tract [63]. Besides, other factors are suspected important in the pathogenesis of the virus as immunological mecha‐ nisms, mainly by cytokines produced by Th2 lymphocytes. It has been suggested that the

**11.2. Respiratory syncytial virus (RSV)**

tein (which induces the formation of syncytia) and G protein.

The virus can evade the immune response in different ways. One is the constant genetic var‐ iation of HA and NA glycoproteins, which are the first that are recognized by antibodies. Another mechanism involves the NS1 Protein, which can block the role of interferon [15].

Until 2009, the origin of influenza pandemics was mainly due to the transmission of virus from birds to humans, by the transfer of genes from avian virus to the seasonal influenza virus that is recognized as human. When a new virus emerges, the body does not recognize it, which can lead to a pandemic. In April 2009, an epidemic arose from the emergence of a new influenza A virus. The first cases occurred in Mexico. Numerous patients with Influen‐ za-like severe symptoms were attended at the Instituto Nacional de Enfermedades Respira‐ torias Ismael Cosio Villegas (INER), Mexico, and in many cases required hospitalization. Patients arrived in a serious, advanced conditions and, for this reason many of them died.

It has been observed that when an epidemic emerges and new viruses are detected, the hu‐ man body quickly begins to produce the antibodies needed to contain the new disease. Some people develop these antibodies faster than others, so that the chance of infection in them is lower, because their immune system acts quickly creating antibodies, while those that fail to quickly develop antibodies start to develop the disease. Despite that, the viruses that cause ARI do not induce a good immune response. If after some time there is antibodies production, many patients will become before producing antibodies against the new virus. Other antibodies have neutralizing capacity.

We decided to carry out a study with the following objectives: To determine the titer of antiinfluenza virus in serum samples from patients infected with influenza A (H1N1), as well as household contacts of patients infected with this virus by the method of inhibition of he‐ magglutination.

To determine the presence and titer of antibodies were used 196 samples of sera, of which 110 were in patients with influenza A H1N1 confirmed by RT-PCR assays and to household contacts 86 patients with influenza A H1N1. In this work, antibody titer of each sample was deyterming using the technique of hemagglutination inhibition.

The figure 6 shows the results of some sera, as can be seen, the positive titers appear as a red spot in the center of the well as a result of inhibition of hemagglutination while negatives occur in a uniform color of lower intensity.

90% of patients confirmed influenza A (H1N1) had antibodies, the highest title was 1:1024 and the lowest was 1:16. In the case of the sera of household contacts the highest antibody titer was 1:256 and the lowest was 1:4. In the case of the sera of healthy household contacts, antibodies are detected in only 84% of the sera.

In conclusion, the antibodies were detected during an acute late phase in the diseases acute phase but late in the disease, since patients usually arrived after two weeks of onset of the disease, and in serious condition. It is possible that after this stage titers have increased. An‐ tibody synthesis is generally low at the beginning of the disease, and it is increased as the immune system responds to the infective agent, that is, after approximately two weeks. Up to that moment the antibodies could not stop the damage the virus had caused.

**Figure 6.** Hemagglutination inhibition test for the detection of antibodies against influenza A (H1N1) virus.

#### **11.2. Respiratory syncytial virus (RSV)**

In pandemics and severe cases, it has been observed that the immune response is exacerbated

The virus can evade the immune response in different ways. One is the constant genetic var‐ iation of HA and NA glycoproteins, which are the first that are recognized by antibodies. Another mechanism involves the NS1 Protein, which can block the role of interferon [15].

Until 2009, the origin of influenza pandemics was mainly due to the transmission of virus from birds to humans, by the transfer of genes from avian virus to the seasonal influenza virus that is recognized as human. When a new virus emerges, the body does not recognize it, which can lead to a pandemic. In April 2009, an epidemic arose from the emergence of a new influenza A virus. The first cases occurred in Mexico. Numerous patients with Influen‐ za-like severe symptoms were attended at the Instituto Nacional de Enfermedades Respira‐ torias Ismael Cosio Villegas (INER), Mexico, and in many cases required hospitalization. Patients arrived in a serious, advanced conditions and, for this reason many of them died. It has been observed that when an epidemic emerges and new viruses are detected, the hu‐ man body quickly begins to produce the antibodies needed to contain the new disease. Some people develop these antibodies faster than others, so that the chance of infection in them is lower, because their immune system acts quickly creating antibodies, while those that fail to quickly develop antibodies start to develop the disease. Despite that, the viruses that cause ARI do not induce a good immune response. If after some time there is antibodies production, many patients will become before producing antibodies against the new virus.

We decided to carry out a study with the following objectives: To determine the titer of antiinfluenza virus in serum samples from patients infected with influenza A (H1N1), as well as household contacts of patients infected with this virus by the method of inhibition of he‐

To determine the presence and titer of antibodies were used 196 samples of sera, of which 110 were in patients with influenza A H1N1 confirmed by RT-PCR assays and to household contacts 86 patients with influenza A H1N1. In this work, antibody titer of each sample was

The figure 6 shows the results of some sera, as can be seen, the positive titers appear as a red spot in the center of the well as a result of inhibition of hemagglutination while negatives

90% of patients confirmed influenza A (H1N1) had antibodies, the highest title was 1:1024 and the lowest was 1:16. In the case of the sera of household contacts the highest antibody titer was 1:256 and the lowest was 1:4. In the case of the sera of healthy household contacts,

In conclusion, the antibodies were detected during an acute late phase in the diseases acute phase but late in the disease, since patients usually arrived after two weeks of onset of the disease, and in serious condition. It is possible that after this stage titers have increased. An‐ tibody synthesis is generally low at the beginning of the disease, and it is increased as the

and may cause a greater damage [61].

24 Respiratory Disease and Infection - A New Insight

Other antibodies have neutralizing capacity.

occur in a uniform color of lower intensity.

antibodies are detected in only 84% of the sera.

deyterming using the technique of hemagglutination inhibition.

magglutination.

RSV belongs to the *Paramyxoviridae* family is not segmented and is capable of performing polymerase mediated mRNA synthesis and is characterized by two glycoproteins, the F pro‐ tein (which induces the formation of syncytia) and G protein.

In the case of RSV, this virus infects children under 2 years of age causing high rates of mor‐ bidity and mortality [61]. Usually, children between one and two years of age have had a virus infection [62]; however, reinfections are frequent, as the immune response is incom‐ plete or immature. As all the respiratory viruses, RSV enters through respiratory airways, taking place the first replication in the nasopharyngeal region. For the ability to make syncy‐ tia, RSV virus can move from cell to cell without leaving, which prevents antibody attack. Thus, secretions or by dragging can reach lower airways, causing bronchiolitis and pneumo‐ nia with mucus production. As a child tracks are narrow, respiratory obstruction can be ob‐ served, which can be very serious. The most important mechanism of pathogenesis of the virus is its capacity to infect their terminal paths of the lung during childhood, when the di‐ ameter is quite small. The RSV virus has a propensity to infect the bronchiolar epithelium in comparison with the infection of the rest of the airways; even if the cause is unknown, pathological studies indicate that the inflammatory response is much greater in the area of the terminal airways that in the upper portions of the respiratory tract [63]. Besides, other factors are suspected important in the pathogenesis of the virus as immunological mecha‐ nisms, mainly by cytokines produced by Th2 lymphocytes. It has been suggested that the nature of the immune response to RSV, is determined by the pattern of cytokines produced sequentially by different cells [63]. There are high-risk groups such as immunocompromised children, premature infants, infants with heart, kidney and lung problems, where mortality is high.

**Author details**

**References**

Ma. Eugenia Manjarrez-Zavala1

Luis Horacio Gutiérrez-González1

dades Respiratorias Ismael Cosio Villegas, D. F., México

, Dora Patricia Rosete-Olvera1

1 Departamento de Investigación en Virología y Micología, Instituto Nacional de Enferme‐

[1] Flint SJ, Enquist LW, KrugRM, Rocaniello VR, Skalka AM. Principles of Virology. Molecular Biology Pathogenesis and Control. ASM Press Washington D.C. 2000. [2] Douglas RG Jr, Couch RB, Baxter BD, Gough M. Attenuation of rhinovirus type 15: relation of illness to plaque size. Infection and Immunity. 1974; 9(3) 519-523.

[3] Gottwein E and Cullen BR. Viral and cellular microRNAs as determinants of viral

[4] Ghosh Z, Mallick B and Chakrabarti J. Cellular versus viral microRNAs in host–virus

[5] Manjarrez ME, Rosete DP, Higuera A, Ocadiz-Delgado R, P{erez-Padilla JR, Cabello C. Start a Pandemic: Influenza A H1N1 Virus. In Respiratory Diseases. Mostafa Cha‐

[6] Schneider-Schaulies J. Cellular receptors for viruses: links to tropism and pathogene‐

[7] Corjon S., Gonzalez G, Henning P, Grichine A, Lindholm L, Boulanger P, Fender P and Hong S. Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction. 2011; 6

[8] Landry ML , Mayo DR and Hsiung GD. Comparison of guinea pig embryo cells, rab‐ bit kidney cells, and human embryonic lung fibroblast cell strains for isolation of her‐

[9] Albrecht T, Fons M, Boldogh I, et al. Effects on Cells. In: Baron S, editor. Medical Mi‐ crobiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Gal‐ veston; 1996. Chapter 44. Available from: http://www.ncbi.nlm.nih.gov/books/

[10] Baron S, Fons M, Albrecht T. Viral Pathogenesis. In: Baron S, editor. Medical Microbi‐ ology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 45. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8149/.

pes simplex virus.Journal Clinical Microbiology.1982; 15(5): 842-847.

pathogenesis and immunity. Cell Host Microbe. 2008; 12; 3(6) 375–387.

Interaction. Nucleic Acids Research. 2009; 37(4): 1035–1048.

nei, Intech Open Access Publisher, Rieja, Croatia 2012.215-242.

sis J Gen Virol. 2000; (81):1413–1429.

(5): e18205.

NBK7979/.

2 Departamento de Biología Molecular y Genética. CINVESTAV, IPN, D.F., México

, Rodolfo Ocadiz-Delgado2

,

and Carlos Cabello-Gutiérrez1

Pathogenesis of Viral Respiratory Infection http://dx.doi.org/10.5772/54287 27

#### **11.3. Parainfluenza virus (PIV)**

PIV is of the same family as the RSV, so that their characteristics are similar, except that one glycoprotein has the hemagglutinin and neuraminidase activities in a single structure. IN addition, PIV has an F fusion protein.

In the case of PI virus, and despite its freqeuncy, little is known regarding to their mecha‐ nism of pathogenesis. The PI virus also infects children causing the same syndromes. Small tracts of children, when ignited, conduce to the obstruction of the air flow allows the accu‐ mulation of secretions, so there are cases with severe obstruction [64, 65].

For either exist a vaccine, although in the case of RSV there is a monoclonal antibody which can be an important resource in the treatment of young children. However, but its cost, is only used in high-risk groups.

#### **12. Conclusions**

The viral pathogenesis represents a world of mysteries and extraordinary surprises, where apparently questions have not been able to answer. One of the main purposes of the patho‐ genesis study is to know and understand the molecular mechanisms by which viruses acti‐ vate in the cells when generating a , and how they avoid the immune response; with that information, trying to eliminate or control the diseases that they provoke in humans.

Although there have been significant advances in different fields of medical virology, there are still many mysteries to solve, questions to answer and so many fields to explore in the patho‐ genesis of viral acute respiratory infections (ARI) and the viral pathogenesis in general. In spite of moving forward in the pathogenesis study, mainly in aspects such as: molecular interactions between viral factors and the host, and identification and comprehension of many of the bio‐ logical, molecular, biochemical and even genetic mechanisms involved in the disease's devel‐ opment, many of these processes remain to be clearly understood. However, thanks to steady developments and improvements in many fields of biological sciences and technology, we hope to attain a deeper knowledge and comprehension of viral pathogenesis development.

#### **Acknowledgements**

The authors acknowledge the financial support from CONACYT (SALUD-2009- C02-126832).

#### **Author details**

nature of the immune response to RSV, is determined by the pattern of cytokines produced sequentially by different cells [63]. There are high-risk groups such as immunocompromised children, premature infants, infants with heart, kidney and lung problems, where mortality

PIV is of the same family as the RSV, so that their characteristics are similar, except that one glycoprotein has the hemagglutinin and neuraminidase activities in a single structure. IN

In the case of PI virus, and despite its freqeuncy, little is known regarding to their mecha‐ nism of pathogenesis. The PI virus also infects children causing the same syndromes. Small tracts of children, when ignited, conduce to the obstruction of the air flow allows the accu‐

For either exist a vaccine, although in the case of RSV there is a monoclonal antibody which can be an important resource in the treatment of young children. However, but its cost, is

The viral pathogenesis represents a world of mysteries and extraordinary surprises, where apparently questions have not been able to answer. One of the main purposes of the patho‐ genesis study is to know and understand the molecular mechanisms by which viruses acti‐ vate in the cells when generating a , and how they avoid the immune response; with that

Although there have been significant advances in different fields of medical virology, there are still many mysteries to solve, questions to answer and so many fields to explore in the patho‐ genesis of viral acute respiratory infections (ARI) and the viral pathogenesis in general. In spite of moving forward in the pathogenesis study, mainly in aspects such as: molecular interactions between viral factors and the host, and identification and comprehension of many of the bio‐ logical, molecular, biochemical and even genetic mechanisms involved in the disease's devel‐ opment, many of these processes remain to be clearly understood. However, thanks to steady developments and improvements in many fields of biological sciences and technology, we hope to attain a deeper knowledge and comprehension of viral pathogenesis development.

The authors acknowledge the financial support from CONACYT (SALUD-2009-

information, trying to eliminate or control the diseases that they provoke in humans.

mulation of secretions, so there are cases with severe obstruction [64, 65].

is high.

**11.3. Parainfluenza virus (PIV)**

26 Respiratory Disease and Infection - A New Insight

only used in high-risk groups.

**12. Conclusions**

**Acknowledgements**

C02-126832).

addition, PIV has an F fusion protein.

Ma. Eugenia Manjarrez-Zavala1 , Dora Patricia Rosete-Olvera1 , Luis Horacio Gutiérrez-González1 , Rodolfo Ocadiz-Delgado2 and Carlos Cabello-Gutiérrez1

1 Departamento de Investigación en Virología y Micología, Instituto Nacional de Enferme‐ dades Respiratorias Ismael Cosio Villegas, D. F., México

2 Departamento de Biología Molecular y Genética. CINVESTAV, IPN, D.F., México

#### **References**


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Response against Human Respiratory Syncytial Virus. Journal of Biological Chemis‐

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[65] Glezen WP, Danny FW. Epidemiology of acute lower respiratory tract disease in chil‐ dren. N Engl J Med 1973; 288:498-505.

**Chapter 2**

**Virology and Molecular Epidemiology of Respiratory**

Human respiratory syncytial virus (RSV) is a ubiquitous virus of worldwide distribution and is the leading cause of infant morbidity from respiratory infections. By the age of two years nearly all children have been infected and can cause severe bronchiolitis and pneumo‐ nia in this age group (Hall et al., 2009). Nearly 100% of children in the USA are infected with the virus by 2 to 3 years of age, several hundred infants may die directly from the infection, while the deaths of an additional several thousand may be attributed to RSV-related compli‐ cations (Nair et al, 2010). The World Health Organization estimates that (RSV) is responsible for 64 million infections worldwide and 160,000 deaths per annum (Openshaw, 2002).

Although mostly young infants are affected, it is increasingly recognized as a significant cause of disease in the elderly population and can often be fatal for patients with impaired immune systems (Collins and Malero., 2011). The incubation period of RSV respiratory dis‐ ease is estimated to be three to five days (Black, 2003). The virus can remain viable on hard surfaces (e.g., countertops) for up to 6hr, on rubber gloves for 90min, and on skin for 20min. (Hall et al., 1980). This prolonged survival highlights the need (and effectiveness) for hand washing and contact precautions in limiting the spread of RSV infection (Koetz et al, 2006). Viral shedding is significantly prolonged in immunocompromised individuals, and can con‐

The spectrum of clinical manifestations ranges from mild upper tract illness, infection in middle ear which progresses to acute otitis media, croup, to apnoea in premature infants,

and reproduction in any medium, provided the original work is properly cited.

© 2013 Al Johani and Akhter; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Syncytial Virus (RSV)**

http://dx.doi.org/10.5772/53916

**1. Introduction**

Sameera Al Johani and Javed Akhter

Additional information is available at the end of the chapter

tinue for several months (Hall et al., 1981; Simoes, 2003).

**2. Spectrum of respiratory disease**

## **Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)**

Sameera Al Johani and Javed Akhter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53916

#### **1. Introduction**

[65] Glezen WP, Danny FW. Epidemiology of acute lower respiratory tract disease in chil‐

dren. N Engl J Med 1973; 288:498-505.

32 Respiratory Disease and Infection - A New Insight

Human respiratory syncytial virus (RSV) is a ubiquitous virus of worldwide distribution and is the leading cause of infant morbidity from respiratory infections. By the age of two years nearly all children have been infected and can cause severe bronchiolitis and pneumo‐ nia in this age group (Hall et al., 2009). Nearly 100% of children in the USA are infected with the virus by 2 to 3 years of age, several hundred infants may die directly from the infection, while the deaths of an additional several thousand may be attributed to RSV-related compli‐ cations (Nair et al, 2010). The World Health Organization estimates that (RSV) is responsible for 64 million infections worldwide and 160,000 deaths per annum (Openshaw, 2002).

Although mostly young infants are affected, it is increasingly recognized as a significant cause of disease in the elderly population and can often be fatal for patients with impaired immune systems (Collins and Malero., 2011). The incubation period of RSV respiratory dis‐ ease is estimated to be three to five days (Black, 2003). The virus can remain viable on hard surfaces (e.g., countertops) for up to 6hr, on rubber gloves for 90min, and on skin for 20min. (Hall et al., 1980). This prolonged survival highlights the need (and effectiveness) for hand washing and contact precautions in limiting the spread of RSV infection (Koetz et al, 2006). Viral shedding is significantly prolonged in immunocompromised individuals, and can con‐ tinue for several months (Hall et al., 1981; Simoes, 2003).

#### **2. Spectrum of respiratory disease**

The spectrum of clinical manifestations ranges from mild upper tract illness, infection in middle ear which progresses to acute otitis media, croup, to apnoea in premature infants,

pneumonia and bronchiolitis (Hall et al., 1975). At the beginning of the illness, the virus rep‐ licates in the nasopharynx. Common symptoms of an upper respiratory tract infection in‐ clude a productive cough and mild to moderate nasal congestion with clear rhinorrhea.. A low-grade fever presents in the early stages of the infection and symptoms may persist for one to three weeks before complete recovery (Welliver, 2003).

modification by the addition of N-linked sugars. The G protein is of particular interest because variability in this protein is greater than that in the other proteins both between

Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)

http://dx.doi.org/10.5772/53916

35

RSV does not normally replicate outside of the bronchopulmonary tree and is restricted to the respiratory mucosa (Othumganpat et al, 2009). The G protein initiates attachment of the virus to the epithelial cell. The virus fuses with the epithelial cell membrane and enters the cytoplasm after the F protein is cleaved by proteolytic enzymes of the infected cell. If the precursor is not cleaved it has no fusion activity, virion penetration will not occur and the virus particle is unable to initiate infection. Fusion by F1 occurs at the neutral PH of the ex‐ tra cellular environment, allowing release of the viral nucleocapsid directly into the cell. This enables the virus to bypass internalization through endosomes. After replication the vi‐ rus matures by budding from the cell surface. The new progeny nucleocapsids form in the cytoplasm and move to the cell surface. The M protein is essential for particle formation, probably serving to link the viral envelope to the nucleocapsid. During budding, most host proteins are excluded from the membrane. If appropriate host cell proteases are present, precursor proteins in the plasma membrane will be activated by cleavage. Activated fusion protein will then cause fusion of adjacent cell membranes, resulting in formation of large syncytia (Jawetz et al., 2004). The viral RNA can spread without forming complete viral par‐ ticles. The infection results in the destruction of the epithelial cells of the upper respiratory tract. Exposure to RSV triggers humoral immune responses. Primary RSV infection results in only a weak antibody response with IgM, IgG, and IgA produced. This response is not sufficient to completely destroy the virus or to prevent upper respiratory tract replication of

the virus, thus an upper respiratory tract illness develops (Smith et al., 2009).

Although, RSV mostly infects the nasal epithelial cells. However, development of extrapul‐ monary disease has been observed in certain T and B cell immunodeficiency states. The as‐ sociation of RSV with asthma and reversible reactive airway disease in early childhood has attracted significant attention (Mohapatra et al., 2008). Recurrent wheezing for up to 5 to 7 years of age and established airway disease has been observed in a significant number of children with a strong family history of allergy, after primary infection or reinfection with RSV (LeManske, 2004). Immune response to primary infection is relatively small but on rein‐ fection, a significant booster effect with sustained immunologic reactivity is observed in se‐ rum and respiratory mucosa. Both CD4- and CD8-specific as well as Th1- and Th2-cell specific immune responses have been observed during human infection. In addition, proin‐ flammatory as well as immunoregulatory cytokines and chemokines are induced in the res‐ piratory tract after natural and induced (in vitro) infection. In vulnerable patients, the RSV infection will spread into the lower respiratory tract (Becker et al., 2006). In premature in‐ fants and immunocompromised hosts, the infection quickly progresses to the LRT. As the virus is destroyed within the lung by T cells, immunopathological responses cause further lung injury. Infected cells release proinflammatory cytokines and chemokines, including in‐

and within the major antigenic groups of RSV (Sullender, 2000).

**4. Mechanism of infection**

Symptoms of lower respiratory tract disease include tachypnea (ie rapid breathing great‐ er than 60 to 70 breaths/min), wheezing, and/or rales, that usually appear up to three days following onset of rhinorrhea. These symptoms are indicative of the virus spread‐ ing into the bronchi and bronchioles. A chest radiograph exhibits hyperinflation with flattened diaphragms. If the RSV further spreads to the alveoli, an interstitial pneumonia may develop, with middle and upper lobes affected. In such patients, tachypnea be‐ comes severe respiratory distress, with deep retractions and grunting respirations. The risk for cardiovascular failure secondary to hypoxemia, acidosis, and dehydration in‐ creases significantly (Greenough, 2002). Immunocompromised and young infants, who have very narrow bronchioles, are at particularly high risk for complete bronchiolar ob‐ struction. The risk for vomiting also increases, which is often related to respiratory dis‐ tress and can increase the likelihood and of gastroesophogeal reflux. This may result in decreased oral intake with dehydration. Premature babies born at 30–35 weeks of gesta‐ tion, HIV-infected patients, infants with cyanotic congenital heart disease,, and other im‐ munosuppressive therapy such as bone marrow transplant are at increased risk for morbidity and mortality during RSV infection (Resch, 2011).

#### **3. The structure of respiratory syncytial virus**

RSV is a member of the subfamily *Pneumovirinae* in the family *Paramyxoviridae*, order *Mono‐ negavirales*. RSV is closely related to several other RNA viruses, including measles, mumps, and parainfluenza types 1, 2, and 3. Respiratory syncytial virus is a medium-sized (120-200nm) enveloped virus that contains a lipoprotein coat and a linear minus-sense RNA genome. The entire genome of RSV is composed of approximately 15,000 nucleotides long (Dickens et al., 1984).

The genome contains 10 mRNAs, each coding for an individual protein (McIntosh,.1997). The structural proteins are divided into three functional groups, the nucleocapsid (N) protein, phosphoprotein (P) and viral polymerase (L). These proteins together, have been demonstrated to function as the RSV replicase. The outer envelope is lined internally with matrix (M) protein and is spiked externally with fusion (F) and attachment (G) gly‐ coprotein projections, these are responsible for the initiation and propagation of an RSV infection (Empey et al., 2010). Another protein (M2) is also present in the viral envelope. The viral capsid is made up of a nucleoprotein, a phosphoprotein, and a polymerase protein (C). The F protein is a type 1 transmembrane glycoprotein with a Cleaved N-ter‐ minal signal sequence and a transmembrane anchor near the C terminus. It is cleaved in‐ to two subunits, F1 and F2 that are linked by disulfide bonds, after synthesis and modification by the addition of N-linked sugars. The G protein is of particular interest because variability in this protein is greater than that in the other proteins both between and within the major antigenic groups of RSV (Sullender, 2000).

#### **4. Mechanism of infection**

pneumonia and bronchiolitis (Hall et al., 1975). At the beginning of the illness, the virus rep‐ licates in the nasopharynx. Common symptoms of an upper respiratory tract infection in‐ clude a productive cough and mild to moderate nasal congestion with clear rhinorrhea.. A low-grade fever presents in the early stages of the infection and symptoms may persist for

Symptoms of lower respiratory tract disease include tachypnea (ie rapid breathing great‐ er than 60 to 70 breaths/min), wheezing, and/or rales, that usually appear up to three days following onset of rhinorrhea. These symptoms are indicative of the virus spread‐ ing into the bronchi and bronchioles. A chest radiograph exhibits hyperinflation with flattened diaphragms. If the RSV further spreads to the alveoli, an interstitial pneumonia may develop, with middle and upper lobes affected. In such patients, tachypnea be‐ comes severe respiratory distress, with deep retractions and grunting respirations. The risk for cardiovascular failure secondary to hypoxemia, acidosis, and dehydration in‐ creases significantly (Greenough, 2002). Immunocompromised and young infants, who have very narrow bronchioles, are at particularly high risk for complete bronchiolar ob‐ struction. The risk for vomiting also increases, which is often related to respiratory dis‐ tress and can increase the likelihood and of gastroesophogeal reflux. This may result in decreased oral intake with dehydration. Premature babies born at 30–35 weeks of gesta‐ tion, HIV-infected patients, infants with cyanotic congenital heart disease,, and other im‐ munosuppressive therapy such as bone marrow transplant are at increased risk for

RSV is a member of the subfamily *Pneumovirinae* in the family *Paramyxoviridae*, order *Mono‐ negavirales*. RSV is closely related to several other RNA viruses, including measles, mumps, and parainfluenza types 1, 2, and 3. Respiratory syncytial virus is a medium-sized (120-200nm) enveloped virus that contains a lipoprotein coat and a linear minus-sense RNA genome. The entire genome of RSV is composed of approximately 15,000 nucleotides long

The genome contains 10 mRNAs, each coding for an individual protein (McIntosh,.1997). The structural proteins are divided into three functional groups, the nucleocapsid (N) protein, phosphoprotein (P) and viral polymerase (L). These proteins together, have been demonstrated to function as the RSV replicase. The outer envelope is lined internally with matrix (M) protein and is spiked externally with fusion (F) and attachment (G) gly‐ coprotein projections, these are responsible for the initiation and propagation of an RSV infection (Empey et al., 2010). Another protein (M2) is also present in the viral envelope. The viral capsid is made up of a nucleoprotein, a phosphoprotein, and a polymerase protein (C). The F protein is a type 1 transmembrane glycoprotein with a Cleaved N-ter‐ minal signal sequence and a transmembrane anchor near the C terminus. It is cleaved in‐ to two subunits, F1 and F2 that are linked by disulfide bonds, after synthesis and

one to three weeks before complete recovery (Welliver, 2003).

34 Respiratory Disease and Infection - A New Insight

morbidity and mortality during RSV infection (Resch, 2011).

**3. The structure of respiratory syncytial virus**

(Dickens et al., 1984).

RSV does not normally replicate outside of the bronchopulmonary tree and is restricted to the respiratory mucosa (Othumganpat et al, 2009). The G protein initiates attachment of the virus to the epithelial cell. The virus fuses with the epithelial cell membrane and enters the cytoplasm after the F protein is cleaved by proteolytic enzymes of the infected cell. If the precursor is not cleaved it has no fusion activity, virion penetration will not occur and the virus particle is unable to initiate infection. Fusion by F1 occurs at the neutral PH of the ex‐ tra cellular environment, allowing release of the viral nucleocapsid directly into the cell. This enables the virus to bypass internalization through endosomes. After replication the vi‐ rus matures by budding from the cell surface. The new progeny nucleocapsids form in the cytoplasm and move to the cell surface. The M protein is essential for particle formation, probably serving to link the viral envelope to the nucleocapsid. During budding, most host proteins are excluded from the membrane. If appropriate host cell proteases are present, precursor proteins in the plasma membrane will be activated by cleavage. Activated fusion protein will then cause fusion of adjacent cell membranes, resulting in formation of large syncytia (Jawetz et al., 2004). The viral RNA can spread without forming complete viral par‐ ticles. The infection results in the destruction of the epithelial cells of the upper respiratory tract. Exposure to RSV triggers humoral immune responses. Primary RSV infection results in only a weak antibody response with IgM, IgG, and IgA produced. This response is not sufficient to completely destroy the virus or to prevent upper respiratory tract replication of the virus, thus an upper respiratory tract illness develops (Smith et al., 2009).

Although, RSV mostly infects the nasal epithelial cells. However, development of extrapul‐ monary disease has been observed in certain T and B cell immunodeficiency states. The as‐ sociation of RSV with asthma and reversible reactive airway disease in early childhood has attracted significant attention (Mohapatra et al., 2008). Recurrent wheezing for up to 5 to 7 years of age and established airway disease has been observed in a significant number of children with a strong family history of allergy, after primary infection or reinfection with RSV (LeManske, 2004). Immune response to primary infection is relatively small but on rein‐ fection, a significant booster effect with sustained immunologic reactivity is observed in se‐ rum and respiratory mucosa. Both CD4- and CD8-specific as well as Th1- and Th2-cell specific immune responses have been observed during human infection. In addition, proin‐ flammatory as well as immunoregulatory cytokines and chemokines are induced in the res‐ piratory tract after natural and induced (in vitro) infection. In vulnerable patients, the RSV infection will spread into the lower respiratory tract (Becker et al., 2006). In premature in‐ fants and immunocompromised hosts, the infection quickly progresses to the LRT. As the virus is destroyed within the lung by T cells, immunopathological responses cause further lung injury. Infected cells release proinflammatory cytokines and chemokines, including in‐ terlukins (IL-1, IL-6, and IL-8) and tumor necrosis factor-alpha (TNF-α). These actions result in activation of inflammatory cells, including macrophages, eosinophils, neutrophils, and T lymphocytes, into the airway lining and surrounding tissues.

1999 to 2007 seasons. During the same periods, mainly RSV group A dominated in sev‐

Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)

http://dx.doi.org/10.5772/53916

37

The subgroup prevalence and genotype distribution patterns of RSV strains were investigat‐ ed in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regu‐ lar 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant RSV-A seasons, followed by a single RSV-B-dominant year. RSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene (Zlateva et al, 2007). A study in India reported on RSV group A dominance for three consecutive epi‐ demic seasons. RSV group B predominated for a single season in the countries investigated and at the same or similar time as observed in Germany. Thus, it can be concluded that RSV group A predominated within similar seasons, implicating that most RSV infections were

The results of the first study to investigate the circulation and genetic diversity of RSV in Cambodia among different age ranges of population over 5 consecutive years (Arnott, et al., 2011). Circulation of RSV was seasonal, coinciding with the rainy season between July and November. The majority of RSV group B strains belonged to the BA genotype, with the ex‐

In an Iranian study during the season 2009, samples were obtained from several provinces: Tehran, Hamadan, Isfahan, Kordestan, Zanjan, Lorestan and West Azarbayjan, and were tested for G protein gene of RSV by RT-PCR (Faghihloo et al., 2011). Of the respiratory sam‐ ples tested, 22% were positive for RSV, of which 67% belonged to subgroup A and 33% to subgroup B. Phylogenetic analysis revealed that subgroup A strains fell in two genotypes GA1 and GA2, whereas subgroup B strains clustered in genotype BA. This study revealed that multiple genotypes of RSV cocirculate in Iran. Subgroup A strains are more prevalent

There has been a distinct lack of data from the Middle East in terms of molecular typing of RSV. For example, In Saudi Arabia, only few reports have described the prevalence of RSV infection in sporadic districts of the kingdom including Riyadh, Al-Quassim and Abha (Jam‐ joom et al., 1993; Bakir et al., 1998; Al-Shehri et al., 2006; Meqdam and Subiah, 2006, Akhter and Johani., 2011). These reports covered short periods of time extending from 1991-1996, 2003-2004 and 2004-2010. Both virus subtypes in RSV infection of Saudi Arabia children are found to occur with a greater dominance of type A viruses in a three year cyclical pattern. However, a greater interest in RSV is needed for proper virus characterization. Molecular typing studies are required to elucidate the nature of RSV spread in these populations.

Similar viruses were first isolated around the same time in Europe, USA, The Gambia, Ma‐ laysia, Uruguay and Australia (Cane and Pringle, 1995). Further studies in North America (Peret et al., 2000), Europe (Lukic-Grlic et al., 1999), and Africa (Cane et al., 1999, Venter et al., 2002) have shown that similar strains of RSV appear simultaneously in indistinct geo‐ graphic locations in these areas. It is clear that infections with very similar viruses may be

ception of 10 strains classified as belonging to a novel RSV group B genotype,

eral other countries.

caused by RSV group A worldwide at the same time.

than subgroup B strains, with genotype GA1 predominant.

#### **5. Molecular epidemiology of RSV**

Variability between RSV strains contributes to the ability of the virus to infect people repeat‐ edly and cause annual outbreaks. Consistent shifts in RSV group dominance have been re‐ ported worldwide in which RSV group A viruses are more frequently detected. A new BA genotype was identified in Buenos Aires in 1999 that is characterized by a 60-nucleotide du‐ plication starting after residue 791 of the G protein. Subsequently, strains with this duplica‐ tion have been found in clinical specimens from distantly related places in the world, including Kenya in East

**Figure 1.** Mechanism of RSV Virus Entry and Replication in Respiratory Virus Infection

Africa. This BA genotype was first discovered in South Africa during the investigation of a nosocomial outbreak in Pretoria in 2006. (Niekerk and Venter, 2011). In Germany, RSV group A was dominant in seven out of nine epidemic seasons predominating between 1999 to 2007 seasons. During the same periods, mainly RSV group A dominated in sev‐ eral other countries.

terlukins (IL-1, IL-6, and IL-8) and tumor necrosis factor-alpha (TNF-α). These actions result in activation of inflammatory cells, including macrophages, eosinophils, neutrophils, and T

Variability between RSV strains contributes to the ability of the virus to infect people repeat‐ edly and cause annual outbreaks. Consistent shifts in RSV group dominance have been re‐ ported worldwide in which RSV group A viruses are more frequently detected. A new BA genotype was identified in Buenos Aires in 1999 that is characterized by a 60-nucleotide du‐ plication starting after residue 791 of the G protein. Subsequently, strains with this duplica‐ tion have been found in clinical specimens from distantly related places in the world,

lymphocytes, into the airway lining and surrounding tissues.

**Figure 1.** Mechanism of RSV Virus Entry and Replication in Respiratory Virus Infection

Africa. This BA genotype was first discovered in South Africa during the investigation of a nosocomial outbreak in Pretoria in 2006. (Niekerk and Venter, 2011). In Germany, RSV group A was dominant in seven out of nine epidemic seasons predominating between

**5. Molecular epidemiology of RSV**

36 Respiratory Disease and Infection - A New Insight

including Kenya in East

The subgroup prevalence and genotype distribution patterns of RSV strains were investigat‐ ed in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regu‐ lar 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant RSV-A seasons, followed by a single RSV-B-dominant year. RSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene (Zlateva et al, 2007). A study in India reported on RSV group A dominance for three consecutive epi‐ demic seasons. RSV group B predominated for a single season in the countries investigated and at the same or similar time as observed in Germany. Thus, it can be concluded that RSV group A predominated within similar seasons, implicating that most RSV infections were caused by RSV group A worldwide at the same time.

The results of the first study to investigate the circulation and genetic diversity of RSV in Cambodia among different age ranges of population over 5 consecutive years (Arnott, et al., 2011). Circulation of RSV was seasonal, coinciding with the rainy season between July and November. The majority of RSV group B strains belonged to the BA genotype, with the ex‐ ception of 10 strains classified as belonging to a novel RSV group B genotype,

In an Iranian study during the season 2009, samples were obtained from several provinces: Tehran, Hamadan, Isfahan, Kordestan, Zanjan, Lorestan and West Azarbayjan, and were tested for G protein gene of RSV by RT-PCR (Faghihloo et al., 2011). Of the respiratory sam‐ ples tested, 22% were positive for RSV, of which 67% belonged to subgroup A and 33% to subgroup B. Phylogenetic analysis revealed that subgroup A strains fell in two genotypes GA1 and GA2, whereas subgroup B strains clustered in genotype BA. This study revealed that multiple genotypes of RSV cocirculate in Iran. Subgroup A strains are more prevalent than subgroup B strains, with genotype GA1 predominant.

There has been a distinct lack of data from the Middle East in terms of molecular typing of RSV. For example, In Saudi Arabia, only few reports have described the prevalence of RSV infection in sporadic districts of the kingdom including Riyadh, Al-Quassim and Abha (Jam‐ joom et al., 1993; Bakir et al., 1998; Al-Shehri et al., 2006; Meqdam and Subiah, 2006, Akhter and Johani., 2011). These reports covered short periods of time extending from 1991-1996, 2003-2004 and 2004-2010. Both virus subtypes in RSV infection of Saudi Arabia children are found to occur with a greater dominance of type A viruses in a three year cyclical pattern. However, a greater interest in RSV is needed for proper virus characterization. Molecular typing studies are required to elucidate the nature of RSV spread in these populations.

Similar viruses were first isolated around the same time in Europe, USA, The Gambia, Ma‐ laysia, Uruguay and Australia (Cane and Pringle, 1995). Further studies in North America (Peret et al., 2000), Europe (Lukic-Grlic et al., 1999), and Africa (Cane et al., 1999, Venter et al., 2002) have shown that similar strains of RSV appear simultaneously in indistinct geo‐ graphic locations in these areas. It is clear that infections with very similar viruses may be occurring world-wide during the same season. Overall there appears to be very little geo‐ graphic clustering of RSV strains and where this has been reported it may often be due to inadequate sampling or delayed reporting of strain variability, particularly from less devel‐ oped parts of the world. As the virus is constantly accumulating genetic and antigenic change, this implies that when a new strain arises it is able to spread very rapidly around the world.

**8. Sub unit vaccines**

2001; Johnson et al., 2004)

particularly in young infants.

halted further development (Kneyber and Kimpen., 2003).

**9. Live attenuated respiratory syncytial virus vaccines**

A large number of subunit vaccines have been studied in preclinical trials, and some have progressed into clinical trials. Most of these candidate vaccines consist of either or both of the RSV surface glycoproteins that mediate membrane fusion (F) and virus attachment (G). These vaccines were administered in various ways, such as using alum phosphate or alum hydroxide. Others were evaluated conjugated to bacterial toxins, as ISCOMs (immunosti‐ mulating complex), or with adjuvants such as CpGs (CpG-DNA), monophosphoryl lipid A, saponines, or oil-in-water emulsions (Piedra, 2003; Kneber and Kimpen, 2004). The use of in‐ activated and subunit vaccines seems safe in RSV seropositive individuals, however, two ex‐ ceptions have been noted in seronegative infants: FI-RSV and subunit vaccines induced high RSV-binding antibody titers with low neutralizing activity and the enhanced disease seen with FI-RSV can be replicated in rodent models using subunit vaccines.(Openshaw et al.,

Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)

http://dx.doi.org/10.5772/53916

39

One of the more recent trials evaluated PFP-3 (purified F protein with aluminum phosphate) in seropositive children 1 to 12 years of age with cystic fibrosis. In this trial, PFP-3 was found to be safe and immunogenic but not protective. Similarly, in a trial of PFP-2 in healthy pregnant women and their offspring, PFP-2 was found to be safe but there was no signifi‐ cant increase in RSV neutralizing IgG titers following vaccination in the third trimester so that a protective effect in the offspring could not be expected (Munoz et al., 2003). A differ‐ ent subunit vaccine was tested consisting of copurified F, G, and M proteins from RSV sub‐ group A (sanofi-aventis, Bridgewater, NJ) in a phase 1 trial in healthy adult volunteers. Of those vaccinated, 80% developed a greater than fourfold increase in neutralizing antibody titers to RSV A and RSV B. However, these titers did not persist so that annual vaccination would be necessary to maintain potentially protective antibody titers with this candidate vaccine (Wright et al., 2000). BBG2Na (Pierre Fabre, Castres, France), a promising vaccine candidate that was developed by fusing the conserved central domain of the RSV G protein to the albumin-binding region of streptococcal protein G, was found to be safe and immuno‐ genic in phase 1 and phase 2 studies. But in the phase 3 trial purpura / type III hypersensi‐ tivity resulted as an unexpected side-effect in a small number of vaccine recipients and so

Several live attenuated RSV vaccines have been identified by passage of RSV under different conditions such as lower temperatures, temperature- sensitive (*ts*), chemical mutagenesis and non-*ts* attenuating mutations. Several candidate vaccines went into clinical trials, but it was not possible to find an appropriate balance between immunogenicity and attenuation,

The development of a method to generate infectious virus from a cDNA copy of the nega‐ tive-sense single-stranded virus genome initiated the era of rational RSV vaccine design. It

#### **6. Vaccines**

There have been exhaustive attempts to identify pharmacological therapies to improve the clinical course and outcomes of this disease. However, presently, there is no licensed vac‐ cine against RSV. High-risk infants can be substantially protected by monthly intramuscular injections of a commercially available RSV-neutralizing antibody (palivizumab) adminis‐ tered during the RSV epidemic season (American Academy of Pediatrics, 2003). RSV is the focus of antiviral- and vaccine-development programmes because of the morbidity and mor‐ tality associated with bronchiolitis early in life. However, these goals are now being aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated (Meisner and Long, 2003).

The reasons why an RSV vaccine is not yet available emanate from numerous problems with its development. First, is the possibility that vaccination will potentiate naturally occurring RSV disease, as observed with the formalin-inactivated vaccine (Domachowske and Rosen‐ berg, 1996). Second, young infants exhibit relative immunologic immaturity or because of suppression of their immune response due to circulating maternally derived anti-RSV anti‐ bodies (Falsey and Walsh, 1996). Another important consideration is the need to provide protection against multiple antigenic strains of RSV in the two major groups, A and B. A number of strategies have been implemented recently to generate safe and effective subunit, inactivated, and live attenuated virus vaccines (Falsey and Walsh, 1997).

#### **7. Inactivated and subunit respiratory syncytial virus vaccines**

The first attempt at an RSV vaccine in the 1960s employed formalin-inactivated RSV parti‐ cles (FIRSV). This vaccine failed to induce a protective immune response and led to en‐ hanced disease upon natural infection with wild type (*wt)* RSV. As a result, 80% of the children vaccinated needed to be hospitalized following *wt* RSV infection, and two children died (Kim et al., 1969). Consequently, there was great apprehension to study any nonrepli‐ cating RSV vaccine in seronegative infants and children. Enhanced disease has never been observed with live attenuated vaccines and it is not seen in seropositive subjects, neither with inactivated nor with subunit RSV vaccines (Collins et al., 2001).

#### **8. Sub unit vaccines**

occurring world-wide during the same season. Overall there appears to be very little geo‐ graphic clustering of RSV strains and where this has been reported it may often be due to inadequate sampling or delayed reporting of strain variability, particularly from less devel‐ oped parts of the world. As the virus is constantly accumulating genetic and antigenic change, this implies that when a new strain arises it is able to spread very rapidly around

There have been exhaustive attempts to identify pharmacological therapies to improve the clinical course and outcomes of this disease. However, presently, there is no licensed vac‐ cine against RSV. High-risk infants can be substantially protected by monthly intramuscular injections of a commercially available RSV-neutralizing antibody (palivizumab) adminis‐ tered during the RSV epidemic season (American Academy of Pediatrics, 2003). RSV is the focus of antiviral- and vaccine-development programmes because of the morbidity and mor‐ tality associated with bronchiolitis early in life. However, these goals are now being aided by an understanding of the virus genome architecture and the mechanisms by which it is

The reasons why an RSV vaccine is not yet available emanate from numerous problems with its development. First, is the possibility that vaccination will potentiate naturally occurring RSV disease, as observed with the formalin-inactivated vaccine (Domachowske and Rosen‐ berg, 1996). Second, young infants exhibit relative immunologic immaturity or because of suppression of their immune response due to circulating maternally derived anti-RSV anti‐ bodies (Falsey and Walsh, 1996). Another important consideration is the need to provide protection against multiple antigenic strains of RSV in the two major groups, A and B. A number of strategies have been implemented recently to generate safe and effective subunit,

inactivated, and live attenuated virus vaccines (Falsey and Walsh, 1997).

with inactivated nor with subunit RSV vaccines (Collins et al., 2001).

**7. Inactivated and subunit respiratory syncytial virus vaccines**

The first attempt at an RSV vaccine in the 1960s employed formalin-inactivated RSV parti‐ cles (FIRSV). This vaccine failed to induce a protective immune response and led to en‐ hanced disease upon natural infection with wild type (*wt)* RSV. As a result, 80% of the children vaccinated needed to be hospitalized following *wt* RSV infection, and two children died (Kim et al., 1969). Consequently, there was great apprehension to study any nonrepli‐ cating RSV vaccine in seronegative infants and children. Enhanced disease has never been observed with live attenuated vaccines and it is not seen in seropositive subjects, neither

the world.

38 Respiratory Disease and Infection - A New Insight

**6. Vaccines**

expressed and replicated (Meisner and Long, 2003).

A large number of subunit vaccines have been studied in preclinical trials, and some have progressed into clinical trials. Most of these candidate vaccines consist of either or both of the RSV surface glycoproteins that mediate membrane fusion (F) and virus attachment (G). These vaccines were administered in various ways, such as using alum phosphate or alum hydroxide. Others were evaluated conjugated to bacterial toxins, as ISCOMs (immunosti‐ mulating complex), or with adjuvants such as CpGs (CpG-DNA), monophosphoryl lipid A, saponines, or oil-in-water emulsions (Piedra, 2003; Kneber and Kimpen, 2004). The use of in‐ activated and subunit vaccines seems safe in RSV seropositive individuals, however, two ex‐ ceptions have been noted in seronegative infants: FI-RSV and subunit vaccines induced high RSV-binding antibody titers with low neutralizing activity and the enhanced disease seen with FI-RSV can be replicated in rodent models using subunit vaccines.(Openshaw et al., 2001; Johnson et al., 2004)

One of the more recent trials evaluated PFP-3 (purified F protein with aluminum phosphate) in seropositive children 1 to 12 years of age with cystic fibrosis. In this trial, PFP-3 was found to be safe and immunogenic but not protective. Similarly, in a trial of PFP-2 in healthy pregnant women and their offspring, PFP-2 was found to be safe but there was no signifi‐ cant increase in RSV neutralizing IgG titers following vaccination in the third trimester so that a protective effect in the offspring could not be expected (Munoz et al., 2003). A differ‐ ent subunit vaccine was tested consisting of copurified F, G, and M proteins from RSV sub‐ group A (sanofi-aventis, Bridgewater, NJ) in a phase 1 trial in healthy adult volunteers. Of those vaccinated, 80% developed a greater than fourfold increase in neutralizing antibody titers to RSV A and RSV B. However, these titers did not persist so that annual vaccination would be necessary to maintain potentially protective antibody titers with this candidate vaccine (Wright et al., 2000). BBG2Na (Pierre Fabre, Castres, France), a promising vaccine candidate that was developed by fusing the conserved central domain of the RSV G protein to the albumin-binding region of streptococcal protein G, was found to be safe and immuno‐ genic in phase 1 and phase 2 studies. But in the phase 3 trial purpura / type III hypersensi‐ tivity resulted as an unexpected side-effect in a small number of vaccine recipients and so halted further development (Kneyber and Kimpen., 2003).

#### **9. Live attenuated respiratory syncytial virus vaccines**

Several live attenuated RSV vaccines have been identified by passage of RSV under different conditions such as lower temperatures, temperature- sensitive (*ts*), chemical mutagenesis and non-*ts* attenuating mutations. Several candidate vaccines went into clinical trials, but it was not possible to find an appropriate balance between immunogenicity and attenuation, particularly in young infants.

The development of a method to generate infectious virus from a cDNA copy of the nega‐ tive-sense single-stranded virus genome initiated the era of rational RSV vaccine design. It allowed the use of site-directed mutagenesis to introduce desired mutations into the RSV ge‐ nome and to evaluate the contribution of each mutation to the attenuation phenotype by in‐ troducing individual mutations or sets of mutations into the RSV genome. Reverse genetics has also been used to generate attenuated deletion mutants that are useful as live attenuated RSV vaccines. Deletion of the genes for the interferon antagonists NS1 or xlink, the small hy‐ drophobic gene SH, or the M2–2 regulator of transcription and replication produced viable viruses that displayed restricted replication in nonhuman primates. Candidate vaccines bearing these deletions with or without additional attenuating point mutations were identi‐ fied as suitable for clinical development (Collins and Murphy, 2005).

impaired immune system or other chronic conditions. LRIs are a common cause of hospital admission and excess mortality. RSV infection is the most frequent etiology of a child's first LRI. Presently, passive protection against RSV is achieved successfully through monthly in‐ tramuscular injection of the humanized monoclonal anti-RSV antibody palivizumab How‐

Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)

http://dx.doi.org/10.5772/53916

41

Attempts to develop a vaccine against RSV have been unsuccessful to date. A formalin-inac‐ tivated RSV vaccine was developed in the 1960s. Although initial serological responses to this vaccine appeared promising, children who received this vaccine developed more severe disease, with a number of deaths, when exposed to natural RSV infection. The development of a successful RSV vaccine must address this issue and achieve protection of very young children if it is to have an impact on severe RSV disease. Recent progress in this area has included development of stable, live-attenuated RSV vaccines that can be administered as nasal spray. Despite progress in this area, a vaccine that is ready for use in clinical practice is

Another approach is the development of an RSV vaccine that involves use of cloned RSV surface proteins as potential subunit vaccines. RSV fusion (F) and glycoprotein (G) can in‐ duce neutralizing and protective antibodies and are the components in development. Phase 1 trials of Fusion (F) protein nanoparticle RSV vaccine candidates have shown that they are generally well-tolerated, highly immunogenic and produce functional antibodies that neu‐ tralize RSV. These are being evaluated for potential immunization of young children and al‐ so for administration to pregnant women during the last trimester to boost anti-RSV

1 Microbiology Section, Department of Pathology and Laboratory Medicine, King AbdulA‐

2 Microbiology, College of Medicine, King Saud Bin AbdulAziz University for Health Scien‐

[1] Akhter J, Johani S. Epidemiology and diagnosis of human respiratory syncytial virus infections. In Human Respiratory syncytial virus infection edited by Resch B. 2011,

Part 2, Chapter 8: 161-176 Intech open access publishers

ever, immunoglobulin products are expensive to administer

antibody levels transferred to the infant (Schmidt, 2007).

still many years away.

**Author details**

Sameera Al Johani1,2 and Javed Akhter1

ziz Medical City, Riyadh, Saudi Arabia

ces, Riyadh, Saudi Arabia

**References**

A promising candidate vaccine is an RSV subgroup A mutant designated *cp*248/404/1030ΔSH. This mutated virus has 11 mutations identified in the biologically de‐ rived *cpts*248/ 404RSV, a further mutation in the large polymerase protein L (the 1030 muta‐ tion), and a deletion of the SH protein. It was was evaluated in a phase 1 trial in 1- to 2 month-old infants, replication was highly restricted in seronegative infants and the vaccine was well tolerated (Karron et al., 2005). Although neither systemic nor mucosal antibody re‐ sponses were repeatedly observed in the youngest vaccinated infants, but complete restric‐ tion of a second dose of vaccine given 4 weeks after the first dose indicated that protective immunity could be induced.

#### **10. Future strategies**

One of the most promising current strategies for protection against respiratory tract infec‐ tion is intranasal treatment with vectors capable of generating RNAs that block viral replica‐ tion. RNA interference (RNAi) is a natural defense of the innate immune system against viruses (Dallas and Vlassov, 2006). Following viral replication, double-stranded viral RNA is recognized by the host RNAi system which cuts it into short oligoribonucleotides, 20–30 bases long. These short sequences activate the cell's RNA cleavage machinery (the RNA in‐ terference silencing complex, or RISC) to destroy the viral RNA. By introducing siRNAs complementary to specific viral mRNAs, double-stranded activating RNA can be generated that activates the RISC cleavage system and destroys the viral message. This approach using antiviral siRNA has been reviewed by Manjunath et al, 2009. The use of siRNA as an antivi‐ ral agent involves a relatively straightforward attack on one or more key viral genes and should be effective against many human pathogens. The use of non-integrating plasmid vec‐ tors removes the risk of mutagenesis caused by some viral vectors.

#### **11. Summary**

The burden of acute respiratory infections (ARIs) caused by viral pathogens is impressive and leaves no doubt that effective and affordable vaccines are urgently needed. The impact of respiratory viral infection is greatest in the very young, the elderly, and people with an impaired immune system or other chronic conditions. LRIs are a common cause of hospital admission and excess mortality. RSV infection is the most frequent etiology of a child's first LRI. Presently, passive protection against RSV is achieved successfully through monthly in‐ tramuscular injection of the humanized monoclonal anti-RSV antibody palivizumab How‐ ever, immunoglobulin products are expensive to administer

Attempts to develop a vaccine against RSV have been unsuccessful to date. A formalin-inac‐ tivated RSV vaccine was developed in the 1960s. Although initial serological responses to this vaccine appeared promising, children who received this vaccine developed more severe disease, with a number of deaths, when exposed to natural RSV infection. The development of a successful RSV vaccine must address this issue and achieve protection of very young children if it is to have an impact on severe RSV disease. Recent progress in this area has included development of stable, live-attenuated RSV vaccines that can be administered as nasal spray. Despite progress in this area, a vaccine that is ready for use in clinical practice is still many years away.

Another approach is the development of an RSV vaccine that involves use of cloned RSV surface proteins as potential subunit vaccines. RSV fusion (F) and glycoprotein (G) can in‐ duce neutralizing and protective antibodies and are the components in development. Phase 1 trials of Fusion (F) protein nanoparticle RSV vaccine candidates have shown that they are generally well-tolerated, highly immunogenic and produce functional antibodies that neu‐ tralize RSV. These are being evaluated for potential immunization of young children and al‐ so for administration to pregnant women during the last trimester to boost anti-RSV antibody levels transferred to the infant (Schmidt, 2007).

#### **Author details**

allowed the use of site-directed mutagenesis to introduce desired mutations into the RSV ge‐ nome and to evaluate the contribution of each mutation to the attenuation phenotype by in‐ troducing individual mutations or sets of mutations into the RSV genome. Reverse genetics has also been used to generate attenuated deletion mutants that are useful as live attenuated RSV vaccines. Deletion of the genes for the interferon antagonists NS1 or xlink, the small hy‐ drophobic gene SH, or the M2–2 regulator of transcription and replication produced viable viruses that displayed restricted replication in nonhuman primates. Candidate vaccines bearing these deletions with or without additional attenuating point mutations were identi‐

A promising candidate vaccine is an RSV subgroup A mutant designated *cp*248/404/1030ΔSH. This mutated virus has 11 mutations identified in the biologically de‐ rived *cpts*248/ 404RSV, a further mutation in the large polymerase protein L (the 1030 muta‐ tion), and a deletion of the SH protein. It was was evaluated in a phase 1 trial in 1- to 2 month-old infants, replication was highly restricted in seronegative infants and the vaccine was well tolerated (Karron et al., 2005). Although neither systemic nor mucosal antibody re‐ sponses were repeatedly observed in the youngest vaccinated infants, but complete restric‐ tion of a second dose of vaccine given 4 weeks after the first dose indicated that protective

One of the most promising current strategies for protection against respiratory tract infec‐ tion is intranasal treatment with vectors capable of generating RNAs that block viral replica‐ tion. RNA interference (RNAi) is a natural defense of the innate immune system against viruses (Dallas and Vlassov, 2006). Following viral replication, double-stranded viral RNA is recognized by the host RNAi system which cuts it into short oligoribonucleotides, 20–30 bases long. These short sequences activate the cell's RNA cleavage machinery (the RNA in‐ terference silencing complex, or RISC) to destroy the viral RNA. By introducing siRNAs complementary to specific viral mRNAs, double-stranded activating RNA can be generated that activates the RISC cleavage system and destroys the viral message. This approach using antiviral siRNA has been reviewed by Manjunath et al, 2009. The use of siRNA as an antivi‐ ral agent involves a relatively straightforward attack on one or more key viral genes and should be effective against many human pathogens. The use of non-integrating plasmid vec‐

The burden of acute respiratory infections (ARIs) caused by viral pathogens is impressive and leaves no doubt that effective and affordable vaccines are urgently needed. The impact of respiratory viral infection is greatest in the very young, the elderly, and people with an

fied as suitable for clinical development (Collins and Murphy, 2005).

tors removes the risk of mutagenesis caused by some viral vectors.

immunity could be induced.

40 Respiratory Disease and Infection - A New Insight

**10. Future strategies**

**11. Summary**

Sameera Al Johani1,2 and Javed Akhter1

1 Microbiology Section, Department of Pathology and Laboratory Medicine, King AbdulA‐ ziz Medical City, Riyadh, Saudi Arabia

2 Microbiology, College of Medicine, King Saud Bin AbdulAziz University for Health Scien‐ ces, Riyadh, Saudi Arabia

#### **References**

[1] Akhter J, Johani S. Epidemiology and diagnosis of human respiratory syncytial virus infections. In Human Respiratory syncytial virus infection edited by Resch B. 2011, Part 2, Chapter 8: 161-176 Intech open access publishers

[2] Al-Shehri, M.A., A. Sadeq, and K. Quli, 2006. Bronchiolitis in Abha, Southwest Saudi Arabia: Viral etiology and predictors for hospital admission. West. Afr. J. Med., 24: 299-304.

[16] Empey, KM., Peebles Jr, RS, Kolls JK. Pharmacologic Advances in the Treatment and Prevention of Respiratory Syncytial Virus *Clin Infect Dis. (2010) 50 (9): 1258-1267. doi:*

Virology and Molecular Epidemiology of Respiratory Syncytial Virus (RSV)

http://dx.doi.org/10.5772/53916

43

[17] Faghihloo, E.; Salimi, V.; Rezaei, F.; Naseri, M.; Mamishi, S.; Mahmoodi, M.; Mokh‐ tari-Azad, T.Genetic diversity in the G protein gene of human respiratory syncytial virus among Iranian children with acute respiratory symptoms. Iranian Journal of

[18] Falsey,A.R.,andWalsh,E.E.(1996).Safety And Immunogenicity OF A Respiratory Syn‐ cytial Virus Subunit Vaccine (PFP- 2) In Ambulatory Adults Over Age 60. Vaccine 14:

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[20] Greenough A. Respiratory syncytial virus infection: Clinical features, management,

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[2] Al-Shehri, M.A., A. Sadeq, and K. Quli, 2006. Bronchiolitis in Abha, Southwest Saudi Arabia: Viral etiology and predictors for hospital admission. West. Afr. J. Med., 24:

[3] American Academy of Pediatrics, C. O. I. D. A. C. O. F. A. N. Revised indications for theuse of palivizumab and respiratory syncytial virus immune globulin intravenous for theprevention of respiratory syncytial virus infections. Pediatrics 2003; 112: 1442.

[4] Arnott A, Vong S, Mardy S, Chu S, Naughtin M, Sovann L, Buecher C, Beauté J, Rith

[5] A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype. J Clin Microbiol.

[6] Becker Y. Respiratory syncytial virus (RSV) evades the human adaptive immune sys‐ tem by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cyto‐

[7] Black CP. Systematic review of the biology and medical management of respiratory

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**Chapter 3**

**Structural and Functional Aspects**

Wahyu Surya, Montserrat Samsó and Jaume Torres

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53957

**1. Introduction**

**of Viroporins in Human Respiratory Viruses:**

**Respiratory Syncytial Virus and Coronaviruses**

Viroporins are an increasingly recognized class of small viral membrane proteins (~60-120 amino acids) which oligomerize to produce hydrophilic pores at the membranes of virusinfected cells [1]. The existence of 'viroporins' was proposed more than 30 years ago after observing enhanced membrane permeability in infected cells [2]. These proteins form oligomers of defined size, and can act as proton or ion channels, and in general enhancing membrane permeability in the host [3]. Even though viroporins are not essential for the rep‐ lication of viruses, their absence results in attenuated or weakened viruses or changes in

In addition to having one – sometimes two – α-helical transmembrane (TM) domain(s), viro‐ porins usually contain additional extramembrane regions that are able to make contacts with viral or host proteins. Indeed, the network of interactions of viroporins with other viral or cellular proteins is key to understand the regulation of viral protein trafficking through

In general, viroporins participate in the entry or release of viral particles into or out of cells, and membrane permeabilization may be a desirable functionality for the virus. Indeed, sev‐ eral viral proteins that are not viroporins are known to affect membrane permeabilization, e.g., A38L protein of vaccinia virus, a 33-kDa glycoprotein that allows Ca2+ influx and indu‐ ces necrosis in infected cells [6]. In viruses that lack typical viroporins, their function may be replaced by such pore-forming glycoproteins. For example, HIV-2 lacks typical viroporins, and ROD10 Env is an envelope glycoprotein that enhances viral particle release. In HIV-1,

> © 2013 Surya et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

tropism (organ localization) and therefore diminished pathological effects [4, 5].

the vesicle system, viral morphogenesis and pathogenicity.

this function is attributed to the viroporin Vpu [7].


## **Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and Coronaviruses**

Wahyu Surya, Montserrat Samsó and Jaume Torres

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53957

#### **1. Introduction**

[57] Welliver RC. Respiratory syncytial virus and other respiratory viruses. Pediatr Infect

[58] Wright PF, Karron RA, Belshe RB, et al. Evaluation of a live, cold-passaged, tempera‐ ture-sensitive, respiratory syncytial virus vaccine candidate in infancy. J Infect Dis

[59] Wright PF, Karron RA, Madhi SA, et al. The interferon antagonist xlink protein of respiratory syncytial virus is an important virulence determinant for humans. J Infect

[60] Zlateva KT, Vijgen L, Dekeersmaeker N, Naranjo C, Van Ranst M. Subgroup preva‐ lence and genotype circulation patterns of human respiratory syncytial virus in Bel‐ gium during ten successive epidemic seasons. J Clin Microbiol. 2007 45(9):3022-30.

Dis J 2003;22:S6-S10.

46 Respiratory Disease and Infection - A New Insight

2000; 182:1331–1342

Dis 2006;193:573–581

Viroporins are an increasingly recognized class of small viral membrane proteins (~60-120 amino acids) which oligomerize to produce hydrophilic pores at the membranes of virusinfected cells [1]. The existence of 'viroporins' was proposed more than 30 years ago after observing enhanced membrane permeability in infected cells [2]. These proteins form oligomers of defined size, and can act as proton or ion channels, and in general enhancing membrane permeability in the host [3]. Even though viroporins are not essential for the rep‐ lication of viruses, their absence results in attenuated or weakened viruses or changes in tropism (organ localization) and therefore diminished pathological effects [4, 5].

In addition to having one – sometimes two – α-helical transmembrane (TM) domain(s), viro‐ porins usually contain additional extramembrane regions that are able to make contacts with viral or host proteins. Indeed, the network of interactions of viroporins with other viral or cellular proteins is key to understand the regulation of viral protein trafficking through the vesicle system, viral morphogenesis and pathogenicity.

In general, viroporins participate in the entry or release of viral particles into or out of cells, and membrane permeabilization may be a desirable functionality for the virus. Indeed, sev‐ eral viral proteins that are not viroporins are known to affect membrane permeabilization, e.g., A38L protein of vaccinia virus, a 33-kDa glycoprotein that allows Ca2+ influx and indu‐ ces necrosis in infected cells [6]. In viruses that lack typical viroporins, their function may be replaced by such pore-forming glycoproteins. For example, HIV-2 lacks typical viroporins, and ROD10 Env is an envelope glycoprotein that enhances viral particle release. In HIV-1, this function is attributed to the viroporin Vpu [7].

© 2013 Surya et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

An important point that needs to be established, in view of the observed channel activi‐ ty of viroporins, is whether the channels they form are selective, with a controlled gating mechanism, or whether permeabilization is non selective, like in some antimicrobial pep‐ tides [8]. Viroporins have also been found to modulate endogenous cellular channels [9-12] and this activity may also have an important regulatory role during the life cycle of the virus.

**2. Envelope (E) and Small Hydrophobic (SH) proteins in respiratory**

**Medical impact of the human respiratory syncytial virus (hRSV) infection.** hRSV is a member of the *Paramyxoviridae* family, and is the leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide [37]. hRSV infection is the most fre‐ quent cause of hospitalization of infants and young children in industrialized countries. In the USA alone, around 100,000 infants with hRSV infection are hospitalized annually. hRSV also is a significant problem in the elderly, patients with cardiopulmonary diseases and in immunocompromised individuals. hRSV accounts for approximately 10,000 deaths per year in the group of >64 years of age in the US. Globally, hRSV infection results in 64 million cases and 160,000 deaths every year (http://www.who.int/vaccine\_research/

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and

Coronaviruses

49

http://dx.doi.org/10.5772/53957

There is currently no effective vaccine available to prevent hRSV infection. Development of vaccines has been complicated by the fact that host immune responses appear to play a sig‐ nificant role in the pathogenesis of the disease [38]. Naturally acquired immunity to hRSV is neither complete nor durable, and recurrent infections occur frequently during the first three years of life. Palivizumab, a humanized monoclonal antibody directed against hRSV surface fusion F protein (Synagis, by MedImmune), is moderately effective but very expen‐ sive. It is currently available as prophylactic drug for infants at high risk. Cost of prevention limits its use in many parts of the world. The only licensed drug for use in infected people is ribavirin, but its efficacy is limited. Antibodies against both F (fusion) and G (attachment) proteins have been found in the serum of hRSV infected patients, but only provide tempora‐ ry protection. Therefore, low immunoprotection and lack of suitable antivirals leads to‐ wards the search and characterization of new drug targets for the effective treatments of hRSV infection. A possible suitable target is the SH protein as will be elaborated below.

**The viral particle formation in RSV**. Based on the reactive patterns to monoclonal antibod‐ ies, there are two hRSV strains that co-circulate in human populations, subtypes A and B. The hRSV genome comprises a nonsegmented negative-stranded RNA of ~15 kb that tran‐ scribes 11 proteins, including the three membrane proteins fusion (F), attachment (G), and small hydrophobic (SH) [39, 40]. The F protein is sufficient for mediating viral entry into cells *in vitro*, and the G protein plays a role in viral attachment [41, 42]. In contrast, the pre‐

hRSV also contains six internal structural proteins: the matrix (M) protein, which provides structure for the virus particle, nucleoprotein (N), phosphoprotein (P) and large (L) poly‐ merase protein form the ribonucleoprotein (RNP) complex, which encapsidates the RSV ge‐ nome and functions as the RNA-dependent RNA polymerase. Lastly, two isoforms of matrix protein 2 (M2-1 and M2-2) are accessory proteins that control transcription and repli‐ cation [43]. Viral proteins traffic to the apical surface of polarized epithelial cells, where they

**viruses**

**2.1. SH protein in hRSV**

diseases/ari/en/index2.html).

cise role of SH protein is still unclear.

Viroporins can be found in all kinds of viruses, RNA, DNA, enveloped and non-enveloped. Examples of viroporins are picornavirus 2B [13], alphavirus 6K [14-16], HIV-1 Vpu [17, 18], influenza virus A M2, (also called AM2) [19], RSV SH protein [20], p10 protein of avian reo‐ virus [21], Human hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) p7 [22, 23], Paramecium bursaria chlorella virus (PBCV-1) Kcv [24], and coronavirus envelope pro‐ teins, e.g., SARS-CoV E [25, 26]. Recent reviews [27, 28] provide more examples and possible functional roles.

The most extensively studied viroporin to date is probably the M2 protein from influen‐ za A virus (AM2). AM2 protein is 97-residue long, with one transmembrane (TM) do‐ main and a C-terminal cytoplasmic amphiphilic helix. AM2 forms homotetramers and is located in the viral envelope, where it enables protons from the endosome to enter the viral particle (virion). This lowers the pH inside the viral particle, causing dissociation of the viral matrix protein M1 from the ribonucleoprotein RNP, uncoating of the virus and exposure of the content to the cytoplasm of the host cell. AM2 also delays acidification of the late Golgi in some strains [29, 30].

The proton channel activity of AM2 can be inhibited by antiviral drugs amantadine and ri‐ mantadine, which block the virus from taking over the host cell. Two different high-resolu‐ tion structures of truncated forms of AM2 have been reported: the structure of a mutated form of its TM region (residues 22-46) [31], and a slightly longer form (residues 18-60) con‐ taining the TM region and a segment of the C-terminal domain [32, 33]. These studies sug‐ gest that the known AM2 adamantane inhibitors, amantadine and rimantadine, act by either blocking the pore [31, 34] or by an allosteric mechanism [32]. New AM2 inhibitors have been reported [35], but their effectiveness against adamantane-resistant viruses remains to be es‐ tablished. The use of these drugs presents a classical example of targeting viral channels to treat viral infection infection [31, 32, 36].

The case of AM2 protein in influenza A represents a link between viroporin activity and structure to viral pathogenesis. Unfortunately, for many viroporins even rudimentary structural models are lacking due to high hydrophobicity, conformational flexibility and tendency to aggregate. For some viroporins however, increasing degrees of structural in‐ formation can be obtained due to availability of high quality purified protein. Examples of these are the viroporins present in coronaviruses (CoV) and in the respiratory syncy‐ tial virus (RSV), envelope (E) protein and the small hydrophobic (SH) protein, respective‐ ly. Both types of virus infect the upper and lower respiratory tract of humans, and their viroporins are the subject of this chapter.

### **2. Envelope (E) and Small Hydrophobic (SH) proteins in respiratory viruses**

#### **2.1. SH protein in hRSV**

An important point that needs to be established, in view of the observed channel activi‐ ty of viroporins, is whether the channels they form are selective, with a controlled gating mechanism, or whether permeabilization is non selective, like in some antimicrobial pep‐ tides [8]. Viroporins have also been found to modulate endogenous cellular channels [9-12] and this activity may also have an important regulatory role during the life cycle

Viroporins can be found in all kinds of viruses, RNA, DNA, enveloped and non-enveloped. Examples of viroporins are picornavirus 2B [13], alphavirus 6K [14-16], HIV-1 Vpu [17, 18], influenza virus A M2, (also called AM2) [19], RSV SH protein [20], p10 protein of avian reo‐ virus [21], Human hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) p7 [22, 23], Paramecium bursaria chlorella virus (PBCV-1) Kcv [24], and coronavirus envelope pro‐ teins, e.g., SARS-CoV E [25, 26]. Recent reviews [27, 28] provide more examples and possible

The most extensively studied viroporin to date is probably the M2 protein from influen‐ za A virus (AM2). AM2 protein is 97-residue long, with one transmembrane (TM) do‐ main and a C-terminal cytoplasmic amphiphilic helix. AM2 forms homotetramers and is located in the viral envelope, where it enables protons from the endosome to enter the viral particle (virion). This lowers the pH inside the viral particle, causing dissociation of the viral matrix protein M1 from the ribonucleoprotein RNP, uncoating of the virus and exposure of the content to the cytoplasm of the host cell. AM2 also delays acidification

The proton channel activity of AM2 can be inhibited by antiviral drugs amantadine and ri‐ mantadine, which block the virus from taking over the host cell. Two different high-resolu‐ tion structures of truncated forms of AM2 have been reported: the structure of a mutated form of its TM region (residues 22-46) [31], and a slightly longer form (residues 18-60) con‐ taining the TM region and a segment of the C-terminal domain [32, 33]. These studies sug‐ gest that the known AM2 adamantane inhibitors, amantadine and rimantadine, act by either blocking the pore [31, 34] or by an allosteric mechanism [32]. New AM2 inhibitors have been reported [35], but their effectiveness against adamantane-resistant viruses remains to be es‐ tablished. The use of these drugs presents a classical example of targeting viral channels to

The case of AM2 protein in influenza A represents a link between viroporin activity and structure to viral pathogenesis. Unfortunately, for many viroporins even rudimentary structural models are lacking due to high hydrophobicity, conformational flexibility and tendency to aggregate. For some viroporins however, increasing degrees of structural in‐ formation can be obtained due to availability of high quality purified protein. Examples of these are the viroporins present in coronaviruses (CoV) and in the respiratory syncy‐ tial virus (RSV), envelope (E) protein and the small hydrophobic (SH) protein, respective‐ ly. Both types of virus infect the upper and lower respiratory tract of humans, and their

of the virus.

48 Respiratory Disease and Infection - A New Insight

functional roles.

of the late Golgi in some strains [29, 30].

treat viral infection infection [31, 32, 36].

viroporins are the subject of this chapter.

**Medical impact of the human respiratory syncytial virus (hRSV) infection.** hRSV is a member of the *Paramyxoviridae* family, and is the leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide [37]. hRSV infection is the most fre‐ quent cause of hospitalization of infants and young children in industrialized countries. In the USA alone, around 100,000 infants with hRSV infection are hospitalized annually. hRSV also is a significant problem in the elderly, patients with cardiopulmonary diseases and in immunocompromised individuals. hRSV accounts for approximately 10,000 deaths per year in the group of >64 years of age in the US. Globally, hRSV infection results in 64 million cases and 160,000 deaths every year (http://www.who.int/vaccine\_research/ diseases/ari/en/index2.html).

There is currently no effective vaccine available to prevent hRSV infection. Development of vaccines has been complicated by the fact that host immune responses appear to play a sig‐ nificant role in the pathogenesis of the disease [38]. Naturally acquired immunity to hRSV is neither complete nor durable, and recurrent infections occur frequently during the first three years of life. Palivizumab, a humanized monoclonal antibody directed against hRSV surface fusion F protein (Synagis, by MedImmune), is moderately effective but very expen‐ sive. It is currently available as prophylactic drug for infants at high risk. Cost of prevention limits its use in many parts of the world. The only licensed drug for use in infected people is ribavirin, but its efficacy is limited. Antibodies against both F (fusion) and G (attachment) proteins have been found in the serum of hRSV infected patients, but only provide tempora‐ ry protection. Therefore, low immunoprotection and lack of suitable antivirals leads to‐ wards the search and characterization of new drug targets for the effective treatments of hRSV infection. A possible suitable target is the SH protein as will be elaborated below.

**The viral particle formation in RSV**. Based on the reactive patterns to monoclonal antibod‐ ies, there are two hRSV strains that co-circulate in human populations, subtypes A and B. The hRSV genome comprises a nonsegmented negative-stranded RNA of ~15 kb that tran‐ scribes 11 proteins, including the three membrane proteins fusion (F), attachment (G), and small hydrophobic (SH) [39, 40]. The F protein is sufficient for mediating viral entry into cells *in vitro*, and the G protein plays a role in viral attachment [41, 42]. In contrast, the pre‐ cise role of SH protein is still unclear.

hRSV also contains six internal structural proteins: the matrix (M) protein, which provides structure for the virus particle, nucleoprotein (N), phosphoprotein (P) and large (L) poly‐ merase protein form the ribonucleoprotein (RNP) complex, which encapsidates the RSV ge‐ nome and functions as the RNA-dependent RNA polymerase. Lastly, two isoforms of matrix protein 2 (M2-1 and M2-2) are accessory proteins that control transcription and repli‐ cation [43]. Viral proteins traffic to the apical surface of polarized epithelial cells, where they assemble into virus filaments at the plasma membrane [44], although the mechanisms that drive assembly into filaments and budding are not well understood.

deletion of SH protein gene from RSV did not result in increased apoptosis in infected

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and

Coronaviruses

51

http://dx.doi.org/10.5772/53957

**Localization and post-translational modifications of SH protein during viral infection.** In infected cells, some SH protein is found in plasma membrane and cytoplasm, but most of the SH protein accumulates at the membranes of the Golgi complex and only very low amounts are found in the viral envelope [64]. Several forms of the SH protein, glycosylated and non-glycosylated, are present during infection [65], but the non-glycosylated form ap‐ pears to be the most abundant [66]. SH protein is also modified by tyrosine phosphorylation [61], and increased accumulation of SH in the Golgi complex was observed in the presence of a kinase inhibitor. Thus, SH protein is modified by a MAPK p38-dependent tyrosine kin‐ ase activity and this modification influences its cellular distribution. Although SH contains one cysteine residue, no palmitoylation has been detected in SH protein in conditions where

**Structural determination of SH protein.** An important step towards the understanding of viroporin function at the molecular level is the availability of these proteins in a highly pure form. This has been so far difficult due to their high hydrophobicity, toxicity to expression hosts, and tendency to aggregate. However, recently we have been able to obtain the full

SH protein after cross-linking has been shown to form multiple oligomers of increasing size in SDS [66, 68]. Later, we showed that the TM domain of SH protein forms only ho‐ mopentamers in perfluoro-octanoic acid (PFO) gels [69]. Reports using purified fulllength SH protein have confirmed the pentameric nature of the oligomer formed by this protein. For example, a bundle formation of a tagged SH protein construct was visual‐ ized under electron microscopy and was interpreted as a pentameric or a hexameric structure [70]. Using a purified tag-free SH protein, we have unequivocally demonstrated the homo-pentameric nature of these oligomers in a variety of detergents using analyti‐ cal ultracentrifugation and electrophoresis (Fig. 1) [71]. Indeed, in the presence of PFO (Fig. 1B) and a variety of other detergents under Blue-native gel electrophoresis (Fig. 1C), the full-length SH protein migrates as a single band with a molecular weight ~40 kDa, consistent with a pentameric oligomer. The pentameric form of SH has been further confirmed by analytical ultracentrifugation sedimentation equilibrium in detergents DPC, C14SB and C8E5 micelles. In these detergents, the species distribution profiles show a

**Secondary structure of SH protein.** The SH protein is predicted to have an α-helical re‐ gion spanning residues 16-46, which includes its predicted TM region (residues ~20-40, Fig. 1A). Fourier Transform infrared (FTIR) data for the full-length SH protein reconsti‐ tuted in model lipid bilayers shows the presence of ~60% α-helical structure, whereas the rest is β-structure [71]. The availability of purified, isotopically labeled protein allowed us to obtain a model for this pentameric oligomer where SH protein was reconstituted in DPC micelles using NMR (Fig. 2). The model shows the lumen of the hypothetic chan‐

length SH protein [67] that allows structural and biophysical studies (Fig. 1A).

best fit to a monomer-pentamer self-association model (Fig. 1 D-E).

nel, sufficient for the passage of ions.

H441 cells [11].

F and G were palmitoylated [48].

Generation of nascent hRSV genomic RNA appears to occur in discrete cytoplasmic inclu‐ sion bodies that contain the hRSV N, P, L, M2-1 and M2-2 proteins but not the F, G, or SH proteins [45]. It is suspected that the RNP complexes form in the inclusions and then traffic to the apical membrane, where they meet with the surface glycoproteins F, G, and SH arriv‐ ing from the Golgi apparatus through the secretory pathway [46]. hRSV proteins and viral RNA assemble into virus filaments at the cell surface. These filaments are thought to con‐ tribute to cell-cell spread of the virus and morphologically resemble the filamentous form of virions seen in electron microscopy (EM) studies of virus produced in polarized cells [47].

**The small hydrophobic (SH) protein.** The SH protein is 65 or 64 amino acids long, in sub‐ type A or B, respectively. SH protein has a single membrane-spanning hydrophobic region [48], and a C-terminal extramembrane tail, oriented extracellularly/lumenally [48]. The se‐ quence of SH protein is highly conserved, especially at the TM domain [49, 50]. hRSV that lacks SH protein, hRSVΔSH, is still viable, and still forms syncytia [51-53]. However, hRSVΔSH was attenuated in *in vivo* mouse and chimpanzee models [4, 5], which indicates that SH protein is important for hRSV pathogenesis. SH protein has been suggested to play an ancillary role in virus-mediated cell fusion [54, 55]. Also, the presence of SH protein has been shown to reduce cytopathic effect (CPE) and apoptosis in L929 and A549 (lung epithe‐ lial cell line) infected cells, at least in part by inhibiting tumor necrosis factor α (TNF-α) pro‐ duction [56], similarly to parainfluenza virus 5 (PIV5).

**Interaction of SH protein with viral and host proteins.** Extensive protein-protein interac‐ tions have been observed between the three membrane proteins on the hRSV envelope, F, G, and SH [51, 57, 58] and these interactions have an effect on fusion activity of hRSV on the host [51, 54]. In cells transiently expressing hRSV membrane proteins, the presence of G and SH proteins enhanced fusion activity mediated by F protein [54, 55]. Thus, SH protein has been suggested to play an ancillary role in virus-mediated cell fusion. However, using virusinfected cells the presence of G protein alone enhanced F-mediated fusion activity [51], whereas SH protein in the absence of G protein inhibited it, suggesting a possible interaction between SH and G [51]. Viruses where the SH protein gene was deleted grew better in HEp-2 cells [55], leading to the suggestion of a negative regulatory effect of SH protein on virus-induced membrane fusion, although direct interaction between SH protein and fusionresponsible F protein has not been observed [58], whereas complexes F-G and G-SH have been detected on the surface of infected cells using immunoprecipitation [58] and heparin agarose affinity chromatography [57]. These three proteins not only form hetero-oligomers, but also homo-oligomers: F forms trimers [59], G forms tetramers [60], and SH forms pen‐ tamers [48, 61, 62]. Thus, a complicated regulatory network of interactions may exist which probably includes both homo- and hetero-oligomeric forms.

In addition to interactions with viral proteins, the fact that SH proteins of hRSV and par‐ ainfluenza virus 5 (PIV5) are necessary for the inhibition of tumor necrosis factor alpha (TNF-α)-induced apoptosis [56, 63] also suggests a possible interaction with host pro‐ teins, although this has not been confirmed experimentally. However, in another study, deletion of SH protein gene from RSV did not result in increased apoptosis in infected H441 cells [11].

assemble into virus filaments at the plasma membrane [44], although the mechanisms that

Generation of nascent hRSV genomic RNA appears to occur in discrete cytoplasmic inclu‐ sion bodies that contain the hRSV N, P, L, M2-1 and M2-2 proteins but not the F, G, or SH proteins [45]. It is suspected that the RNP complexes form in the inclusions and then traffic to the apical membrane, where they meet with the surface glycoproteins F, G, and SH arriv‐ ing from the Golgi apparatus through the secretory pathway [46]. hRSV proteins and viral RNA assemble into virus filaments at the cell surface. These filaments are thought to con‐ tribute to cell-cell spread of the virus and morphologically resemble the filamentous form of virions seen in electron microscopy (EM) studies of virus produced in polarized cells [47]. **The small hydrophobic (SH) protein.** The SH protein is 65 or 64 amino acids long, in sub‐ type A or B, respectively. SH protein has a single membrane-spanning hydrophobic region [48], and a C-terminal extramembrane tail, oriented extracellularly/lumenally [48]. The se‐ quence of SH protein is highly conserved, especially at the TM domain [49, 50]. hRSV that lacks SH protein, hRSVΔSH, is still viable, and still forms syncytia [51-53]. However, hRSVΔSH was attenuated in *in vivo* mouse and chimpanzee models [4, 5], which indicates that SH protein is important for hRSV pathogenesis. SH protein has been suggested to play an ancillary role in virus-mediated cell fusion [54, 55]. Also, the presence of SH protein has been shown to reduce cytopathic effect (CPE) and apoptosis in L929 and A549 (lung epithe‐ lial cell line) infected cells, at least in part by inhibiting tumor necrosis factor α (TNF-α) pro‐

**Interaction of SH protein with viral and host proteins.** Extensive protein-protein interac‐ tions have been observed between the three membrane proteins on the hRSV envelope, F, G, and SH [51, 57, 58] and these interactions have an effect on fusion activity of hRSV on the host [51, 54]. In cells transiently expressing hRSV membrane proteins, the presence of G and SH proteins enhanced fusion activity mediated by F protein [54, 55]. Thus, SH protein has been suggested to play an ancillary role in virus-mediated cell fusion. However, using virusinfected cells the presence of G protein alone enhanced F-mediated fusion activity [51], whereas SH protein in the absence of G protein inhibited it, suggesting a possible interaction between SH and G [51]. Viruses where the SH protein gene was deleted grew better in HEp-2 cells [55], leading to the suggestion of a negative regulatory effect of SH protein on virus-induced membrane fusion, although direct interaction between SH protein and fusionresponsible F protein has not been observed [58], whereas complexes F-G and G-SH have been detected on the surface of infected cells using immunoprecipitation [58] and heparin agarose affinity chromatography [57]. These three proteins not only form hetero-oligomers, but also homo-oligomers: F forms trimers [59], G forms tetramers [60], and SH forms pen‐ tamers [48, 61, 62]. Thus, a complicated regulatory network of interactions may exist which

In addition to interactions with viral proteins, the fact that SH proteins of hRSV and par‐ ainfluenza virus 5 (PIV5) are necessary for the inhibition of tumor necrosis factor alpha (TNF-α)-induced apoptosis [56, 63] also suggests a possible interaction with host pro‐ teins, although this has not been confirmed experimentally. However, in another study,

drive assembly into filaments and budding are not well understood.

50 Respiratory Disease and Infection - A New Insight

duction [56], similarly to parainfluenza virus 5 (PIV5).

probably includes both homo- and hetero-oligomeric forms.

**Localization and post-translational modifications of SH protein during viral infection.** In infected cells, some SH protein is found in plasma membrane and cytoplasm, but most of the SH protein accumulates at the membranes of the Golgi complex and only very low amounts are found in the viral envelope [64]. Several forms of the SH protein, glycosylated and non-glycosylated, are present during infection [65], but the non-glycosylated form ap‐ pears to be the most abundant [66]. SH protein is also modified by tyrosine phosphorylation [61], and increased accumulation of SH in the Golgi complex was observed in the presence of a kinase inhibitor. Thus, SH protein is modified by a MAPK p38-dependent tyrosine kin‐ ase activity and this modification influences its cellular distribution. Although SH contains one cysteine residue, no palmitoylation has been detected in SH protein in conditions where F and G were palmitoylated [48].

**Structural determination of SH protein.** An important step towards the understanding of viroporin function at the molecular level is the availability of these proteins in a highly pure form. This has been so far difficult due to their high hydrophobicity, toxicity to expression hosts, and tendency to aggregate. However, recently we have been able to obtain the full length SH protein [67] that allows structural and biophysical studies (Fig. 1A).

SH protein after cross-linking has been shown to form multiple oligomers of increasing size in SDS [66, 68]. Later, we showed that the TM domain of SH protein forms only ho‐ mopentamers in perfluoro-octanoic acid (PFO) gels [69]. Reports using purified fulllength SH protein have confirmed the pentameric nature of the oligomer formed by this protein. For example, a bundle formation of a tagged SH protein construct was visual‐ ized under electron microscopy and was interpreted as a pentameric or a hexameric structure [70]. Using a purified tag-free SH protein, we have unequivocally demonstrated the homo-pentameric nature of these oligomers in a variety of detergents using analyti‐ cal ultracentrifugation and electrophoresis (Fig. 1) [71]. Indeed, in the presence of PFO (Fig. 1B) and a variety of other detergents under Blue-native gel electrophoresis (Fig. 1C), the full-length SH protein migrates as a single band with a molecular weight ~40 kDa, consistent with a pentameric oligomer. The pentameric form of SH has been further confirmed by analytical ultracentrifugation sedimentation equilibrium in detergents DPC, C14SB and C8E5 micelles. In these detergents, the species distribution profiles show a best fit to a monomer-pentamer self-association model (Fig. 1 D-E).

**Secondary structure of SH protein.** The SH protein is predicted to have an α-helical re‐ gion spanning residues 16-46, which includes its predicted TM region (residues ~20-40, Fig. 1A). Fourier Transform infrared (FTIR) data for the full-length SH protein reconsti‐ tuted in model lipid bilayers shows the presence of ~60% α-helical structure, whereas the rest is β-structure [71]. The availability of purified, isotopically labeled protein allowed us to obtain a model for this pentameric oligomer where SH protein was reconstituted in DPC micelles using NMR (Fig. 2). The model shows the lumen of the hypothetic chan‐ nel, sufficient for the passage of ions.

**Figure 2. NMR-based pentameric model of RSV SH protein in detergent DPC micelles** [71]. (A) Side view; (B) Nterminal (cytoplasmic) view; (C) C-terminal (extracellular or lumenal) view; (D) Electrostatic surface of the assembly showing the mostly hydrophobic central lumen. The SH protein assembly spans the entire bilayer with an overall

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**Ion channel activity of SH protein.** The presence of SH protein at the plasma membrane of HEK293 transfected cells allowed the study of SH protein channel activity [71], which was reported to be pH sensitive. Mutants where both histidines, H22 and H51 (Fig. 1A), were changed to phenylalanine (FF mutant, Fig. 1C), were found to be channel inactive. In a Bluenative gel electrophoresis, this FF mutant showed similar electrophoretic mobility (Fig. 1C) and similar plasma membrane localization to the wild type [71], which suggests that the ob‐ served channel activity is not mediated by direct or indirect interaction of SH protein with

One of the two histidines, His22, was suggested to face the lumen of the pentameric oligom‐ er using site-specific infrared dichroism of the isotopically labeled TM domain reconstituted in model lipid bilayers [69]. In the NMR based model of the full length protein (Fig. 2), al‐ though His22 adopts a lumenal orientation, the second histidine, His51, appears in an extra‐ membrane location, at the tip of the C-terminal extended loop (Fig. 2A), which is difficult to reconcile with an activation role based on His protonation. Thus, it is possible that the struc‐ ture of this C-terminal domain in detergent micelles, used for the NMR experiment, does not represent accurately the structure of SH protein in lipid bilayers, where we obtained the patch clamp data. Nevertheless, the pH-activated channel activity observed, and the histi‐ dine-less inactive mutant strongly suggests that protonation of histidines may be involved in channel activity. Indeed, the presence of a lumenal histidine sidechain is reminiscent of the one found in the TM domain of the influenza A AM2 proton channel, which is also acti‐

Despite the similarities between AM2 and SH protein, we have been unable to observe strong proton channel activity of SH protein *in vitro* (unpublished observations). In addi‐ tion, the different life cycle of hRSV and influenza virus A does not provide a rationale for this hypothetic proton channel activity. Equally, no obvious rationale can be assigned to pH mediated activation. The use of specific channel inhibitors for SH protein could contribute to clarify the precise role of channel activity in this protein, disentangled from

length of about 45 Å.

host-endogenous channels.

other effects.

vated at low pH via histidine protonation [33].

**Figure 1. Sequence and oligomerization of SH protein from hRSV subtype A.** (A) Amino acid sequence of full-length SH protein, with three additional N-terminal residues, SNA, as a result of tobacco etch virus (TEV) protease cleavage to re‐ move the expression tag [71]; (B) Gel electrophoresis analysis of SH protein in PFO shows a band consistent with pentamers (arrow); (C) Blue-native gel electrophoresis of wild type SH protein (WT) and double mutant H22F/H51F (FF) presolubi‐ lized in a variety of detergents show one band migrating consistent with pentamers (arrows); (D) Analytical ultracentrifu‐ gation sedimentation equilibrium data for 50 μM SH (WT) protein collected at three different speeds: 16,000 (blue), 19,500 (green), and 24,000 (red) rpm. Sedimentation profile was globally best-fitted to a monomer-pentamer equilibrium model. The graph shows both data points (black filled circles) and fitted function (in color). Lower panels represent fit resid‐ uals; (E) SH (WT) oligomeric species distribution in C14SB, C8E5 and DPC detergents, where the thick bar on the curve rep‐ resents the range of protein:detergent molar ratios used in the AUC experiment. The dotted line indicates the protein:DPC molar ratio (1:200) used in the NMR experiments [71] and its corresponding pentamer fraction (~90%). C14SB, DPC and C8E5 detergent concentrations were 5, 15 and 33 mM, respectively.

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and Coronaviruses http://dx.doi.org/10.5772/53957 53

**Figure 2. NMR-based pentameric model of RSV SH protein in detergent DPC micelles** [71]. (A) Side view; (B) Nterminal (cytoplasmic) view; (C) C-terminal (extracellular or lumenal) view; (D) Electrostatic surface of the assembly showing the mostly hydrophobic central lumen. The SH protein assembly spans the entire bilayer with an overall length of about 45 Å.

**Ion channel activity of SH protein.** The presence of SH protein at the plasma membrane of HEK293 transfected cells allowed the study of SH protein channel activity [71], which was reported to be pH sensitive. Mutants where both histidines, H22 and H51 (Fig. 1A), were changed to phenylalanine (FF mutant, Fig. 1C), were found to be channel inactive. In a Bluenative gel electrophoresis, this FF mutant showed similar electrophoretic mobility (Fig. 1C) and similar plasma membrane localization to the wild type [71], which suggests that the ob‐ served channel activity is not mediated by direct or indirect interaction of SH protein with host-endogenous channels.

One of the two histidines, His22, was suggested to face the lumen of the pentameric oligom‐ er using site-specific infrared dichroism of the isotopically labeled TM domain reconstituted in model lipid bilayers [69]. In the NMR based model of the full length protein (Fig. 2), al‐ though His22 adopts a lumenal orientation, the second histidine, His51, appears in an extra‐ membrane location, at the tip of the C-terminal extended loop (Fig. 2A), which is difficult to reconcile with an activation role based on His protonation. Thus, it is possible that the struc‐ ture of this C-terminal domain in detergent micelles, used for the NMR experiment, does not represent accurately the structure of SH protein in lipid bilayers, where we obtained the patch clamp data. Nevertheless, the pH-activated channel activity observed, and the histi‐ dine-less inactive mutant strongly suggests that protonation of histidines may be involved in channel activity. Indeed, the presence of a lumenal histidine sidechain is reminiscent of the one found in the TM domain of the influenza A AM2 proton channel, which is also acti‐ vated at low pH via histidine protonation [33].

**Figure 1. Sequence and oligomerization of SH protein from hRSV subtype A.** (A) Amino acid sequence of full-length SH protein, with three additional N-terminal residues, SNA, as a result of tobacco etch virus (TEV) protease cleavage to re‐ move the expression tag [71]; (B) Gel electrophoresis analysis of SH protein in PFO shows a band consistent with pentamers (arrow); (C) Blue-native gel electrophoresis of wild type SH protein (WT) and double mutant H22F/H51F (FF) presolubi‐ lized in a variety of detergents show one band migrating consistent with pentamers (arrows); (D) Analytical ultracentrifu‐ gation sedimentation equilibrium data for 50 μM SH (WT) protein collected at three different speeds: 16,000 (blue), 19,500 (green), and 24,000 (red) rpm. Sedimentation profile was globally best-fitted to a monomer-pentamer equilibrium model. The graph shows both data points (black filled circles) and fitted function (in color). Lower panels represent fit resid‐ uals; (E) SH (WT) oligomeric species distribution in C14SB, C8E5 and DPC detergents, where the thick bar on the curve rep‐ resents the range of protein:detergent molar ratios used in the AUC experiment. The dotted line indicates the protein:DPC molar ratio (1:200) used in the NMR experiments [71] and its corresponding pentamer fraction (~90%). C14SB, DPC and

C8E5 detergent concentrations were 5, 15 and 33 mM, respectively.

52 Respiratory Disease and Infection - A New Insight

Despite the similarities between AM2 and SH protein, we have been unable to observe strong proton channel activity of SH protein *in vitro* (unpublished observations). In addi‐ tion, the different life cycle of hRSV and influenza virus A does not provide a rationale for this hypothetic proton channel activity. Equally, no obvious rationale can be assigned to pH mediated activation. The use of specific channel inhibitors for SH protein could contribute to clarify the precise role of channel activity in this protein, disentangled from other effects.

#### **2.2. E protein in coronaviruses**

**Medical impact of coronaviruses.** Coronaviruses (family *Coronaviridae*, genus *Coronavirus* [72]) are enveloped viruses that cause common cold in humans and a variety of lethal dis‐ eases in birds and mammals [73]. The species in the genus *Coronavirus* have been organized into 3 groups with genetic and antigenic criteria [74]: α-coronaviruses include the porcine *Transmissible gastroenteritis virus* (TGEV) and *Human coronaviruses 229E* (HCoV-229E) or NL63 (HCoV-NL63). β-coronaviruses include *Murine hepatitis virus* (MHV) and *Human coro‐ navirus OC43* (HCoV-OC43). γ-coronaviruses include the avian *Infectious bronchitis virus* (IBV) and the Turkey coronavirus (TCoV). The virus responsible for the severe acute respira‐ tory syndrome (SARS-CoV), a respiratory disease in humans, is close to the β-coronaviruses, formerly group 2 [75].

teins from several species using a hidden Markov model (e.g., http://phobius.sbc.su.se/) [91] shows that they have at least one α-helical TM domain. In some cases a second TM domain is also predicted, e.g., in IBV E and MHV E (Fig 3). However, in none of these coronavirus E proteins this second putative TM has a predicted α-helical conformation. Instead, a β-coil-β motif appears to predominate in that part of the sequence, with a totally conserved Pro resi‐

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**Figure 3. Secondary structure and TM prediction of E proteins of coronaviruses.** E proteins from four representa‐ tives of coronavirus are presented: TGEV, MHV, SARS-CoV and IBV E proteins. Regions predicted to be α-helical, βsheet, or random coil are marked in blue, red, and yellow, respectively. Red bars show the probability of a region being

**Topology of E proteins.** The topology of coronavirus envelope proteins is an issue still un‐ der debate. Experimental determination of E protein orientation in infected cells [88, 92, 93] has shown that in TGEV E, the N-terminus is exposed to the cytoplasm, with the C-terminus facing the Golgi lumen (NcytoCexo). In MHV E, both N and C-terminal ends were found to face the cytoplasm (NcytoCcyto). For SARS-CoV E, an NcytoCcyto topology, similar to MHV E, was reported in transfected cells [94], consistent with two TM domains (NcytoCcyto), although a small fraction of the population (~10%) was found to be glycosylated at residue N66. As glycosylation must have occurred in the Golgi lumen, the authors suggested the existence of

a TM domain. The location of the conserved Pro residue in each protein is indicated by a 'P'.

due in a central position ('P' in Fig. 3).

SARS produced a near pandemic in 2003, with 8,096 infected cases and 774 deaths world‐ wide (fatality rate of 9.6%). Mortality was 6% for those aged 25-44, 15 % for the 45-64 group and >50% for those over 65 (*http://www.who.int/csr/sarsarchive/2003\_05\_07a/en/*). For compari‐ son, the case fatality rate for influenza A is usually around 0.6% (primarily among the elder‐ ly) and 33% in locally severe epidemics of new strains. SARS-CoV was enzootic in an unknown animal or bird species, probably a bat [76], before suddenly emerging as a virulent virus in humans. A similar crossing of the animal-human species barrier is thought to have occurred between the bovine coronavirus (BCoV) and human coronavirus OC43 (HCoV-OC43) more than 100 years ago [77]. Such interspecies jumps, from animal hosts to humans, are likely to reoccur.

Protective efficacy of candidate vaccines against coronaviruses in humans has been mainly studied in animals so far, and only few vaccines have entered Phase 1 human trials [78]. Rib‐ avirin [79], interferons [80], unconventional agents [81-83] and non-steroidal anti-inflamma‐ tory agents [84] have shown activity against SARS-CoV and HCoV-229E, but there is no data from animal studies or clinical trials [85]. Studies of antiviral therapy against coronavi‐ ruses other than SARS-CoV have been scarce; *in vitro* data show that several chemicals may have inhibitory activities on HCoV-NL63 and HCoV-229E [86, 87].

In addition to the genes involved in viral RNA replication and transcription, other essential genes in coronaviruses encode the common viral structural proteins, S (spike), E (envelope), M (membrane) and N (nucleocapsid). Of these, S, E, and M are incorporated into the virion lipidic envelope, and S protein is involved in fusion with host membranes during entry into cells. The M protein is the most abundant constituent of coronaviruses and gives the virion envelopes their shape; the E protein is only a minor constituent of the virion but is abun‐ dantly expressed inside the infected cell [88-90].

The E protein in SARS-CoV is the shortest, with only 76 amino acids, whereas that of IBV E is one of the longest (109 amino acids). E protein sequences are extremely divergent in their sequence, but the same general architecture is found in all of them: a short hydrophilic Nterminus (8–12 residues), an N-terminal TM domain (21–29 residues) followed by a cluster of 2-3 cysteines which are likely to be palmitoylated, and finally a less hydrophobic C-termi‐ nal tail (39–76 residues). Prediction of TM domains of representatives of coronavirus E pro‐

#### Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and Coronaviruses http://dx.doi.org/10.5772/53957 55

teins from several species using a hidden Markov model (e.g., http://phobius.sbc.su.se/) [91] shows that they have at least one α-helical TM domain. In some cases a second TM domain is also predicted, e.g., in IBV E and MHV E (Fig 3). However, in none of these coronavirus E proteins this second putative TM has a predicted α-helical conformation. Instead, a β-coil-β motif appears to predominate in that part of the sequence, with a totally conserved Pro resi‐ due in a central position ('P' in Fig. 3).

**2.2. E protein in coronaviruses**

54 Respiratory Disease and Infection - A New Insight

formerly group 2 [75].

are likely to reoccur.

**Medical impact of coronaviruses.** Coronaviruses (family *Coronaviridae*, genus *Coronavirus* [72]) are enveloped viruses that cause common cold in humans and a variety of lethal dis‐ eases in birds and mammals [73]. The species in the genus *Coronavirus* have been organized into 3 groups with genetic and antigenic criteria [74]: α-coronaviruses include the porcine *Transmissible gastroenteritis virus* (TGEV) and *Human coronaviruses 229E* (HCoV-229E) or NL63 (HCoV-NL63). β-coronaviruses include *Murine hepatitis virus* (MHV) and *Human coro‐ navirus OC43* (HCoV-OC43). γ-coronaviruses include the avian *Infectious bronchitis virus* (IBV) and the Turkey coronavirus (TCoV). The virus responsible for the severe acute respira‐ tory syndrome (SARS-CoV), a respiratory disease in humans, is close to the β-coronaviruses,

SARS produced a near pandemic in 2003, with 8,096 infected cases and 774 deaths world‐ wide (fatality rate of 9.6%). Mortality was 6% for those aged 25-44, 15 % for the 45-64 group and >50% for those over 65 (*http://www.who.int/csr/sarsarchive/2003\_05\_07a/en/*). For compari‐ son, the case fatality rate for influenza A is usually around 0.6% (primarily among the elder‐ ly) and 33% in locally severe epidemics of new strains. SARS-CoV was enzootic in an unknown animal or bird species, probably a bat [76], before suddenly emerging as a virulent virus in humans. A similar crossing of the animal-human species barrier is thought to have occurred between the bovine coronavirus (BCoV) and human coronavirus OC43 (HCoV-OC43) more than 100 years ago [77]. Such interspecies jumps, from animal hosts to humans,

Protective efficacy of candidate vaccines against coronaviruses in humans has been mainly studied in animals so far, and only few vaccines have entered Phase 1 human trials [78]. Rib‐ avirin [79], interferons [80], unconventional agents [81-83] and non-steroidal anti-inflamma‐ tory agents [84] have shown activity against SARS-CoV and HCoV-229E, but there is no data from animal studies or clinical trials [85]. Studies of antiviral therapy against coronavi‐ ruses other than SARS-CoV have been scarce; *in vitro* data show that several chemicals may

In addition to the genes involved in viral RNA replication and transcription, other essential genes in coronaviruses encode the common viral structural proteins, S (spike), E (envelope), M (membrane) and N (nucleocapsid). Of these, S, E, and M are incorporated into the virion lipidic envelope, and S protein is involved in fusion with host membranes during entry into cells. The M protein is the most abundant constituent of coronaviruses and gives the virion envelopes their shape; the E protein is only a minor constituent of the virion but is abun‐

The E protein in SARS-CoV is the shortest, with only 76 amino acids, whereas that of IBV E is one of the longest (109 amino acids). E protein sequences are extremely divergent in their sequence, but the same general architecture is found in all of them: a short hydrophilic Nterminus (8–12 residues), an N-terminal TM domain (21–29 residues) followed by a cluster of 2-3 cysteines which are likely to be palmitoylated, and finally a less hydrophobic C-termi‐ nal tail (39–76 residues). Prediction of TM domains of representatives of coronavirus E pro‐

have inhibitory activities on HCoV-NL63 and HCoV-229E [86, 87].

dantly expressed inside the infected cell [88-90].

**Figure 3. Secondary structure and TM prediction of E proteins of coronaviruses.** E proteins from four representa‐ tives of coronavirus are presented: TGEV, MHV, SARS-CoV and IBV E proteins. Regions predicted to be α-helical, βsheet, or random coil are marked in blue, red, and yellow, respectively. Red bars show the probability of a region being a TM domain. The location of the conserved Pro residue in each protein is indicated by a 'P'.

**Topology of E proteins.** The topology of coronavirus envelope proteins is an issue still un‐ der debate. Experimental determination of E protein orientation in infected cells [88, 92, 93] has shown that in TGEV E, the N-terminus is exposed to the cytoplasm, with the C-terminus facing the Golgi lumen (NcytoCexo). In MHV E, both N and C-terminal ends were found to face the cytoplasm (NcytoCcyto). For SARS-CoV E, an NcytoCcyto topology, similar to MHV E, was reported in transfected cells [94], consistent with two TM domains (NcytoCcyto), although a small fraction of the population (~10%) was found to be glycosylated at residue N66. As glycosylation must have occurred in the Golgi lumen, the authors suggested the existence of a minor fraction of E protein in an NcytoCexo topology. However, a study in infected cells de‐ tected the C terminus oriented cytoplasmically and the N-terminus lumenally [95], consis‐ tent with a single TM domain. Lastly, in IBV E, the C-terminus was found exposed to the cytoplasm, but not the N-terminus, suggesting a topology NexoCcyto, with the N-terminus fac‐ ing the lumen of the Golgi [96].

virus [108]. This suggests a role of the SARS-CoV E gene as a virulence factor influencing tissue tropism and pathogenicity. Recently, SARS-CoV lacking E gene has been suggested as vaccine candidate [109]. Studies using the E deleted SARS-CoV E have shown that E protein affects stress and inflammation responses [110], which probably contribute to the attenua‐

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and

In other coronaviruses, it has been found that E protein is involved in viral morphogene‐ sis, e.g., co-expression of M and E is sufficient for formation and release of virus-like particles (VLP) in the host cell [93, 111-115]. Also, mutations in the extramembrane do‐ main of E protein were shown to impair viral assembly and maturation in MHV [116], probably due to a defective interaction with M protein. In TGEV, the absence of E pro‐ tein resulted in a blockade of virus trafficking in the secretory pathway, and the preven‐

**Interaction partners of E proteins.** The interaction of E protein with M in IBV has already been reported by two different labs [96, 119] and involves at least the C-terminal tail of these two proteins, which therefore should be on the same side of the lipid bilayer. Additionally, the extramembrane cytoplasmic tail of SARS-CoV E has also been reported to bind Bcl-XL [120] and the N-terminal domain of non-structural protein 3 (nsp3) [121]. Similar studies have shown that SARS-CoV E via its four last C-terminal amino-acids, interacts with the host protein, PALS1, a tight junction-associated protein. Intercellular tight junctions are a physical barrier that protects underlying tissues from pathogen invasions. In SARS-CoV–in‐ fected Vero E6 cells, PALS1 redistributes to the ERGIC/Golgi region, where E accumulates. Hijacking PALS1 by SARS-CoV E may play a determinant role in the disruption of the lung epithelium in SARS patients [122]. SARS-CoV E has also been found to interact with Na+

**Channel activity in coronavirus E proteins.** Enhanced permeability has been observed in bacterial and mammalian cells expressing MHV E [123] or SARS-CoV E [26]. In addition, E proteins of SARS, human coronavirus 229E, MHV, and IBV, have shown *in vitro* ion channel activity in planar lipid bilayers [124, 125], which in some cases was inhibited by the drug hexamethylene amiloride (HMA) [125]. In a patch clamp study, channel activity was ob‐ served in cells transfected with SARS-CoV E [25], although another study could not detect SARS-CoV E protein in the plasma membrane of transfected or infected cells [95]. Neverthe‐ less, channel activity has been shown in black lipid membranes for purified synthetic TM domains and full length SARS-CoV [67, 126, 127], and inactivating mutations located in the TM domain, N15A and V25F [126], have been confirmed recently in a separate study [128]. In the latter, a significant contribution of lipid composition was observed, and a protein-lip‐ id complex forming pore was proposed. These mutants may help elucidate the contribution

**Structural determination of E protein.** At present, detailed structures of coronavirus enve‐ lope proteins are lacking. This is due difficulties in both expression and purification, and to their high tendency to aggregate which makes crystallization and NMR studies extremely challenging. Recently, we have successfully utilized a modified β-barrel fusion protein con‐ struct to express and subsequently purify full-length SARS-CoV E and IBV E proteins [67].

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tion of the virus observed in the absence of this protein [107].

tion of virus maturation [117, 118].

ATPase α-1 subunit and stomatin [95].

of channel activity to SARS-CoV E protein function.

The results of these experiments should be interpreted with caution, especially compar‐ ing data from transfected cells and infected cells. Equally, the possible lack of accessibili‐ ty to antibodies of parts of the protein plays a part. Indeed, as discussed elsewhere [97], in the case of IBV E, if the entire N-terminal region of IBV E protein was buried within the intracellular membrane, it would have remained inaccessible to the antibodies used. Several coexisting forms may exist for E proteins, which would have different roles in the life cycle of the virus.

The factors that would favour one topology over another are unknown, but one possible candidate is palmitoylation. Indeed, E proteins of SARS [26], IBV [98] and MHV [99] are pal‐ mitoylated at one or more cysteines. This modification is likely to have structural and func‐ tional consequences, because removal of the cysteines in MHV E resulted in deformed viruses [100, 101]. Experimental determination of the topology of these E protein homologs – with or without palmitoylation – in model membranes or membrane-like detergents is crit‐ ical to understand the function of the envelope protein in coronavirus biology. Unfortunate‐ ly, these detailed structural studies are still not available.

The importance of the correct topology in E proteins may be highlighted by a recent study [102] that showed that E protein in MHV could be replaced by some heterologous E pro‐ teins. The MHV virus became viable when the replacement was from groups 2, i.e., β-coro‐ naviruses (SARS-CoV E) and 3, i.e., γ-coronaviruses (IBV E), but not when TGEV E (group 1, or α-coronaviruses) was used. This discrimination may have to do with topology considera‐ tions, because the contribution of E proteins to the formation of viral particles in coronavi‐ ruses could be provided by a broad range of sequences, and not by specific interactions.

**Localization of E protein during viral infection.** E protein can be found between the ER and Golgi compartments inside the cell [103-105]. However, only a small amount ends up in the virion [88-90], suggesting that its main role is inside the cell [95]. In transfected HeLa cells, SARS-CoV E protein is targeted to the Golgi complex, and this localization has been attributed, at least in part, to the β-hairpin motif in its C-terminus [106] (see Fig. 3). In infect‐ ed Vero E6 cells, SARS-CoV E protein accumulates in the ER-Golgi intermediate compart‐ ment (ERGIC) [95]; the latter study could not detect any SARS-CoV E protein in the plasma membrane.

**Effect of coronavirus E gene deletion.** While the absence of S and M protein are clearly del‐ eterious to the virus because of their abundance and key role in envelope formation, the E protein is not essential for *in vitro* or *in vivo* coronavirus replication. However, the absence of E protein results in an attenuated virus, as shown for SARS-CoV [107] and other coronavi‐ ruses (see below). Recently, it has been shown that SARS viruses lacking gene E, in addition to being attenuated, did not grow in the central nervous system, in contrast to the wild type virus [108]. This suggests a role of the SARS-CoV E gene as a virulence factor influencing tissue tropism and pathogenicity. Recently, SARS-CoV lacking E gene has been suggested as vaccine candidate [109]. Studies using the E deleted SARS-CoV E have shown that E protein affects stress and inflammation responses [110], which probably contribute to the attenua‐ tion of the virus observed in the absence of this protein [107].

a minor fraction of E protein in an NcytoCexo topology. However, a study in infected cells de‐ tected the C terminus oriented cytoplasmically and the N-terminus lumenally [95], consis‐ tent with a single TM domain. Lastly, in IBV E, the C-terminus was found exposed to the cytoplasm, but not the N-terminus, suggesting a topology NexoCcyto, with the N-terminus fac‐

The results of these experiments should be interpreted with caution, especially compar‐ ing data from transfected cells and infected cells. Equally, the possible lack of accessibili‐ ty to antibodies of parts of the protein plays a part. Indeed, as discussed elsewhere [97], in the case of IBV E, if the entire N-terminal region of IBV E protein was buried within the intracellular membrane, it would have remained inaccessible to the antibodies used. Several coexisting forms may exist for E proteins, which would have different roles in

The factors that would favour one topology over another are unknown, but one possible candidate is palmitoylation. Indeed, E proteins of SARS [26], IBV [98] and MHV [99] are pal‐ mitoylated at one or more cysteines. This modification is likely to have structural and func‐ tional consequences, because removal of the cysteines in MHV E resulted in deformed viruses [100, 101]. Experimental determination of the topology of these E protein homologs – with or without palmitoylation – in model membranes or membrane-like detergents is crit‐ ical to understand the function of the envelope protein in coronavirus biology. Unfortunate‐

The importance of the correct topology in E proteins may be highlighted by a recent study [102] that showed that E protein in MHV could be replaced by some heterologous E pro‐ teins. The MHV virus became viable when the replacement was from groups 2, i.e., β-coro‐ naviruses (SARS-CoV E) and 3, i.e., γ-coronaviruses (IBV E), but not when TGEV E (group 1, or α-coronaviruses) was used. This discrimination may have to do with topology considera‐ tions, because the contribution of E proteins to the formation of viral particles in coronavi‐ ruses could be provided by a broad range of sequences, and not by specific interactions.

**Localization of E protein during viral infection.** E protein can be found between the ER and Golgi compartments inside the cell [103-105]. However, only a small amount ends up in the virion [88-90], suggesting that its main role is inside the cell [95]. In transfected HeLa cells, SARS-CoV E protein is targeted to the Golgi complex, and this localization has been attributed, at least in part, to the β-hairpin motif in its C-terminus [106] (see Fig. 3). In infect‐ ed Vero E6 cells, SARS-CoV E protein accumulates in the ER-Golgi intermediate compart‐ ment (ERGIC) [95]; the latter study could not detect any SARS-CoV E protein in the plasma

**Effect of coronavirus E gene deletion.** While the absence of S and M protein are clearly del‐ eterious to the virus because of their abundance and key role in envelope formation, the E protein is not essential for *in vitro* or *in vivo* coronavirus replication. However, the absence of E protein results in an attenuated virus, as shown for SARS-CoV [107] and other coronavi‐ ruses (see below). Recently, it has been shown that SARS viruses lacking gene E, in addition to being attenuated, did not grow in the central nervous system, in contrast to the wild type

ing the lumen of the Golgi [96].

56 Respiratory Disease and Infection - A New Insight

the life cycle of the virus.

membrane.

ly, these detailed structural studies are still not available.

In other coronaviruses, it has been found that E protein is involved in viral morphogene‐ sis, e.g., co-expression of M and E is sufficient for formation and release of virus-like particles (VLP) in the host cell [93, 111-115]. Also, mutations in the extramembrane do‐ main of E protein were shown to impair viral assembly and maturation in MHV [116], probably due to a defective interaction with M protein. In TGEV, the absence of E pro‐ tein resulted in a blockade of virus trafficking in the secretory pathway, and the preven‐ tion of virus maturation [117, 118].

**Interaction partners of E proteins.** The interaction of E protein with M in IBV has already been reported by two different labs [96, 119] and involves at least the C-terminal tail of these two proteins, which therefore should be on the same side of the lipid bilayer. Additionally, the extramembrane cytoplasmic tail of SARS-CoV E has also been reported to bind Bcl-XL [120] and the N-terminal domain of non-structural protein 3 (nsp3) [121]. Similar studies have shown that SARS-CoV E via its four last C-terminal amino-acids, interacts with the host protein, PALS1, a tight junction-associated protein. Intercellular tight junctions are a physical barrier that protects underlying tissues from pathogen invasions. In SARS-CoV–in‐ fected Vero E6 cells, PALS1 redistributes to the ERGIC/Golgi region, where E accumulates. Hijacking PALS1 by SARS-CoV E may play a determinant role in the disruption of the lung epithelium in SARS patients [122]. SARS-CoV E has also been found to interact with Na+ /K+ ATPase α-1 subunit and stomatin [95].

**Channel activity in coronavirus E proteins.** Enhanced permeability has been observed in bacterial and mammalian cells expressing MHV E [123] or SARS-CoV E [26]. In addition, E proteins of SARS, human coronavirus 229E, MHV, and IBV, have shown *in vitro* ion channel activity in planar lipid bilayers [124, 125], which in some cases was inhibited by the drug hexamethylene amiloride (HMA) [125]. In a patch clamp study, channel activity was ob‐ served in cells transfected with SARS-CoV E [25], although another study could not detect SARS-CoV E protein in the plasma membrane of transfected or infected cells [95]. Neverthe‐ less, channel activity has been shown in black lipid membranes for purified synthetic TM domains and full length SARS-CoV [67, 126, 127], and inactivating mutations located in the TM domain, N15A and V25F [126], have been confirmed recently in a separate study [128]. In the latter, a significant contribution of lipid composition was observed, and a protein-lip‐ id complex forming pore was proposed. These mutants may help elucidate the contribution of channel activity to SARS-CoV E protein function.

**Structural determination of E protein.** At present, detailed structures of coronavirus enve‐ lope proteins are lacking. This is due difficulties in both expression and purification, and to their high tendency to aggregate which makes crystallization and NMR studies extremely challenging. Recently, we have successfully utilized a modified β-barrel fusion protein con‐ struct to express and subsequently purify full-length SARS-CoV E and IBV E proteins [67]. We showed that both full length proteins form homopentamers, confirming previous results obtained only with the synthetic TM domain [25].

Previous reports have studied the oligomerization of coronavirus E proteins. However, results were not conclusive, partly because these experiments were performed in SDS, a harsh detergent that leads to monomers or to non-specific aggregates. For example, SARS-CoV E oligomerization has been studied in Western Blots after SDS-PAGE and la‐ beling with polyclonal antibodies [99], antibodies against a hemagglutinin-derived C-ter‐ minal tag [100], or using non purified or truncated synthetic E proteins [124, 125]. In the latter approach, a predominanly monomeric form was observed in SDS. In our hands, synthetic SARS-CoV E also produced in SDS mostly monomers, and a minor fraction of dimers (unpublished observations), but several oligomers were observed for the recombinant form (Fig. 4, lane WT). The differences between synthetic and recombi‐ nant E protein may be due to unwanted side reactions that take place during synthesis. Addition of DTT (Fig. 4B) produces bands compatible with monomers and trimers, whereas cysteine-less mutants only produced monomers. Thus, the three cysteines in SARS-CoV E seem to participate in some inter-monomeric contacts. Indeed, sedimenta‐ tion data for SARS-CoV E could only be fitted after addition of reductant [67] in the case of SARS-CoV E. Differences between absence and presence of reductant were ob‐ served even when only one cysteine was available (Fig. 4B), therefore these disulfide bonds may not be specific. Changes in hydrophobicity and local secondary structure seem to play a major role in the results observed. Further, disulfide bonds are not nec‐ essary to form pentamers; sedimentation equilibrium of full-length SARS-CoV E or IBV E in C14SB detergent and in the presence of reducing agent produced best fit to a mon‐ omer-pentamer equilibrium model [67], similar to what has been observed for the TM region alone [127].

The likely orientation of these cysteine residues relative the pentameric bundle can be deter‐ mined on the basis of the available structure formed by synthetic TM8-38 [25]. That structure did not include any of the three cysteines of SARS-CoV E, but if the structural model is pro‐ longed by two turns (Fig. 5), the three cysteines are seen oriented either towards the lumen of the channel or inter-helically.

The juxtamembrane cysteines in coronavirus envelope proteins are well conserved, and have been found to be crucial in the coronavirus cycle. For example, in MHV E, removal of the cysteines resulted in deformed viruses [99-101]. Using the full length infectious clone [99], double- and triple-mutants to alanine produced smaller plaques and decreased virus yields. Single-substitution mutants, in contrast, did not produce anomalous growth, where‐ as replacement of all three cysteines resulted in crippled virus with significantly reduced yields. In these reports, these effects were attributed to the absence of palmitoylation sites, which may direct E proteins towards lipid rafts [129]. E proteins of SARS [26], IBV [98] and MHV [99] have been shown to be palmitoylated at one or more cysteines. It is possible that an additional role of palmitoylation is to drag the C-terminal tail of E proteins towards the membrane and trigger a conformational change.

**Figure 4. SDS-PAGE electrophoresis of SARS-CoV E and IBV E.** (A) SARS-CoV E wild type (WT) and cysteine mutants in the absence of DTT in gel 4-12 % Nu-PAGE in MES/SDS buffer; (B) same as A in presence of DTT. The lane contain‐ ing the molecular weight markers (MW) is indicated. The oligomeric size is indicated by black circles (●); (C) Effect of three reductants (DTT, TCEP and β-mercaptoethanol (β-ME) on the electrophoretic mobility SARS-CoV E WT in 15%

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SDS PAGE gel.

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and Coronaviruses http://dx.doi.org/10.5772/53957 59

We showed that both full length proteins form homopentamers, confirming previous results

Previous reports have studied the oligomerization of coronavirus E proteins. However, results were not conclusive, partly because these experiments were performed in SDS, a harsh detergent that leads to monomers or to non-specific aggregates. For example, SARS-CoV E oligomerization has been studied in Western Blots after SDS-PAGE and la‐ beling with polyclonal antibodies [99], antibodies against a hemagglutinin-derived C-ter‐ minal tag [100], or using non purified or truncated synthetic E proteins [124, 125]. In the latter approach, a predominanly monomeric form was observed in SDS. In our hands, synthetic SARS-CoV E also produced in SDS mostly monomers, and a minor fraction of dimers (unpublished observations), but several oligomers were observed for the recombinant form (Fig. 4, lane WT). The differences between synthetic and recombi‐ nant E protein may be due to unwanted side reactions that take place during synthesis. Addition of DTT (Fig. 4B) produces bands compatible with monomers and trimers, whereas cysteine-less mutants only produced monomers. Thus, the three cysteines in SARS-CoV E seem to participate in some inter-monomeric contacts. Indeed, sedimenta‐ tion data for SARS-CoV E could only be fitted after addition of reductant [67] in the case of SARS-CoV E. Differences between absence and presence of reductant were ob‐ served even when only one cysteine was available (Fig. 4B), therefore these disulfide bonds may not be specific. Changes in hydrophobicity and local secondary structure seem to play a major role in the results observed. Further, disulfide bonds are not nec‐ essary to form pentamers; sedimentation equilibrium of full-length SARS-CoV E or IBV E in C14SB detergent and in the presence of reducing agent produced best fit to a mon‐ omer-pentamer equilibrium model [67], similar to what has been observed for the TM

The likely orientation of these cysteine residues relative the pentameric bundle can be deter‐ mined on the basis of the available structure formed by synthetic TM8-38 [25]. That structure did not include any of the three cysteines of SARS-CoV E, but if the structural model is pro‐ longed by two turns (Fig. 5), the three cysteines are seen oriented either towards the lumen

The juxtamembrane cysteines in coronavirus envelope proteins are well conserved, and have been found to be crucial in the coronavirus cycle. For example, in MHV E, removal of the cysteines resulted in deformed viruses [99-101]. Using the full length infectious clone [99], double- and triple-mutants to alanine produced smaller plaques and decreased virus yields. Single-substitution mutants, in contrast, did not produce anomalous growth, where‐ as replacement of all three cysteines resulted in crippled virus with significantly reduced yields. In these reports, these effects were attributed to the absence of palmitoylation sites, which may direct E proteins towards lipid rafts [129]. E proteins of SARS [26], IBV [98] and MHV [99] have been shown to be palmitoylated at one or more cysteines. It is possible that an additional role of palmitoylation is to drag the C-terminal tail of E proteins towards the

obtained only with the synthetic TM domain [25].

58 Respiratory Disease and Infection - A New Insight

region alone [127].

of the channel or inter-helically.

membrane and trigger a conformational change.

**Figure 4. SDS-PAGE electrophoresis of SARS-CoV E and IBV E.** (A) SARS-CoV E wild type (WT) and cysteine mutants in the absence of DTT in gel 4-12 % Nu-PAGE in MES/SDS buffer; (B) same as A in presence of DTT. The lane contain‐ ing the molecular weight markers (MW) is indicated. The oligomeric size is indicated by black circles (●); (C) Effect of three reductants (DTT, TCEP and β-mercaptoethanol (β-ME) on the electrophoretic mobility SARS-CoV E WT in 15% SDS PAGE gel.

spectrum obtained for full length SARS-CoV E (Fig. 6e, dotted line) reconstituted in the

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**Figure 6.** Secondary structure prediction of coronavirus envelope proteins and correspondence with results obtained for synthetic peptides of SARS-CoV E. (a) Sequences corresponding to E proteins representative of coronavirus groups 1 (TGEV E), 2 (MHV E), 3 (IBV E) and SARS CoV E, and their predicted secondary structure (consensus)[136]. The posi‐ tion of the conserved proline is indicated with a black dot; (b-d) amide I and II bands of the synthetic SARS-CoV E pep‐ tides indicated when incorporated in DMPC bilayers in H2O (solid) or after D2O (dash) hydration. The position of amide II band is indicated by a star; (e) ATR-FTIR spectra corresponding to SARS-CoV E (solid line), and IBV E (broken line) reconstituted in DMPC bilayers, in the lipid ester region (1740 cm-1) and amide I region (C=O stretching, 1650 cm-1). The dotted line resulted from the addition of the spectra corresponding to the TM [130] and fragment 36-76 (panel b); (f) X-ray diffraction pattern for peptides (36-76) or (46-60) after drying from acetonitrile. The inter-sheet spacing is 10.8 Å (inner ring) whereas the hydrogen bond spacing is represented by the outer ring at 4.8 Å, characteristic of amy‐ loid fibrils. Due to poor alignment, the reflection at distance 4.8 Å appeared as a ring; (g) schematic model of full length SARS-CoV E build using prediction tools and experimental data obtained using infrared spectroscopy.

Similar experiments with fragments 46-60 and 59-76 (Fig. 6, c-d) showed that 46-60 forms βsheets resistant to H/D exchange. Indeed, this latter peptide showed limited solubility, simi‐ lar to the 'parent' peptide 36-76. Further, its amide I spectrum in DMPC displayed the

same conditions.

**Figure 5. Cysteine location in the pentameric arrangement of the TM domain of SARS-CoV E.** The scheme is ar‐ ranged according to previous published models [25, 130] after prolonging the helices 2 turns at the C-terminal end, and shows the position of the three cysteines in SARS-CoV E, C40, C43 and C44; A and B, views from the N-terminus of the pentamer, with C40 (A) and C43 and C44 (B); C, Side view, showing only two helices for clarity, and possible inter‐ helical disulfide bonds C43-C40 and C44-C40.

**Dissection of the domains of SARS-CoV E.** The four representatives of CoV E proteins have a predicted α-helical TM domain (Fig. 6a) and a C-terminal region predicted to have βstructure. Synthetic peptide 36-76 encompasses the C-terminal extramembrane domain and was analyzed in the presence of DMPC lipid membranes using FTIR. This peptide presented limited solubility both in water and in organic solvents. The amide I spectrum of this pep‐ tide (Fig. 6b) shows bands assigned to antiparallel β-sheet, because of the splitting of the amide I band caused by strong inter- and intra-strand transition dipole coupling (TDC) [131-133], resulting in a weak band at high frequency (1675–1690 cm-1) and a strong band at lower frequency (1625–1640 cm-1). The band at 1666 cm-1 can be assigned to disordered structure [134, 135]. The amide A frequency was blue-shifted from that observed for the TM domain alone [130] (3,305 cm-1) or full length SARS-CoV E (3294 cm-1, not shown) to 3281 cm-1, again consistent with the presence of β-structure. The intensity of the amide II band decreased upon exposure to D2O (dotted line, Fig. 6b) by about 30 ± 5%, i.e., ~28 residues are resistant to exchange in this peptide. The fast-exchanging fraction is likely due to the region predicted to have random coil conformation. Thus, the 36-76 fragment has an intrinsic ten‐ dency to fold as β-sheet, and presents both β-structure and random coil, in agreement with the secondary structure prediction (Fig. 6a). In fact, the spectrum resulting from the addition of TM α-helix (8-38) [130] and C-terminal tail (36-76), shown in Fig. 6b, is very similar to the spectrum obtained for full length SARS-CoV E (Fig. 6e, dotted line) reconstituted in the same conditions.

**Figure 5. Cysteine location in the pentameric arrangement of the TM domain of SARS-CoV E.** The scheme is ar‐ ranged according to previous published models [25, 130] after prolonging the helices 2 turns at the C-terminal end, and shows the position of the three cysteines in SARS-CoV E, C40, C43 and C44; A and B, views from the N-terminus of the pentamer, with C40 (A) and C43 and C44 (B); C, Side view, showing only two helices for clarity, and possible inter‐

**Dissection of the domains of SARS-CoV E.** The four representatives of CoV E proteins have a predicted α-helical TM domain (Fig. 6a) and a C-terminal region predicted to have βstructure. Synthetic peptide 36-76 encompasses the C-terminal extramembrane domain and was analyzed in the presence of DMPC lipid membranes using FTIR. This peptide presented limited solubility both in water and in organic solvents. The amide I spectrum of this pep‐ tide (Fig. 6b) shows bands assigned to antiparallel β-sheet, because of the splitting of the amide I band caused by strong inter- and intra-strand transition dipole coupling (TDC) [131-133], resulting in a weak band at high frequency (1675–1690 cm-1) and a strong band at lower frequency (1625–1640 cm-1). The band at 1666 cm-1 can be assigned to disordered structure [134, 135]. The amide A frequency was blue-shifted from that observed for the TM domain alone [130] (3,305 cm-1) or full length SARS-CoV E (3294 cm-1, not shown) to 3281 cm-1, again consistent with the presence of β-structure. The intensity of the amide II band decreased upon exposure to D2O (dotted line, Fig. 6b) by about 30 ± 5%, i.e., ~28 residues are resistant to exchange in this peptide. The fast-exchanging fraction is likely due to the region predicted to have random coil conformation. Thus, the 36-76 fragment has an intrinsic ten‐ dency to fold as β-sheet, and presents both β-structure and random coil, in agreement with the secondary structure prediction (Fig. 6a). In fact, the spectrum resulting from the addition of TM α-helix (8-38) [130] and C-terminal tail (36-76), shown in Fig. 6b, is very similar to the

helical disulfide bonds C43-C40 and C44-C40.

60 Respiratory Disease and Infection - A New Insight

**Figure 6.** Secondary structure prediction of coronavirus envelope proteins and correspondence with results obtained for synthetic peptides of SARS-CoV E. (a) Sequences corresponding to E proteins representative of coronavirus groups 1 (TGEV E), 2 (MHV E), 3 (IBV E) and SARS CoV E, and their predicted secondary structure (consensus)[136]. The posi‐ tion of the conserved proline is indicated with a black dot; (b-d) amide I and II bands of the synthetic SARS-CoV E pep‐ tides indicated when incorporated in DMPC bilayers in H2O (solid) or after D2O (dash) hydration. The position of amide II band is indicated by a star; (e) ATR-FTIR spectra corresponding to SARS-CoV E (solid line), and IBV E (broken line) reconstituted in DMPC bilayers, in the lipid ester region (1740 cm-1) and amide I region (C=O stretching, 1650 cm-1). The dotted line resulted from the addition of the spectra corresponding to the TM [130] and fragment 36-76 (panel b); (f) X-ray diffraction pattern for peptides (36-76) or (46-60) after drying from acetonitrile. The inter-sheet spacing is 10.8 Å (inner ring) whereas the hydrogen bond spacing is represented by the outer ring at 4.8 Å, characteristic of amy‐ loid fibrils. Due to poor alignment, the reflection at distance 4.8 Å appeared as a ring; (g) schematic model of full length SARS-CoV E build using prediction tools and experimental data obtained using infrared spectroscopy.

Similar experiments with fragments 46-60 and 59-76 (Fig. 6, c-d) showed that 46-60 forms βsheets resistant to H/D exchange. Indeed, this latter peptide showed limited solubility, simi‐ lar to the 'parent' peptide 36-76. Further, its amide I spectrum in DMPC displayed the features of antiparallel β-sheet, with bands at 1635 cm-1 and 1685 cm-1 (Fig. 6c) and showed no H/D exchange in the amide II region (Fig. 6c, star). In contrast, fragment 61-76 is predict‐ ed to form random coil (Fig. 6a), and should show complete H/D exchange. Indeed, the hy‐ drophilic fragment 59–76 dissolved readily in water (>5 mg/ml), produced an amide I spectrum in DMPC consistent with random structure (Fig. 6d), with a broad amide I band at 1645 cm-1, and showed complete H/D exchange at the amide II region (star).

attenuate the observed cytopathological effects of SARS-CoV E in cells. Indeed, a similar

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**Figure 7. Electron microscopy and image processing;** (A) Micrograph of a negatively stained sample of peptide 36-76 dissolved in acetonitrile. The ordered mesh is formed by 7-10 nm wide fibrils with 20-40 nm long straight sec‐ tions. Globular structures of 10-50 nm diameter are also present; (B) Control specimen without peptide showing simi‐ lar globular structures; (C, D) Magnified views of fibril branching points; (E) Two thin fibrils merging into a thicker one; (F) Class average of the 8 nm fibril; (G) Histogram of the lengths of the fibril straight sections. (H) Intensity profile along the x-axis of panel (F). The width of the peak above background level indicates filament thickness. The scale bar

is shown in each panel.

strategy led to disruption of Golgi targeting in SARS-CoV E [106].

The two folding domains observed in the C-terminal domain of SARS-CoV E are remi‐ niscent of the two separate domains reported for the amyloid peptide [137], where frag‐ ment 34–42 has limited solubility and adopts antiparallel β-sheet structure, and fragment 26–33 is more soluble in water, and has a disordered conformation. Thus, we tested if peptide (36-76) can form amyloid-like fibrils. The aggregate obtained after drying this peptide from acetonitrile showed intense X-ray reflections at ~4.8 Å and ~10.8 Å (Fig. 6f), which correspond to the distances between hydrogen bonded peptide backbones and β– pleated sheets, respectively, characteristically found in Alzheimer disease amyloid plaque cores [137]. This peptide was monomeric in SDS. Based on the above results, a topologi‐ cal model for SARS-CoV E can be proposed, with one α-helical TM domain and a C-ter‐ minal β-hairpin (Fig. 6g). We have reported previously that SARS-CoV E secondary structure in lipid bilayers is predominantly α-helical [67], in contrast with the results shown in Fig. 6e. However, we have found that the secondary structure of E protein is strongly dependent on the reconstitution conditions. In our previous report [67], the pro‐ tein was presolubilized in hexafluoroisopropanol, an α-helix inducer, whereas in Fig. 6e pre-solubilization was done in methanol. Thus, the β-hairpin prediction for the residues around the highly conserved residue P54, may be correct only in certain experimental conditions. The dual conformation, α-helical and β-hairpin, conformation proposed here is reminiscent of the proposed dual topology of a similar β-hairpin with central con‐ served Pro residue found in stomatin. In that case, secondary structure changed to α-he‐ lix when Pro was mutated to Ser [138].

Examination of the peptide (36-76) precipitate by electron microscopy (Fig. 7A) revealed a protofibrillar morphology [139-141]. These structures were not observed in the control speci‐ men prepared in the absence of peptide (Fig. 7B). The fibrils form an ordered mesh structure characterized by straight sections intervened by bends. The fibril width was 7, 8 and 10 nm, consistent with reports of other filaments derived from β-sheet structures [141-143]. The length of the straight sections was rather homogenous, with most measurements falling be‐ tween 20-40 nm and with an average value of 32 nm.

The formation of fibrils is anticipated by the residue composition in the region around the conserved proline (P54). For example, from the 17 residues in the stretch I46 to V62, 9 resi‐ dues are either V, I or Y. Amino acids with β-branched side chains, e.g. valine and isoleu‐ cine, or bulky residues, have been shown previously to disfavor α-helical conformation, and to pack efficiently along the surface of a β-sheet [144, 145]. Accordingly, a series of hexapep‐ tides containing similar motifs (e.g., VxVx) have been shown to be good amyloid-forming peptides [146]. We speculate that changing some of these residues to non-branched, for ex‐ ample from V to L, would abolish the ability of SARS-CoV E to form fibers, and possibly attenuate the observed cytopathological effects of SARS-CoV E in cells. Indeed, a similar strategy led to disruption of Golgi targeting in SARS-CoV E [106].

features of antiparallel β-sheet, with bands at 1635 cm-1 and 1685 cm-1 (Fig. 6c) and showed no H/D exchange in the amide II region (Fig. 6c, star). In contrast, fragment 61-76 is predict‐ ed to form random coil (Fig. 6a), and should show complete H/D exchange. Indeed, the hy‐ drophilic fragment 59–76 dissolved readily in water (>5 mg/ml), produced an amide I spectrum in DMPC consistent with random structure (Fig. 6d), with a broad amide I band at

The two folding domains observed in the C-terminal domain of SARS-CoV E are remi‐ niscent of the two separate domains reported for the amyloid peptide [137], where frag‐ ment 34–42 has limited solubility and adopts antiparallel β-sheet structure, and fragment 26–33 is more soluble in water, and has a disordered conformation. Thus, we tested if peptide (36-76) can form amyloid-like fibrils. The aggregate obtained after drying this peptide from acetonitrile showed intense X-ray reflections at ~4.8 Å and ~10.8 Å (Fig. 6f), which correspond to the distances between hydrogen bonded peptide backbones and β– pleated sheets, respectively, characteristically found in Alzheimer disease amyloid plaque cores [137]. This peptide was monomeric in SDS. Based on the above results, a topologi‐ cal model for SARS-CoV E can be proposed, with one α-helical TM domain and a C-ter‐ minal β-hairpin (Fig. 6g). We have reported previously that SARS-CoV E secondary structure in lipid bilayers is predominantly α-helical [67], in contrast with the results shown in Fig. 6e. However, we have found that the secondary structure of E protein is strongly dependent on the reconstitution conditions. In our previous report [67], the pro‐ tein was presolubilized in hexafluoroisopropanol, an α-helix inducer, whereas in Fig. 6e pre-solubilization was done in methanol. Thus, the β-hairpin prediction for the residues around the highly conserved residue P54, may be correct only in certain experimental conditions. The dual conformation, α-helical and β-hairpin, conformation proposed here is reminiscent of the proposed dual topology of a similar β-hairpin with central con‐ served Pro residue found in stomatin. In that case, secondary structure changed to α-he‐

Examination of the peptide (36-76) precipitate by electron microscopy (Fig. 7A) revealed a protofibrillar morphology [139-141]. These structures were not observed in the control speci‐ men prepared in the absence of peptide (Fig. 7B). The fibrils form an ordered mesh structure characterized by straight sections intervened by bends. The fibril width was 7, 8 and 10 nm, consistent with reports of other filaments derived from β-sheet structures [141-143]. The length of the straight sections was rather homogenous, with most measurements falling be‐

The formation of fibrils is anticipated by the residue composition in the region around the conserved proline (P54). For example, from the 17 residues in the stretch I46 to V62, 9 resi‐ dues are either V, I or Y. Amino acids with β-branched side chains, e.g. valine and isoleu‐ cine, or bulky residues, have been shown previously to disfavor α-helical conformation, and to pack efficiently along the surface of a β-sheet [144, 145]. Accordingly, a series of hexapep‐ tides containing similar motifs (e.g., VxVx) have been shown to be good amyloid-forming peptides [146]. We speculate that changing some of these residues to non-branched, for ex‐ ample from V to L, would abolish the ability of SARS-CoV E to form fibers, and possibly

1645 cm-1, and showed complete H/D exchange at the amide II region (star).

lix when Pro was mutated to Ser [138].

62 Respiratory Disease and Infection - A New Insight

tween 20-40 nm and with an average value of 32 nm.

**Figure 7. Electron microscopy and image processing;** (A) Micrograph of a negatively stained sample of peptide 36-76 dissolved in acetonitrile. The ordered mesh is formed by 7-10 nm wide fibrils with 20-40 nm long straight sec‐ tions. Globular structures of 10-50 nm diameter are also present; (B) Control specimen without peptide showing simi‐ lar globular structures; (C, D) Magnified views of fibril branching points; (E) Two thin fibrils merging into a thicker one; (F) Class average of the 8 nm fibril; (G) Histogram of the lengths of the fibril straight sections. (H) Intensity profile along the x-axis of panel (F). The width of the peak above background level indicates filament thickness. The scale bar is shown in each panel.

In addition, a sequence of ordered fragments (α-helices or strands) flanking a disordered or turn loop, with Pro at its center, has been described for several fusion peptides, e.g., in EnvA of the Avian sarcoma/leukosis virus subtype A (ASLV-A) [147], Ebola virus GP [148] and mouse or macaque fertilin α (ADAM 1) [149], which suggests that this part of SARS-CoV E is analogous to an internal fusion peptide. This motif has also been observed in a cis-proline turn [150] linking two β-hairpin strands in the structure of an HIV-1IIIB V3 peptide. It was found by mutagenesis of the fusion peptide of Env in ASLV-A, that proline, or a residue of similar intermediate hydrophobicity, are part of an accessible loop and was needed for ini‐ tial interactions of fusion peptides with target membranes.

**3. Conclusion**

viroporins will be elucidated in the near future.

, Montserrat Samsó2

\*Address all correspondence to: jtorres@ntu.edu.sg

**Acknowledgements**

CRP4-2008-02.

**Author details**

Wahyu Surya1

**References**

Viroporins constitute important components of viruses, and we are just beginning to un‐ derstand what is their biological role during the viral life cycle. One of the main prob‐ lems in their *in vitro* structural and functional study is high hydrophobicity and strong tendency to aggregate. This may reflect their likely multifunctional role in the cell, inter‐ acting with several viral and host partners. This multifunctionality seems dictated by ge‐ netic minimalism observed in viruses, in turn forced by the need to rapidly produce new progeny inside an alien environment. Viroporins such as those presented here, SH pro‐ tein and CoV E proteins, form complexes that are still not well characterized that are crit‐ ical for viral eggress. In this context, the biological function of channel activity is still unknown. More data is becoming available with more purified proteins, and inevitably extrapolations will have to be made from easier to handle proteins. For example, we could obtain a reasonably detailed SH protein NMR spectrum in detergents, but that is still not possible for E proteins. Even when structural data can be obtained, efforts will be directed towards environments that best mimic the conditions of natural lipid bilayers, as protein conformation is likely to change. With multidisciplinary action, the key roles of

Structural and Functional Aspects of Viroporins in Human Respiratory Viruses: Respiratory Syncytial Virus and

Coronaviruses

65

http://dx.doi.org/10.5772/53957

J.T. acknowledges the funding of the National Research Foundation grant NRF-

and Jaume Torres1\*

1 School of Biological Sciences, Nanyang Technological University, Singapore

*ment of antiviral agents.* Nature, 1978. 272(5655): p. 694-699.

2 School of Medicine, Virginia Commonwealth University, Richmond, VA, USA

[1] Gonzalez, M.E. and L. Carrasco, Viroporins. FEBS Lett., 2003. 552(1): p. 28-34.

[2] Carrasco, L., *Membrane leakiness after viral infection and a new approach to the develop‐*

Amyloid fiber formation has been reported for fragments of many non pathogenic proteins [151], and they have been found in a variety of proteins which are not associated with dis‐ ease [152, 153]. Therefore, this finding may not have relevance for the toxicity of the virus. Nevertheless, this possibility cannot be discarded in view of other roles of similar semenderived fibers in HIV viral entry which dramatically enhance HIV infection [154]. A more likely possibility, however, is that this conformational plasticity is needed during membrane fusion; a transition form a α-helical conformation to an antiparallel β-structure, with Pro as a hinge, could drive membrane fusion by pulling the two membranes in close apposition.

**NMR studies: towards the high-resolution structure of SARS-CoV E.** Full-length SARS-CoV E protein shows a high tendency to aggregate when solubilized in detergents, making it difficult to find a suitable condition for structural determination. While the TM region could be studied in DPC [25], 2D-HSQC spectra of full-length SARS-CoV E protein show poor quality in DPC-solubilized samples, even when SDS is included to improve spectral quality (Fig. 8A). Some degree of improvement can be observed with a truncated version of SARS-CoV E, which is lacking ~10 amino acids at both termini (Fig. 8B). We have also ob‐ tained a good, well-dispersed spectrum for this construct in SDS (Fig. 8C), allowing us to begin the structural determination of the extramembrane regions of SARS-CoV E.

**Figure 8. TROSY-HSQC of SARS-CoV E protein in various detergent micelles.** (A) full-length SARS-CoV E in a mix‐ ture of DPC and SDS, (B) truncated SARS-CoV E in DPC and (C) in SDS.

#### **3. Conclusion**

In addition, a sequence of ordered fragments (α-helices or strands) flanking a disordered or turn loop, with Pro at its center, has been described for several fusion peptides, e.g., in EnvA of the Avian sarcoma/leukosis virus subtype A (ASLV-A) [147], Ebola virus GP [148] and mouse or macaque fertilin α (ADAM 1) [149], which suggests that this part of SARS-CoV E is analogous to an internal fusion peptide. This motif has also been observed in a cis-proline turn [150] linking two β-hairpin strands in the structure of an HIV-1IIIB V3 peptide. It was found by mutagenesis of the fusion peptide of Env in ASLV-A, that proline, or a residue of similar intermediate hydrophobicity, are part of an accessible loop and was needed for ini‐

Amyloid fiber formation has been reported for fragments of many non pathogenic proteins [151], and they have been found in a variety of proteins which are not associated with dis‐ ease [152, 153]. Therefore, this finding may not have relevance for the toxicity of the virus. Nevertheless, this possibility cannot be discarded in view of other roles of similar semenderived fibers in HIV viral entry which dramatically enhance HIV infection [154]. A more likely possibility, however, is that this conformational plasticity is needed during membrane fusion; a transition form a α-helical conformation to an antiparallel β-structure, with Pro as a hinge, could drive membrane fusion by pulling the two membranes in close apposition.

**NMR studies: towards the high-resolution structure of SARS-CoV E.** Full-length SARS-CoV E protein shows a high tendency to aggregate when solubilized in detergents, making it difficult to find a suitable condition for structural determination. While the TM region could be studied in DPC [25], 2D-HSQC spectra of full-length SARS-CoV E protein show poor quality in DPC-solubilized samples, even when SDS is included to improve spectral quality (Fig. 8A). Some degree of improvement can be observed with a truncated version of SARS-CoV E, which is lacking ~10 amino acids at both termini (Fig. 8B). We have also ob‐ tained a good, well-dispersed spectrum for this construct in SDS (Fig. 8C), allowing us to

**Figure 8. TROSY-HSQC of SARS-CoV E protein in various detergent micelles.** (A) full-length SARS-CoV E in a mix‐

ture of DPC and SDS, (B) truncated SARS-CoV E in DPC and (C) in SDS.

begin the structural determination of the extramembrane regions of SARS-CoV E.

tial interactions of fusion peptides with target membranes.

64 Respiratory Disease and Infection - A New Insight

Viroporins constitute important components of viruses, and we are just beginning to un‐ derstand what is their biological role during the viral life cycle. One of the main prob‐ lems in their *in vitro* structural and functional study is high hydrophobicity and strong tendency to aggregate. This may reflect their likely multifunctional role in the cell, inter‐ acting with several viral and host partners. This multifunctionality seems dictated by ge‐ netic minimalism observed in viruses, in turn forced by the need to rapidly produce new progeny inside an alien environment. Viroporins such as those presented here, SH pro‐ tein and CoV E proteins, form complexes that are still not well characterized that are crit‐ ical for viral eggress. In this context, the biological function of channel activity is still unknown. More data is becoming available with more purified proteins, and inevitably extrapolations will have to be made from easier to handle proteins. For example, we could obtain a reasonably detailed SH protein NMR spectrum in detergents, but that is still not possible for E proteins. Even when structural data can be obtained, efforts will be directed towards environments that best mimic the conditions of natural lipid bilayers, as protein conformation is likely to change. With multidisciplinary action, the key roles of viroporins will be elucidated in the near future.

#### **Acknowledgements**

J.T. acknowledges the funding of the National Research Foundation grant NRF-CRP4-2008-02.

#### **Author details**

Wahyu Surya1 , Montserrat Samsó2 and Jaume Torres1\*

\*Address all correspondence to: jtorres@ntu.edu.sg

1 School of Biological Sciences, Nanyang Technological University, Singapore

2 School of Medicine, Virginia Commonwealth University, Richmond, VA, USA

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**Section 2**

**Bacterial Infections**

### **Bacterial Infections**

**Chapter 4**

**Biophysical Effects on Chronic**

**Rhinosinusitis Bacterial Biofilms**

Mohammed Al-Haddad, Ahmed Al-Jumaily,

Additional information is available at the end of the chapter

films have been implicated in the pathogenesis of CRS [2-4].

Chronic rhinosinusitis (CRS) is a common debilitating condition. In the United States alone according to recent data from the National Health Interview Survey CRS affects approxi‐ mately 31 million people [1] resulting in an estimated annual cost of \$6 billion. Bacterial bio‐

This literature review focuses on the effect of physical excitation by ultrasound and/or elec‐ tric fields on bacterial biofilms. The associated research is based on the hypothesis that *exter‐ nal forces applied by ultrasound and electric fields can alter the attachment forces of biofilms and*

Firstly, the role of bacterial biofilms in CRS is discussed identifying the major bacterial spe‐ cies involved in CRS (section 2). Secondly, the importance of the cyclic process of biofilm formation is reviewed focusing on the nature of the attachment and detachment process (section 3). Thirdly, treatments are categorized into medication and physical methods (sec‐ tion 4), with a major emphasis on ultrasound and the bioelectric effect. Finally, future direc‐ tions on the use of ultrasound and electric fields, and their potential effects on biofilms are

The respiratory system is divided into the upper and the lower airways. In the upper air‐ ways, the air is filtered, warmed and moistened by the nasal cavity, which is surrounded by

and reproduction in any medium, provided the original work is properly cited.

© 2013 Al-Haddad et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

*possibly help patients with chronic infective diseases such as chronic rhinosinusitis.*

John Brooks and Jim Bartley

http://dx.doi.org/10.5772/53860

**1. Introduction**

discussed (section 5).

**2. Chronic rhinosinusitis**

**Chapter 4**

### **Biophysical Effects on Chronic Rhinosinusitis Bacterial Biofilms**

Mohammed Al-Haddad, Ahmed Al-Jumaily, John Brooks and Jim Bartley

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53860

#### **1. Introduction**

Chronic rhinosinusitis (CRS) is a common debilitating condition. In the United States alone according to recent data from the National Health Interview Survey CRS affects approxi‐ mately 31 million people [1] resulting in an estimated annual cost of \$6 billion. Bacterial bio‐ films have been implicated in the pathogenesis of CRS [2-4].

This literature review focuses on the effect of physical excitation by ultrasound and/or elec‐ tric fields on bacterial biofilms. The associated research is based on the hypothesis that *exter‐ nal forces applied by ultrasound and electric fields can alter the attachment forces of biofilms and possibly help patients with chronic infective diseases such as chronic rhinosinusitis.*

Firstly, the role of bacterial biofilms in CRS is discussed identifying the major bacterial spe‐ cies involved in CRS (section 2). Secondly, the importance of the cyclic process of biofilm formation is reviewed focusing on the nature of the attachment and detachment process (section 3). Thirdly, treatments are categorized into medication and physical methods (sec‐ tion 4), with a major emphasis on ultrasound and the bioelectric effect. Finally, future direc‐ tions on the use of ultrasound and electric fields, and their potential effects on biofilms are discussed (section 5).

### **2. Chronic rhinosinusitis**

The respiratory system is divided into the upper and the lower airways. In the upper air‐ ways, the air is filtered, warmed and moistened by the nasal cavity, which is surrounded by

a ring of air-filled cavities called the paranasal sinuses. These consist of the maxillary, sphe‐ noid, frontal and ethmoid sinuses (Fig. 1). The proposed functions of the sinuses include moisturizing and humidifying ambient air, acting as resonators for speech, lightening the weight of the skull bones, providing protection to the brain from trauma, and producing ni‐ tric oxide.

Bacteria in a biofilm communicate chemically by using molecular chemical signals, a phe‐ nomenon called quorum sensing. It plays a major role in structuring complex communities and regulates a variety of physiological functions in both Gram-negative and Gram-positive bacteria [14]. Physical signals may also be involved in bacterial communication [15], but this

Biophysical Effects on Chronic Rhinosinusitis Bacterial Biofilms

http://dx.doi.org/10.5772/53860

81

The knowledge relating to the structure, function and mechanisms of quorum sensing is limited. It has been proposed that quorum sensing generates and responds to physical sig‐ nals including sound waves, electric current and electromagnetic radiation [15]. Production of sound waves has been observed with *Bacillus subtilis* [16], however, measurable data have not been reported in the literature. Intensity limitations and time scale requirements are

Generally, bacteria are described as negatively charged under most physiological conditions [17]. However, the net charge varies from species to species and is most likely influenced by culture conditions [18], age of the culture [19], ionic strength [20] and pH [21]. During the formation of biofilms, the net negative charge of the bacteria is reduced in order to modify their cellular surface charge [22]. This phenomenon has been observed with *Staphylococcus aureus* [23, 24]. Similarly, the measurement of surface charge of *Staphylococcus epidermidis* shows a variation in the net charge suggesting that different regions of the cell on the sur‐ face have different surface charges although the overall net charge may be negative [25]. The

In addition to the biological and physical attributes of bacterial biofilms, they have a resist‐ ance to antibiotics 10-1000 times higher than planktonic bacteria of the same species [26]. A simple description of the biofilm resistance is the formation of a physical barrier to prevent antibiotics from penetrating biofilms [27]. Specifically, it has been reported that mechanisms include restricted penetration, enzymatic destruction of antimicrobial, gene transfer, quo‐ rum sensing, altered growth rate (persister cells), stress response to hostile environmental conditions and over-expression of genes [28]. Multiple mechanisms may work together to

Several studies have investigated the relationship between biofilms and CRS [2, 3, 29, 30]. The most common bacterial biofilms in CRS are *S. aureus*, *Pseudomonas aeruginosa*, coagulasenegative staphylococci, *Streptococcus pneumoniae*, *Moraxella catarrhalis* and *Haemophilus influ‐ enzae*. Using scanning electron microscopy, Cryer et al. obtained the first evidence of biofilms in CRS showing the presence of *P. aeruginosa* biofilm in specimens taken from max‐ illary and ethmoid sinus mucosa [31]. Healy et al. investigated the presence of bioflms in CRS by using microscopic fluorescent *in situ* hybridization and showed that *H. influenzae* was most frequently present (80%) among the species identified, which included *S. aureus*, *P. aeruginosa, S. pneumoniae* and fungi [32]. However, confocal scanning microscopy and transmission electron microscopy studies have shown that *S. aureus* and *P. aeruginosa* respec‐ tively are the most common biofilm bacteria in CRS [33, 34]. Despite the uncertainty with respect to the dominant bacterial species of biofilms in CRS, they have been reported as the major cause of CRS. In addition, biofilms are found attached to sinus surfaces and/or aggre‐ gated with a surrounding mucus layer or fluid [35-37]. However, regarding the aggregation,

communication is not well understood.

challenges in acquiring physical quantitative data from biofilms.

net charge has been decreased by increasing pH [21].

increase bacterial biofilm resistance to antibiotics.

Rhinosinusitis is defined as a group of disorders characterized by inflammation of the nose and paranasal sinuses. Based on the duration of inflammation, rhinosinusitis is classified as acute (< 4 weeks), subacute (4-12 weeks) or chronic (>12 weeks). CRS symptoms have been classified as major, which includes facial pain or pressure, nasal obstruction or blockage, discolored postnasal drainage, hyposmia (a reduced ability to smell) and purulence in nasal cavity, or minor, which includes headache, fever, dental pain and ear pain [5].

**Figure 1.** The paranasal sinuses from (a) front and (b) side view [6]

While the pathology of CRS remains unclear and is described as multifactorial, bacterial bio‐ films have been implicated as a major contributing factor [2-4]. Bacteria are now recognized as existing in two forms: free floating (planktonic) or in sophisticated communities called bi‐ ofilms. They have been defined as "a structured community of bacterial cells enclosed in a self-produced polymeric matrix adherent to an inert or living surface" [7].

#### **2.1. Biofilms**

Biofilms are composed of two major components: microorganism cells that account for less than 10% and a matrix of extracellular polymer substances (EPS) that account for more than 90% of biofilms [8-10]. The characteristics of the cells in biofilms differ from their planktonic counterparts [11]. EPS is considered the primary matrix material of biofilms and is com‐ prised of 50-90% organic carbon [10]. The biofilm matrix is composed of secreted polymers (polysaccharides), lipids, proteins, DNA and RNA [12]. Normally biofilms protect their in‐ habitants against environmental and biological threats [13].

Bacteria in a biofilm communicate chemically by using molecular chemical signals, a phe‐ nomenon called quorum sensing. It plays a major role in structuring complex communities and regulates a variety of physiological functions in both Gram-negative and Gram-positive bacteria [14]. Physical signals may also be involved in bacterial communication [15], but this communication is not well understood.

a ring of air-filled cavities called the paranasal sinuses. These consist of the maxillary, sphe‐ noid, frontal and ethmoid sinuses (Fig. 1). The proposed functions of the sinuses include moisturizing and humidifying ambient air, acting as resonators for speech, lightening the weight of the skull bones, providing protection to the brain from trauma, and producing ni‐

Rhinosinusitis is defined as a group of disorders characterized by inflammation of the nose and paranasal sinuses. Based on the duration of inflammation, rhinosinusitis is classified as acute (< 4 weeks), subacute (4-12 weeks) or chronic (>12 weeks). CRS symptoms have been classified as major, which includes facial pain or pressure, nasal obstruction or blockage, discolored postnasal drainage, hyposmia (a reduced ability to smell) and purulence in nasal

(a) (b)

While the pathology of CRS remains unclear and is described as multifactorial, bacterial bio‐ films have been implicated as a major contributing factor [2-4]. Bacteria are now recognized as existing in two forms: free floating (planktonic) or in sophisticated communities called bi‐ ofilms. They have been defined as "a structured community of bacterial cells enclosed in a

Biofilms are composed of two major components: microorganism cells that account for less than 10% and a matrix of extracellular polymer substances (EPS) that account for more than 90% of biofilms [8-10]. The characteristics of the cells in biofilms differ from their planktonic counterparts [11]. EPS is considered the primary matrix material of biofilms and is com‐ prised of 50-90% organic carbon [10]. The biofilm matrix is composed of secreted polymers (polysaccharides), lipids, proteins, DNA and RNA [12]. Normally biofilms protect their in‐

self-produced polymeric matrix adherent to an inert or living surface" [7].

habitants against environmental and biological threats [13].

**Figure 1.** The paranasal sinuses from (a) front and (b) side view [6]

cavity, or minor, which includes headache, fever, dental pain and ear pain [5].

tric oxide.

80 Respiratory Disease and Infection - A New Insight

**2.1. Biofilms**

The knowledge relating to the structure, function and mechanisms of quorum sensing is limited. It has been proposed that quorum sensing generates and responds to physical sig‐ nals including sound waves, electric current and electromagnetic radiation [15]. Production of sound waves has been observed with *Bacillus subtilis* [16], however, measurable data have not been reported in the literature. Intensity limitations and time scale requirements are challenges in acquiring physical quantitative data from biofilms.

Generally, bacteria are described as negatively charged under most physiological conditions [17]. However, the net charge varies from species to species and is most likely influenced by culture conditions [18], age of the culture [19], ionic strength [20] and pH [21]. During the formation of biofilms, the net negative charge of the bacteria is reduced in order to modify their cellular surface charge [22]. This phenomenon has been observed with *Staphylococcus aureus* [23, 24]. Similarly, the measurement of surface charge of *Staphylococcus epidermidis* shows a variation in the net charge suggesting that different regions of the cell on the sur‐ face have different surface charges although the overall net charge may be negative [25]. The net charge has been decreased by increasing pH [21].

In addition to the biological and physical attributes of bacterial biofilms, they have a resist‐ ance to antibiotics 10-1000 times higher than planktonic bacteria of the same species [26]. A simple description of the biofilm resistance is the formation of a physical barrier to prevent antibiotics from penetrating biofilms [27]. Specifically, it has been reported that mechanisms include restricted penetration, enzymatic destruction of antimicrobial, gene transfer, quo‐ rum sensing, altered growth rate (persister cells), stress response to hostile environmental conditions and over-expression of genes [28]. Multiple mechanisms may work together to increase bacterial biofilm resistance to antibiotics.

Several studies have investigated the relationship between biofilms and CRS [2, 3, 29, 30]. The most common bacterial biofilms in CRS are *S. aureus*, *Pseudomonas aeruginosa*, coagulasenegative staphylococci, *Streptococcus pneumoniae*, *Moraxella catarrhalis* and *Haemophilus influ‐ enzae*. Using scanning electron microscopy, Cryer et al. obtained the first evidence of biofilms in CRS showing the presence of *P. aeruginosa* biofilm in specimens taken from max‐ illary and ethmoid sinus mucosa [31]. Healy et al. investigated the presence of bioflms in CRS by using microscopic fluorescent *in situ* hybridization and showed that *H. influenzae* was most frequently present (80%) among the species identified, which included *S. aureus*, *P. aeruginosa, S. pneumoniae* and fungi [32]. However, confocal scanning microscopy and transmission electron microscopy studies have shown that *S. aureus* and *P. aeruginosa* respec‐ tively are the most common biofilm bacteria in CRS [33, 34]. Despite the uncertainty with respect to the dominant bacterial species of biofilms in CRS, they have been reported as the major cause of CRS. In addition, biofilms are found attached to sinus surfaces and/or aggre‐ gated with a surrounding mucus layer or fluid [35-37]. However, regarding the aggregation, it is not well known whether they detach from surfaces, or whether aggregated biofilms form in the fluids themselves [38].

The transition from weak interactions (forces) of the cell with the substratum to a permanent bonding is mediated by the presence of EPS [51], which increases the strength of attachment [13]. In this transition, a process of attachment transfers from the reversible to the irreversi‐ ble stage. Several forces, including covalent and hydrogen bonding as well as hydrophobic interactions, are generated [52]. In addition, electron transfer has been suggested as an extra step involved, by either donating electrons to, or accepting electrons from the substratum [53]. This might explain why the production of EPS is suggested to be mediated by electro‐ static charges [54]. Electrostatic interaction forces contribute to the cross-linkers of the bio‐ film matrix [55]. Aggregated interaction (forces) between positive and negative charges are shown between exo-polysaccharide alginate of *P. aeruginosa* and extracellular enzymes [56]. Electrostatic interaction and hydrogen bonding forces are suggested to be the dominant

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Bacterial adhesion forces change depending on the distance between the cells and the sub‐ stratum. Force-distance relations are proposed to consist of three steps: van der Waals forces at a distance greater than 50 nm, van der Waals and electrostatic interaction between 10 and 20 nm and van der Waals, electrostatic interaction and other interactions at a distance less than 1.5 nm [58]. However, these physical forces have been further classified as long and short range interaction forces [59]. Long range occurs at a distance greater than 50 nm while short range occurs at less than 5 nm. Long and short range physical forces are related forces

Adhesion forces have recently been measured using an atomic force microscope and optical tweezers [60-62]. However, a clear quantitative measurement of the bacterial forces has not yet been defined. Although all microscopic techniques including scanning electron micro‐ scopy, optical microscopy, and confocal laser microscopy do not quantitatively measure the adhesion forces, they qualitatively do observe the morphology of biofilms. Hence, a new

Although most of the above techniques were based on the experimental approaches, the the‐ oretical analysis of bacterial adhesion is determined by three approaches: the Derjaguin-Landau-Verwey-Overbeek (DLVO) model, the thermodynamic approach and the extended DLVO theory. The DLVO model calculates the net energy of interaction between a cell and a

However, this approach does not consider molecular and acid-base interactions. The ther‐ modynamic approach calculates the surface free energies, assuming that the process is re‐ versible only while it is described as distance dependent in the DLVO model. In addition, the thermodynamic system is assumed to be a closed system without any external input en‐ ergy. Furthermore, electrostatic interactions are not taken into account in the surface free en‐ ergies [63]. Neither the DLVO model nor the thermodynamic approach explain clearly the bacterial adhesion. Extended DLVO theory has been proposed recently by calculating the to‐

tal adhesion energy in which the acid base interactions are included and given by:

*net energy of interaction* =*van der Waals energy* + *electrostatic energy* (1)

and highly dependent on the distance between the biofilm and a surface.

technique is needed for accurately quantifying bacterial forces.

forces in biofilm formation [57].

surface [50]:

#### **3. Biofilm formation**

Before biofilms form, microorganisms move or are moved to a surface. Planktonic cells con‐ ventionally are thought to initiate contact with surfaces. However, this phenomenon and the mechanism behind it are not well understood. It is hypothesized to include random contact caused by several factors. These include Brownian motion, sedimentation owing to differen‐ ces in specific gravity between the bacteria and the bulk liquid, convective mass transport in which cells are physically transported towards the surface, and active transport owing to the activity of the filamentous cell appendages such as flagella, in which chemotaxis may or may not be included [39].

The formation of a biofilm is divided into attachment, growth and detachment. Although the growth is a part of the cyclical process of biofilm formation, the focus will be on the physical mechanisms influencing the attachment and detachment of biofilms to surfaces.

#### **3.1. Attachment**

Several experimental and theoretical approaches have been used to investigate the attach‐ ment process. The process of attachment is generally divided into two stages, reversible and irreversible, or similarly named as docking and locking [13]. Numbers of environmental, physical, chemical and biological variables initiate the attachment, including extracellular DNA (eDNA), temperature, electrostatic charges and acid base interaction forces. The role of eDNA in the initial cell attachment is not fully understood, despite its role in the growth and development of *P. aeruginosa* biofilm [40, 41]. However, eDNA enhances the adhesion of Gram-positive bacteria [42].

Temperature effects appear to be different depending on the microorganisms. *P. aeruginosa* has been observed to adhere better to hydrophilic contact lenses at 37 o C than at 26 o C [43]. On the other hand, *S. pneumoniae* adhere better at 24o C than at 10 o C [44]. The temperature may cause an increase in nutrient intake by increasing the activity of enzymes in the produc‐ tion of EPS [45]. It has been observed that the temperature has an impact on the presence of bacterial surface appendages and a decrease in temperature may initiate adhesion [46]. The temperature may also change the environmental conditions surrounding the bacteria by changing the ionic strength of the polysaccharides [47].

A number of physical and chemical forces, including van der Waals, electrostatic charges and acid base interactions, initiate the attachment during the reversible stage [48]. Generally, van der Waals forces are described as an attractive force, while electrostatic forces are a re‐ pulsive force [49]. If the repulsive forces are greater than the attractive forces, small shear forces can detach bacteria from surfaces, unless a conditioning film on the substrate is formed by the presence of EPS [49, 50].

The transition from weak interactions (forces) of the cell with the substratum to a permanent bonding is mediated by the presence of EPS [51], which increases the strength of attachment [13]. In this transition, a process of attachment transfers from the reversible to the irreversi‐ ble stage. Several forces, including covalent and hydrogen bonding as well as hydrophobic interactions, are generated [52]. In addition, electron transfer has been suggested as an extra step involved, by either donating electrons to, or accepting electrons from the substratum [53]. This might explain why the production of EPS is suggested to be mediated by electro‐ static charges [54]. Electrostatic interaction forces contribute to the cross-linkers of the bio‐ film matrix [55]. Aggregated interaction (forces) between positive and negative charges are shown between exo-polysaccharide alginate of *P. aeruginosa* and extracellular enzymes [56]. Electrostatic interaction and hydrogen bonding forces are suggested to be the dominant forces in biofilm formation [57].

it is not well known whether they detach from surfaces, or whether aggregated biofilms

Before biofilms form, microorganisms move or are moved to a surface. Planktonic cells con‐ ventionally are thought to initiate contact with surfaces. However, this phenomenon and the mechanism behind it are not well understood. It is hypothesized to include random contact caused by several factors. These include Brownian motion, sedimentation owing to differen‐ ces in specific gravity between the bacteria and the bulk liquid, convective mass transport in which cells are physically transported towards the surface, and active transport owing to the activity of the filamentous cell appendages such as flagella, in which chemotaxis may or

The formation of a biofilm is divided into attachment, growth and detachment. Although the growth is a part of the cyclical process of biofilm formation, the focus will be on the physical mechanisms influencing the attachment and detachment of biofilms to surfaces.

Several experimental and theoretical approaches have been used to investigate the attach‐ ment process. The process of attachment is generally divided into two stages, reversible and irreversible, or similarly named as docking and locking [13]. Numbers of environmental, physical, chemical and biological variables initiate the attachment, including extracellular DNA (eDNA), temperature, electrostatic charges and acid base interaction forces. The role of eDNA in the initial cell attachment is not fully understood, despite its role in the growth and development of *P. aeruginosa* biofilm [40, 41]. However, eDNA enhances the adhesion of

Temperature effects appear to be different depending on the microorganisms. *P. aeruginosa*

may cause an increase in nutrient intake by increasing the activity of enzymes in the produc‐ tion of EPS [45]. It has been observed that the temperature has an impact on the presence of bacterial surface appendages and a decrease in temperature may initiate adhesion [46]. The temperature may also change the environmental conditions surrounding the bacteria by

A number of physical and chemical forces, including van der Waals, electrostatic charges and acid base interactions, initiate the attachment during the reversible stage [48]. Generally, van der Waals forces are described as an attractive force, while electrostatic forces are a re‐ pulsive force [49]. If the repulsive forces are greater than the attractive forces, small shear forces can detach bacteria from surfaces, unless a conditioning film on the substrate is

C than at 10 o

C than at 26 o

C [44]. The temperature

C [43].

has been observed to adhere better to hydrophilic contact lenses at 37 o

On the other hand, *S. pneumoniae* adhere better at 24o

changing the ionic strength of the polysaccharides [47].

formed by the presence of EPS [49, 50].

form in the fluids themselves [38].

82 Respiratory Disease and Infection - A New Insight

**3. Biofilm formation**

may not be included [39].

Gram-positive bacteria [42].

**3.1. Attachment**

Bacterial adhesion forces change depending on the distance between the cells and the sub‐ stratum. Force-distance relations are proposed to consist of three steps: van der Waals forces at a distance greater than 50 nm, van der Waals and electrostatic interaction between 10 and 20 nm and van der Waals, electrostatic interaction and other interactions at a distance less than 1.5 nm [58]. However, these physical forces have been further classified as long and short range interaction forces [59]. Long range occurs at a distance greater than 50 nm while short range occurs at less than 5 nm. Long and short range physical forces are related forces and highly dependent on the distance between the biofilm and a surface.

Adhesion forces have recently been measured using an atomic force microscope and optical tweezers [60-62]. However, a clear quantitative measurement of the bacterial forces has not yet been defined. Although all microscopic techniques including scanning electron micro‐ scopy, optical microscopy, and confocal laser microscopy do not quantitatively measure the adhesion forces, they qualitatively do observe the morphology of biofilms. Hence, a new technique is needed for accurately quantifying bacterial forces.

Although most of the above techniques were based on the experimental approaches, the the‐ oretical analysis of bacterial adhesion is determined by three approaches: the Derjaguin-Landau-Verwey-Overbeek (DLVO) model, the thermodynamic approach and the extended DLVO theory. The DLVO model calculates the net energy of interaction between a cell and a surface [50]:

$$\text{Input energy of interaction} = \text{van der Waals energy} + \text{electrostatic energy} \tag{1}$$

However, this approach does not consider molecular and acid-base interactions. The ther‐ modynamic approach calculates the surface free energies, assuming that the process is re‐ versible only while it is described as distance dependent in the DLVO model. In addition, the thermodynamic system is assumed to be a closed system without any external input en‐ ergy. Furthermore, electrostatic interactions are not taken into account in the surface free en‐ ergies [63]. Neither the DLVO model nor the thermodynamic approach explain clearly the bacterial adhesion. Extended DLVO theory has been proposed recently by calculating the to‐ tal adhesion energy in which the acid base interactions are included and given by:

$$
\Delta G^{\text{all}} = \Delta G^{\text{vdlW}} + \Delta G^{\text{dl}} + \Delta G^{AB} \tag{2}
$$

**4. Methods of disrupting biofilms**

**4.1. Medication**

icate bacteria in a biofilm state.

against biofilms [82].

ue in the study.

*4.2.1. Ultrasound*

**4.2. Biophysical approach**

tive method for treating biofilms [86].

conventional medication (biochemical) and biophysical.

Available methods of treatment for CRS bacterial biofilms are classified into two categories;

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Medication is the current recommended treatment method for CRS. However, they do not seem to improve long-term outcomes [77]. In addition, there are side effects [78] and there is a possibility of changing pathogens and resistance patterns that result in the persistence of CRS [79]. A problem of CRS antibiotic treatment is that these treatments are not able to erad‐

Alternative natural substances similar to antibiotics have been proposed such as honey and garlic. Manuka honey has been reported to be an effective treatment for CRS with‐ out damaging respiratory epithelium [80]. However, it does not block biofilm quorum sensing; in contrast, garlic has been shown to be effective in blocking quorum sensing [81]. Both conventional antibiotics and alternative natural substances are often ineffective

Vitamin D recently has been proposed as a treatment for CRS and to decrease the viability of *P. aeroginosa* biofilms [83]. However, the mechanism of action is unclear. As another treat‐ ment, baby shampoo as simple chemotherapy has been proposed for treating CRS [84]. However, side effects were observed in more than 10% of the patients who could not contin‐

Physical methods are classified into two categories: ultrasound and bioelectric effect.

Ultrasound is a cyclic sound pressure utilizing frequencies higher than 20 kHz. It has been employed in a variety of medical applications ranging from diagnostic imaging to physical therapy [85]. Variables in ultrasound therapy include the type of ultrasound (continuous or pulsed), frequency, intensity, duty cycle, individual treatment duration and overall treat‐ ment length. Recently, it has been suggested that ultrasound could be a promising alterna‐

*In vitro* [87] and *in vivo* research [88] has shown that ultrasound has a role in disrupting bac‐ terial biofilms. It disrupts *Escherichia coli* [89], *S. epidermidis* [88]*, P. aerginosa* [90], coagulasenegative Staphylococci [87] and *S. aureus* [87] bacterial biofilms. Bacterial viability is further reduced when ultrasound is combined with antibiotics [91, 92]. The susceptibility of biofilms from different bacterial species to ultrasound appears to vary. Under identical conditions, ultrasound at 67 kHz was effective against Gram-negative *E. coli* and *P. aeruginosa* cultures but not against Gram-positive *S. aureus* and *S. epidermidis* cultures [92]. However, *S. epider‐*

Where ∆*Gadh* : Total adhesion energy

∆*GvdW* :van der Waals interaction∆*Gdl* :Electric double layer interaction∆*GAB* :Acid-base interactions

Although extended DLVO seems to be a promising theoretical approach, further investiga‐ tion is needed to explain the exact mechanism of bacterial adhesion.

#### **3.2. Detachment**

The detachment of biofilms is influenced by several physical, chemical and biological condi‐ tions. This may lead to changes in the biofilm cells. Physical conditions play a major role in the bulk liquid surrounding biofilms and physical forces have been reported to change the structure of biofilms [64]. In addition, high shear forces have been shown to change the strength and weakness of biofilms [65]. Increasing the velocity and particle concentration of the liquid normally increases the detachment rate [66]. Shear forces influence biofilm thick‐ nesses [51]; however variations in these forces depend on the biofilm formation [67, 68]. It has been observed that under steady shear forces that biofilms roll along surfaces [69].

A direct electric current was reported to enhance the detachment of *S. epidermidis* biofilm from surgical stainless steel [70]. However, a variation in the detachment rate was ob‐ served when the direction of the current was changed between electrodes at the same magnitude [71]. Direct current appears to cause a disruption of the electrical bi-layer on the substrate [72].

In addition to the above physical factors, the detachment of a biofilm can be attributed to changes in the chemical properties of the biofilm or surrounding media. pH has been shown to influence the structure of biofilms in a form of expansion and contraction [73]. The effect of nutrients on biofilm detachment is not well understood. Limitation of nutrients at the bio‐ film-liquid interface increases the detachment rate [74]; however, another study has shown that the increased availability of nutrients also increases the detachment rate [75].

Apart from the above physical and chemical changes, there are biological factors influencing the detachment process. These include polysaccharide, enzymes, genes and quorum sens‐ ing. While these factors have shown a marginal effect, surface-protein-releasing-enzyme produced by *Streptococcus mutans* has caused the release of biofilms from tooth surfaces at rates 20% higher than control samples [76].

Physical and biological parameters appear to be more significant than chemical ones despite the fact that the latter have an important role in the detachment process. The significance of the physical detachment is attributed to attachment forces; however, quorum sensing is bio‐ logically significant in structuring the biofilm community. A question worth raising is would a change in the physical parameters, such as frequency, break down and disrupt bio‐ film attachment to surfaces? Would these changes block quorum sensing in biofilms?

#### **4. Methods of disrupting biofilms**

Available methods of treatment for CRS bacterial biofilms are classified into two categories; conventional medication (biochemical) and biophysical.

#### **4.1. Medication**

∆*Gadh* = ∆*GvdW* + ∆*Gdl* + ∆*GAB* (2)

Although extended DLVO seems to be a promising theoretical approach, further investiga‐

The detachment of biofilms is influenced by several physical, chemical and biological condi‐ tions. This may lead to changes in the biofilm cells. Physical conditions play a major role in the bulk liquid surrounding biofilms and physical forces have been reported to change the structure of biofilms [64]. In addition, high shear forces have been shown to change the strength and weakness of biofilms [65]. Increasing the velocity and particle concentration of the liquid normally increases the detachment rate [66]. Shear forces influence biofilm thick‐ nesses [51]; however variations in these forces depend on the biofilm formation [67, 68]. It has been observed that under steady shear forces that biofilms roll along surfaces [69].

A direct electric current was reported to enhance the detachment of *S. epidermidis* biofilm from surgical stainless steel [70]. However, a variation in the detachment rate was ob‐ served when the direction of the current was changed between electrodes at the same magnitude [71]. Direct current appears to cause a disruption of the electrical bi-layer on

In addition to the above physical factors, the detachment of a biofilm can be attributed to changes in the chemical properties of the biofilm or surrounding media. pH has been shown to influence the structure of biofilms in a form of expansion and contraction [73]. The effect of nutrients on biofilm detachment is not well understood. Limitation of nutrients at the bio‐ film-liquid interface increases the detachment rate [74]; however, another study has shown

Apart from the above physical and chemical changes, there are biological factors influencing the detachment process. These include polysaccharide, enzymes, genes and quorum sens‐ ing. While these factors have shown a marginal effect, surface-protein-releasing-enzyme produced by *Streptococcus mutans* has caused the release of biofilms from tooth surfaces at

Physical and biological parameters appear to be more significant than chemical ones despite the fact that the latter have an important role in the detachment process. The significance of the physical detachment is attributed to attachment forces; however, quorum sensing is bio‐ logically significant in structuring the biofilm community. A question worth raising is would a change in the physical parameters, such as frequency, break down and disrupt bio‐

film attachment to surfaces? Would these changes block quorum sensing in biofilms?

that the increased availability of nutrients also increases the detachment rate [75].

tion is needed to explain the exact mechanism of bacterial adhesion.

:Electric double layer interaction∆*GAB* :Acid-base

Where ∆*Gadh* : Total adhesion energy ∆*GvdW* :van der Waals interaction∆*Gdl*

84 Respiratory Disease and Infection - A New Insight

interactions

**3.2. Detachment**

the substrate [72].

rates 20% higher than control samples [76].

Medication is the current recommended treatment method for CRS. However, they do not seem to improve long-term outcomes [77]. In addition, there are side effects [78] and there is a possibility of changing pathogens and resistance patterns that result in the persistence of CRS [79]. A problem of CRS antibiotic treatment is that these treatments are not able to erad‐ icate bacteria in a biofilm state.

Alternative natural substances similar to antibiotics have been proposed such as honey and garlic. Manuka honey has been reported to be an effective treatment for CRS with‐ out damaging respiratory epithelium [80]. However, it does not block biofilm quorum sensing; in contrast, garlic has been shown to be effective in blocking quorum sensing [81]. Both conventional antibiotics and alternative natural substances are often ineffective against biofilms [82].

Vitamin D recently has been proposed as a treatment for CRS and to decrease the viability of *P. aeroginosa* biofilms [83]. However, the mechanism of action is unclear. As another treat‐ ment, baby shampoo as simple chemotherapy has been proposed for treating CRS [84]. However, side effects were observed in more than 10% of the patients who could not contin‐ ue in the study.

#### **4.2. Biophysical approach**

Physical methods are classified into two categories: ultrasound and bioelectric effect.

#### *4.2.1. Ultrasound*

Ultrasound is a cyclic sound pressure utilizing frequencies higher than 20 kHz. It has been employed in a variety of medical applications ranging from diagnostic imaging to physical therapy [85]. Variables in ultrasound therapy include the type of ultrasound (continuous or pulsed), frequency, intensity, duty cycle, individual treatment duration and overall treat‐ ment length. Recently, it has been suggested that ultrasound could be a promising alterna‐ tive method for treating biofilms [86].

*In vitro* [87] and *in vivo* research [88] has shown that ultrasound has a role in disrupting bac‐ terial biofilms. It disrupts *Escherichia coli* [89], *S. epidermidis* [88]*, P. aerginosa* [90], coagulasenegative Staphylococci [87] and *S. aureus* [87] bacterial biofilms. Bacterial viability is further reduced when ultrasound is combined with antibiotics [91, 92]. The susceptibility of biofilms from different bacterial species to ultrasound appears to vary. Under identical conditions, ultrasound at 67 kHz was effective against Gram-negative *E. coli* and *P. aeruginosa* cultures but not against Gram-positive *S. aureus* and *S. epidermidis* cultures [92]. However, *S. epider‐* *midis* responded favourably at a lower frequency (28.48 kHz) and a longer treatment time [88]. A variety of frequencies ranging from 28.48 kHz to 10 MHz has been utilised on bacte‐ rial biofilms [91, 93, 94]. A comparison between frequencies (70 kHz, 500 kHz, 2.25 MHz and 10 MHz) shows that lower frequencies appear more effective than higher ones [93, 95]. In both continuous and pulse ultrasound modes, high power intensities (200 mW/cm2 ) appear more efficacious than low power intensities (2 and 20 mW/cm2 ) [95, 96].

does not explain physical changes in biofilms on either the nature of biofilms or attachment processes. Hence, it does not explain electrostatic charges, van der Waals forces, electron

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The effects of application of a variety of currents ranging from 1 to 2000 mA and 15 µA/cm2 to 9 mA/cm2 have been investigated on biofilms [121]. The main variables in the use of bioelectric effects are electric field and current density. Although these variables have been thoroughly investigated on biofilms, their significance has not yet been estab‐ lished. In contrast, in the laboratory, the electric field is probably not the variable that breaks down the biofilms; however, it has been suggested that the current drives charg‐ ed molecules and ions into the biofilm matrix [119]. Applying electric current alone has been reported as not killing biofilms [122, 124]; however, other studies have reported

Results have been analyzed in terms of changes in CFU with respect to time. Application of electric fields alone, have reduced the number of CFU of biofilms for a certain period of time, but they return to the original number of bacteria at the end of the treatment period [118, 119]. Time of treatment has been evaluated at 12, 24 and 48 hours [114, 124, 126]. When time was increased from 12 to 48 hours, electric current showed a slight increase in the via‐ ble count of biofilms compared with the absence of electric current [122]. A dual treatment by bioelectric effect and antibiotics showed a further reduction in biofilms beyond the use antibiotics alone. The reduction is varied by a difference in magnitude ranging from 1-2 to 6-8 log [121]. However, variation in action on biofilms has shown different reactions accord‐ ing to type and concentration of antibiotics at the same level of electric field on the same species [114]. The morphology of biofilms could show the exact changes due to bioelectric effects if the bacterial biofilms behaviour were analysed by the changes of CFU in real time

Ultrasound and bioelectric effect are new strategies for breaking down biofilms. However, their mechanisms are not well understood. Ultrasound frequencies that disturb bacterial bi‐ ofilms need further investigation. The roles of time in the treatment and overall time of treatment have not been investigated and its effect with ultrasound has gained little atten‐ tion. Bioelectric effect may disrupt bi-layer; however, the mechanisms through which elec‐ tric currents or applied voltages cause a reduction in the number of viable biofilm cells is not conclusively understood. Evidence suggests that the biophysical approach could prevent the formation of biofilms and thus be clinically applicable to treat many chronic infectious dis‐

Several questions need to be answered. Are attachment bonds broken by ultrasound or does ultrasound disturb the biofilm structures? Is this caused by stretching the biofilm beyond the attachment forces or does the frequency affect the structural resonance of biofilms?

transportation or electromagnetic radiation.

some electric current effects [123, 125].

rather at a discrete point of time.

eases including chronic rhinosinusitis.

**5. Summary**

Most studies describe the effect of ultrasound on bacterial viability in terms of the changes in colony forming unit (CFU) per unit area. Although this provides an indication as to changes in the number of bacterial biofilm cells, it cannot measure physical changes in bio‐ films. Using ultrasound, one study suggests that bioacoustical affect biofilms [97], while an‐ other suggests it affects planktonic bacteria rather than biofilms [92]. It has also been suggested that ultrasound also enhances the transport of antimicrobial agents to bacteria [98], increases the transport of oxygen and nutrient to the cell and waste product away from the cell increasing bacterial growth rate [99], disturbs the cations in biofilms [100], and in‐ creases the permeability of the cell membrane [101-103].

Continuous [104] and pulsed [105] ultrasound at a frequency of 1 MHz with power intensi‐ ties of 1 and 0.5 W/cm2 for maxillary and frontal sinuses respectively have been evaluated in CRS treatment. Continuous ultrasound is more likely to cause a thermal effect [105], howev‐ er, no thermal effect has been observed with pulsed ultrasound. In acute rhinosinusitis the efficacy of ultrasound is comparable to antibiotics and when compared to antibiotics was al‐ so the preferred treatment option [106]. Therapeutic ultrasound is also effective in treating CRS [86, 104, 105, 107, 108].

#### *4.2.2. Bioelectric effect*

In the last decade the influence of high and low intensity electric fields and current densities on biofilms has been studied. High intensity electric fields influence the organization of bio‐ logical membranes [109], membrane analogues [110], the shape of the cell [111], cell behav‐ iour [112] and the dimensions of the bacterial glycocalyx [113]. However, low electrical current has been shown to enhance the efficacy of antimicrobial agents against *P. aeruginosa* [114], *S. epidermidis* [114], *Streptocoocus gordonii* [115], *E. coli* [116] and *S. aureus* [114] biofilms. This phenomenon has been referred to as the bioelectric effect.

Several methodologies of bioelectric effect on bacterial biofilms have been investigated, in‐ cluding direct current [117-119], radio frequency [116] and electromagnetic pulses [120]. The mechanism(s) of bioelectric actions on bacterial biofilms is not understood yet. A number of hypotheses have been proposed to explain the action(s) on biofilms [121]. These include in‐ creased bacterial growth due to electrolytic oxygen generation [122, 123], increased connec‐ tive transport due to expansion and contraction of the biofilms [73], electrochemical generation of oxygen [119], and increased membrane permeability [124]. Most of the bioelec‐ tric studies on biofilms have analyzed the effects in terms of the changes in the number of CFU. Although this provides an indication of the effects on the number of biofilm cells, it does not measure chemical changes in biofilms including chemical bonds from covalent and hydrogen bonds, hydrophobic interactions, pH and EPS matrix. In addition, this analysis does not explain physical changes in biofilms on either the nature of biofilms or attachment processes. Hence, it does not explain electrostatic charges, van der Waals forces, electron transportation or electromagnetic radiation.

The effects of application of a variety of currents ranging from 1 to 2000 mA and 15 µA/cm2 to 9 mA/cm2 have been investigated on biofilms [121]. The main variables in the use of bioelectric effects are electric field and current density. Although these variables have been thoroughly investigated on biofilms, their significance has not yet been estab‐ lished. In contrast, in the laboratory, the electric field is probably not the variable that breaks down the biofilms; however, it has been suggested that the current drives charg‐ ed molecules and ions into the biofilm matrix [119]. Applying electric current alone has been reported as not killing biofilms [122, 124]; however, other studies have reported some electric current effects [123, 125].

Results have been analyzed in terms of changes in CFU with respect to time. Application of electric fields alone, have reduced the number of CFU of biofilms for a certain period of time, but they return to the original number of bacteria at the end of the treatment period [118, 119]. Time of treatment has been evaluated at 12, 24 and 48 hours [114, 124, 126]. When time was increased from 12 to 48 hours, electric current showed a slight increase in the via‐ ble count of biofilms compared with the absence of electric current [122]. A dual treatment by bioelectric effect and antibiotics showed a further reduction in biofilms beyond the use antibiotics alone. The reduction is varied by a difference in magnitude ranging from 1-2 to 6-8 log [121]. However, variation in action on biofilms has shown different reactions accord‐ ing to type and concentration of antibiotics at the same level of electric field on the same species [114]. The morphology of biofilms could show the exact changes due to bioelectric effects if the bacterial biofilms behaviour were analysed by the changes of CFU in real time rather at a discrete point of time.

#### **5. Summary**

*midis* responded favourably at a lower frequency (28.48 kHz) and a longer treatment time [88]. A variety of frequencies ranging from 28.48 kHz to 10 MHz has been utilised on bacte‐ rial biofilms [91, 93, 94]. A comparison between frequencies (70 kHz, 500 kHz, 2.25 MHz and 10 MHz) shows that lower frequencies appear more effective than higher ones [93, 95]. In

Most studies describe the effect of ultrasound on bacterial viability in terms of the changes in colony forming unit (CFU) per unit area. Although this provides an indication as to changes in the number of bacterial biofilm cells, it cannot measure physical changes in bio‐ films. Using ultrasound, one study suggests that bioacoustical affect biofilms [97], while an‐ other suggests it affects planktonic bacteria rather than biofilms [92]. It has also been suggested that ultrasound also enhances the transport of antimicrobial agents to bacteria [98], increases the transport of oxygen and nutrient to the cell and waste product away from the cell increasing bacterial growth rate [99], disturbs the cations in biofilms [100], and in‐

Continuous [104] and pulsed [105] ultrasound at a frequency of 1 MHz with power intensi‐ ties of 1 and 0.5 W/cm2 for maxillary and frontal sinuses respectively have been evaluated in CRS treatment. Continuous ultrasound is more likely to cause a thermal effect [105], howev‐ er, no thermal effect has been observed with pulsed ultrasound. In acute rhinosinusitis the efficacy of ultrasound is comparable to antibiotics and when compared to antibiotics was al‐ so the preferred treatment option [106]. Therapeutic ultrasound is also effective in treating

In the last decade the influence of high and low intensity electric fields and current densities on biofilms has been studied. High intensity electric fields influence the organization of bio‐ logical membranes [109], membrane analogues [110], the shape of the cell [111], cell behav‐ iour [112] and the dimensions of the bacterial glycocalyx [113]. However, low electrical current has been shown to enhance the efficacy of antimicrobial agents against *P. aeruginosa* [114], *S. epidermidis* [114], *Streptocoocus gordonii* [115], *E. coli* [116] and *S. aureus* [114] biofilms.

Several methodologies of bioelectric effect on bacterial biofilms have been investigated, in‐ cluding direct current [117-119], radio frequency [116] and electromagnetic pulses [120]. The mechanism(s) of bioelectric actions on bacterial biofilms is not understood yet. A number of hypotheses have been proposed to explain the action(s) on biofilms [121]. These include in‐ creased bacterial growth due to electrolytic oxygen generation [122, 123], increased connec‐ tive transport due to expansion and contraction of the biofilms [73], electrochemical generation of oxygen [119], and increased membrane permeability [124]. Most of the bioelec‐ tric studies on biofilms have analyzed the effects in terms of the changes in the number of CFU. Although this provides an indication of the effects on the number of biofilm cells, it does not measure chemical changes in biofilms including chemical bonds from covalent and hydrogen bonds, hydrophobic interactions, pH and EPS matrix. In addition, this analysis

) appear

) [95, 96].

both continuous and pulse ultrasound modes, high power intensities (200 mW/cm2

more efficacious than low power intensities (2 and 20 mW/cm2

creases the permeability of the cell membrane [101-103].

This phenomenon has been referred to as the bioelectric effect.

CRS [86, 104, 105, 107, 108].

86 Respiratory Disease and Infection - A New Insight

*4.2.2. Bioelectric effect*

Ultrasound and bioelectric effect are new strategies for breaking down biofilms. However, their mechanisms are not well understood. Ultrasound frequencies that disturb bacterial bi‐ ofilms need further investigation. The roles of time in the treatment and overall time of treatment have not been investigated and its effect with ultrasound has gained little atten‐ tion. Bioelectric effect may disrupt bi-layer; however, the mechanisms through which elec‐ tric currents or applied voltages cause a reduction in the number of viable biofilm cells is not conclusively understood. Evidence suggests that the biophysical approach could prevent the formation of biofilms and thus be clinically applicable to treat many chronic infectious dis‐ eases including chronic rhinosinusitis.

Several questions need to be answered. Are attachment bonds broken by ultrasound or does ultrasound disturb the biofilm structures? Is this caused by stretching the biofilm beyond the attachment forces or does the frequency affect the structural resonance of biofilms?

#### **Author details**

Mohammed Al-Haddad1 , Ahmed Al-Jumaily1\*, John Brooks2 and Jim Bartley1,3

\*Address all correspondence to: ahmed.al-jumaily@aut.ac.nz, mohammed.alhad‐ dad@aut.ac.nz

[11] Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G. The Application of Biofilm Science to the Study and Control of Chronic Bacterial Infections. Journal of

Biophysical Effects on Chronic Rhinosinusitis Bacterial Biofilms

http://dx.doi.org/10.5772/53860

89

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[20] Dan N. The Effect of Charge Regulation on Cell Adhesion to Substrates: Salt-induced

[21] Palmer JS, Flint SH, Schmid J, Brooks JD. The Role of Surface Charge and Hydropho‐ bicity in the Attachment of Anoxybacillus Flavithermus Isolated from Milk Powder.

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1 Institute of Biomedical Technologies (IBTec), Auckland University of Technology, Auck‐ land, New Zealand

2 Biotechnology Research Institute, Auckland University of Technology, Auckland, New Zealand

3 Department of Surgery, University of Auckland, Auckland, New Zealand

#### **References**


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**Author details**

dad@aut.ac.nz

Zealand

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Mohammed Al-Haddad1

88 Respiratory Disease and Infection - A New Insight

, Ahmed Al-Jumaily1\*, John Brooks2

\*Address all correspondence to: ahmed.al-jumaily@aut.ac.nz, mohammed.alhad‐

1 Institute of Biomedical Technologies (IBTec), Auckland University of Technology, Auck‐

2 Biotechnology Research Institute, Auckland University of Technology, Auckland, New

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5785-90.


**Chapter 5**

**Clinical Diagnosis and Severity Assessment in**

Community-acquired pneumonia (CAP) is a common and potentially serious illness [1]. It is defined as an acute infection of the pulmonary parenchyma, occurring outside the hospital, with clinical symptoms accompanied by the presence of pulmonary infiltrates on chest ra‐ diograph. With a prevalence estimated at nearly five million cases annually in United States, emergency physicians and general practitioners diagnose and treat CAP on a regular basis [2]; nearly one third of CAP patients arrived by emergency medical services, and half even‐

Community-acquired pneumonia is the major infection-related cause of death in developed countries [4] and ranks as the third leading cause of all deaths in the world after ischaemic heart disease and cerebrovascular disease [5]. Mortality from CAP ranges from less than 1% in patients without risk factors treated as outpatients to 5-15% in hospital admitted patients to greater than 20-30% in intensive care unit patients [6]. Pneumonia increases in frequency with advancing age, and with associated comorbid medical illnesses (specially cardiovascu‐ lar, metabolic, neoplastic, respiratory and neurological disease) there is a significant increase

Acute respiratory infections are a prevalent problem, affecting children, adults and the eld‐ erly, the main pathogens involved are respiratory viruses (*rhinovirus*, *influenza*, *parainfluenza*, *adenovirus*, *respiratory syncytial virus*, *metapneumovirus*) and secondly bacteria (*Streptococcus pneumoniae*, *Haemophilus influenzae*, *Mycoplasma pneumoniae*, *Chlamydophila pneumoniae*, *Le‐*

and reproduction in any medium, provided the original work is properly cited.

© 2013 Saldías et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Immunocompetent Adult Patients with**

Fernando Peñafiel Saldías, Orlando Díaz Patiño and

**Community-Acquired Pneumonia**

Additional information is available at the end of the chapter

Pablo Aguilera Fuenzalida

http://dx.doi.org/10.5772/53475

**1. Introduction**

tually were admitted [3].

in morbidity and mortality [7].

## **Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with Community-Acquired Pneumonia**

Fernando Peñafiel Saldías, Orlando Díaz Patiño and Pablo Aguilera Fuenzalida

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53475

#### **1. Introduction**

Community-acquired pneumonia (CAP) is a common and potentially serious illness [1]. It is defined as an acute infection of the pulmonary parenchyma, occurring outside the hospital, with clinical symptoms accompanied by the presence of pulmonary infiltrates on chest ra‐ diograph. With a prevalence estimated at nearly five million cases annually in United States, emergency physicians and general practitioners diagnose and treat CAP on a regular basis [2]; nearly one third of CAP patients arrived by emergency medical services, and half even‐ tually were admitted [3].

Community-acquired pneumonia is the major infection-related cause of death in developed countries [4] and ranks as the third leading cause of all deaths in the world after ischaemic heart disease and cerebrovascular disease [5]. Mortality from CAP ranges from less than 1% in patients without risk factors treated as outpatients to 5-15% in hospital admitted patients to greater than 20-30% in intensive care unit patients [6]. Pneumonia increases in frequency with advancing age, and with associated comorbid medical illnesses (specially cardiovascu‐ lar, metabolic, neoplastic, respiratory and neurological disease) there is a significant increase in morbidity and mortality [7].

Acute respiratory infections are a prevalent problem, affecting children, adults and the eld‐ erly, the main pathogens involved are respiratory viruses (*rhinovirus*, *influenza*, *parainfluenza*, *adenovirus*, *respiratory syncytial virus*, *metapneumovirus*) and secondly bacteria (*Streptococcus pneumoniae*, *Haemophilus influenzae*, *Mycoplasma pneumoniae*, *Chlamydophila pneumoniae*, *Le‐*

© 2013 Saldías et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*gionella pneumophila*, gram negative bacilli, and others), they are an important cause of school and labor absenteeism, especially during the cold seasons [1,8].

The differential diagnosis of CAP includes several noninfectious causes, including pulmona‐ ry embolism, malignancy and congestive heart failure, among others [21]. The presence of a non-infectious differential diagnosis is usually suspected only after failure of antibiotic ther‐ apy, with the ensuing risks related to untreated, potentially life threatening non-bacterial disease [22]. Conversely, a delay of antibiotic treatment of more than 4-8 hours after hospital admission is associated with increased mortality [23]. Hence, both a rapid diagnosis of CAP and an accurate differentiation from viral respiratory illnesses and non-infectious causes has

Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with...

http://dx.doi.org/10.5772/53475

101

In the initial diagnosis of the patient who complains of acute respiratory symptoms or fever is necessary to establish the correct diagnosis based on clinical manifestations (history and physical examination) and laboratory tests (e.g., blood cell count, chest radiograph, C-reac‐ tive protein, procalcitonin, etc.) that are available in ambulatory practice. This requires knowledge of the epidemiology of respiratory infections in the geographic area, together with the sensitivity and specificity of the clinical history and physical signs abnormalities in

The primary care physicians are often being confronted with patients presenting with non‐ specific constitutional symptoms (e.g., malaise, fever, chills, myalgia, headache, anorexia) or respiratory symptoms (e.g., cough, sputum production, chest pain, dyspnea), and must to establish a presumptive diagnosis based on their knowledge of the local epidemiology of acute respiratory infections and the main findings on clinical history and physical examina‐ tion. Unfortunately clinical manifestations at presentation distinguish poorly between com‐ munity-acquired pneumonia and other causes of respiratory illnesses (view differential diagnosis on Table 1). The likelihood ratio (LR) for these findings ranges between 1 and 3, which is not useful to confirm the diagnosis of pneumonia (Table 2) [25-29]. In general, the presence or absence of preexisting diseases, respiratory or constitutional symptoms does not

Five studies based in emergency departments have assessed the characteristics of individual items in the clinical history in the diagnosis of community-acquired pneumonia in adult pa‐ tients [25-29]. In each of these studies, the reference standard for the diagnosis of pneumonia was a new infiltrate on a chest radiograph with or without clinical monitoring during one month. Table 2 summarizes the likelihood ratios associated to main clinical findings ob‐ tained from the history, including general manifestations, preexisting diseases and respira‐ tory symptoms. Differences on results reflect, in part, variation in the selection criteria for each study. For example, chest radiographs were obtained for all patients presenting with acute cough in one study [25], while the other studies obtained chest radiographs only when the emergency physician previously determined a need for them, often to confirm or ex‐ clude a suspected diagnosis of pneumonia [26-29]. The latter approach provides a more highly selected population of patients with acute respiratory complaints that may change the measured test characteristics of individual clinical findings. Thus, the prevalence of pneumonia in study populations ranged from as low as 2.6% [25] to as high as 38.3% [26].

important therapeutic and prognostic implications [24].

have a substantial effect on the probability of pneumonia.

**2.1. Medical history**

diagnosing pneumonia [18].

The clinical manifestations associated with respiratory infections, such as malaise, fever, chills, myalgia, sore throat, runny nose, cough, sputum production, chest pain and dyspnea, can occur in different clinical contexts that differ in etiology, pathogenesis, clinical course, treatment and prognosis [8,9]. Thus, the clinical picture may correspond to a mild self-limit‐ ed upper respiratory tract infection to a severe lung parenchyma infection that requires spe‐ cific treatment, as in cases of pneumonia and tuberculosis [10].

The reference standard to diagnose CAP is a new infiltrate on chest radiograph in the pres‐ ence of recently acquired respiratory signs and symptoms [11]. These include cough, in‐ creased sputum production, dyspnea, fever and abnormal auscultatory findings [12]. Unfortunately, clinical findings do not reliably predict radiologically confirmed pneumonia [13]. Especially elderly people often present with atypical symptoms and without fever [14]. However physicians, especially in primary care, may not perform chest radiography and re‐ ly on the patient's history and physical examination [15].

The initial management of patients suspected of having community-acquired pneumonia is challenging because of the broad range of clinical presentations, comprehensive differential diagnosis, potential life-threatening nature of the illness, the need for antibiotic treatment and associated high costs of care [16,17]. The initial testing strategies should accurately es‐ tablish a diagnosis and prognosis in order to determine the optimal treatment strategy, such as decisions about the site of care (ambulatory or in-hospital), extension of microbiology and laboratory assessments and antimicrobial recommendations.

The diagnosis is important to implement specific management measures, such as empirical antibiotic treatment and prevention of complications, and the prognosis is important in de‐ termining the site of care (ambulatory, general ward or intensive care facilities) and define treatment strategies to be implemented in each particular case [16,17]. This paper reviews the sensitivity, specificity and accuracy of the history, physical examination, and laboratory findings, individually and in combination, in diagnosing community-acquired pneumonia and predicting short-term risk for complications and death from the infection.

#### **2. Diagnosis of pneumonia**

Primary-care physicians usually rely on patient history, and signs and symptoms to diag‐ nose or exclude pneumonia [10]. However, most signs and symptoms traditionally associat‐ ed with pneumonia (e.g. malaise, fever, cough, sputum production and dyspnea) are not predictive of pneumonia in general practice [18]. Chest radiography (CXR) is the most fre‐ quently performed diagnostic investigation requested by general practitioners in the ambu‐ latory setting (primary care and emergency department) [19]. The history and physical examination cannot provide a high level of certainty in the diagnosis of community-ac‐ quired pneumonia, but the absence of vital sign abnormalities and abnormal chest examina‐ tion findings substantially reduces the probability of the infection [20].

The differential diagnosis of CAP includes several noninfectious causes, including pulmona‐ ry embolism, malignancy and congestive heart failure, among others [21]. The presence of a non-infectious differential diagnosis is usually suspected only after failure of antibiotic ther‐ apy, with the ensuing risks related to untreated, potentially life threatening non-bacterial disease [22]. Conversely, a delay of antibiotic treatment of more than 4-8 hours after hospital admission is associated with increased mortality [23]. Hence, both a rapid diagnosis of CAP and an accurate differentiation from viral respiratory illnesses and non-infectious causes has important therapeutic and prognostic implications [24].

#### **2.1. Medical history**

*gionella pneumophila*, gram negative bacilli, and others), they are an important cause of

The clinical manifestations associated with respiratory infections, such as malaise, fever, chills, myalgia, sore throat, runny nose, cough, sputum production, chest pain and dyspnea, can occur in different clinical contexts that differ in etiology, pathogenesis, clinical course, treatment and prognosis [8,9]. Thus, the clinical picture may correspond to a mild self-limit‐ ed upper respiratory tract infection to a severe lung parenchyma infection that requires spe‐

The reference standard to diagnose CAP is a new infiltrate on chest radiograph in the pres‐ ence of recently acquired respiratory signs and symptoms [11]. These include cough, in‐ creased sputum production, dyspnea, fever and abnormal auscultatory findings [12]. Unfortunately, clinical findings do not reliably predict radiologically confirmed pneumonia [13]. Especially elderly people often present with atypical symptoms and without fever [14]. However physicians, especially in primary care, may not perform chest radiography and re‐

The initial management of patients suspected of having community-acquired pneumonia is challenging because of the broad range of clinical presentations, comprehensive differential diagnosis, potential life-threatening nature of the illness, the need for antibiotic treatment and associated high costs of care [16,17]. The initial testing strategies should accurately es‐ tablish a diagnosis and prognosis in order to determine the optimal treatment strategy, such as decisions about the site of care (ambulatory or in-hospital), extension of microbiology and

The diagnosis is important to implement specific management measures, such as empirical antibiotic treatment and prevention of complications, and the prognosis is important in de‐ termining the site of care (ambulatory, general ward or intensive care facilities) and define treatment strategies to be implemented in each particular case [16,17]. This paper reviews the sensitivity, specificity and accuracy of the history, physical examination, and laboratory findings, individually and in combination, in diagnosing community-acquired pneumonia

Primary-care physicians usually rely on patient history, and signs and symptoms to diag‐ nose or exclude pneumonia [10]. However, most signs and symptoms traditionally associat‐ ed with pneumonia (e.g. malaise, fever, cough, sputum production and dyspnea) are not predictive of pneumonia in general practice [18]. Chest radiography (CXR) is the most fre‐ quently performed diagnostic investigation requested by general practitioners in the ambu‐ latory setting (primary care and emergency department) [19]. The history and physical examination cannot provide a high level of certainty in the diagnosis of community-ac‐ quired pneumonia, but the absence of vital sign abnormalities and abnormal chest examina‐

and predicting short-term risk for complications and death from the infection.

tion findings substantially reduces the probability of the infection [20].

school and labor absenteeism, especially during the cold seasons [1,8].

100 Respiratory Disease and Infection - A New Insight

cific treatment, as in cases of pneumonia and tuberculosis [10].

ly on the patient's history and physical examination [15].

laboratory assessments and antimicrobial recommendations.

**2. Diagnosis of pneumonia**

In the initial diagnosis of the patient who complains of acute respiratory symptoms or fever is necessary to establish the correct diagnosis based on clinical manifestations (history and physical examination) and laboratory tests (e.g., blood cell count, chest radiograph, C-reac‐ tive protein, procalcitonin, etc.) that are available in ambulatory practice. This requires knowledge of the epidemiology of respiratory infections in the geographic area, together with the sensitivity and specificity of the clinical history and physical signs abnormalities in diagnosing pneumonia [18].

The primary care physicians are often being confronted with patients presenting with non‐ specific constitutional symptoms (e.g., malaise, fever, chills, myalgia, headache, anorexia) or respiratory symptoms (e.g., cough, sputum production, chest pain, dyspnea), and must to establish a presumptive diagnosis based on their knowledge of the local epidemiology of acute respiratory infections and the main findings on clinical history and physical examina‐ tion. Unfortunately clinical manifestations at presentation distinguish poorly between com‐ munity-acquired pneumonia and other causes of respiratory illnesses (view differential diagnosis on Table 1). The likelihood ratio (LR) for these findings ranges between 1 and 3, which is not useful to confirm the diagnosis of pneumonia (Table 2) [25-29]. In general, the presence or absence of preexisting diseases, respiratory or constitutional symptoms does not have a substantial effect on the probability of pneumonia.

Five studies based in emergency departments have assessed the characteristics of individual items in the clinical history in the diagnosis of community-acquired pneumonia in adult pa‐ tients [25-29]. In each of these studies, the reference standard for the diagnosis of pneumonia was a new infiltrate on a chest radiograph with or without clinical monitoring during one month. Table 2 summarizes the likelihood ratios associated to main clinical findings ob‐ tained from the history, including general manifestations, preexisting diseases and respira‐ tory symptoms. Differences on results reflect, in part, variation in the selection criteria for each study. For example, chest radiographs were obtained for all patients presenting with acute cough in one study [25], while the other studies obtained chest radiographs only when the emergency physician previously determined a need for them, often to confirm or ex‐ clude a suspected diagnosis of pneumonia [26-29]. The latter approach provides a more highly selected population of patients with acute respiratory complaints that may change the measured test characteristics of individual clinical findings. Thus, the prevalence of pneumonia in study populations ranged from as low as 2.6% [25] to as high as 38.3% [26].

There are no individual items from the clinical history whose presence or absence would re‐ duce or increase the odds of disease sufficiently to exclude or confirm the diagnosis of pneu‐ monia without obtain a chest radiograph [18,20]. Though the presence of fever, comorbidities, history of dementia or immunosuppression may be helpful, they are not con‐ firmatory, particularly given the typically low prevalence of pneumonia in the primary health services (2-5%) [1-3]. For example, when the estimated prevalence of pneumonia in primary care services is around 2-3%, the presence of subjective fever (LR+ =1.3-2.1) had a positive predictive value (PPV) ranging between 2.6% and 6.2% [25,28,29], reflecting the low prevalence of pneumonia in the ambulatory care setting. Meanwhile the presence of odyno‐ phagia or rhinorrhea (LR+ =0.5-0.7) in the same context had a positive predictive value rang‐ ing between 1.0% and 2.1% [25,29].

**Variables Diehr et al Gennis et al Singal et al Heckerling et al Saldías et al Cough** ┄ NS 1.8 NS NS

**Dyspnea** ━ 1.4 NS NS NS **Fever** 2.1 NS ━ 1.7 1.3 **Chills** 1.6 1.3 ━ 1.7 1.4 **Myalgias** 1.3 NS ━ ━ NS **Odynophagia** 0.7 NS ━ ━ 0.5 **Rhinorrhea** 0.7 NS ━ ━ 0.5 **HR > 100/min** NS 1.6 NS 2.3 1.4 **RR > 20/min** ━ 1.2 ━ ━ 1.3 **T > 37.8 °C** 4.4 1.4 2.4 2.4 2.2

**vital signs** ━ 0.2 ━ ━ 0.3

**Ronchi** NS 1.5 ━ 1.4 NS **Bronchophony** ━ ━ ━ 3.5 9.5 **Crackles** 2.7 1.6 1.7 2.6 2.0

**exam** ━ 0.5 ━ ━ 0.4

LR+: positive likelihood ratio for pneumonia (sensitivity/1-specificity). HR: heart rate, RR: respiratory rate, T: tempera‐ ture. Normal vital signs: heart rate < 100 beats/min, respiratory rate < 20 breaths/min and temperature < 37.8 °C. NS:

**Table 2.** Predictive value of clinical manifestations (symptoms and signs) associated with the diagnosis of community-

The effect of vital sign abnormalities (e.g., tachycardia, tachypnea, fever) or pulmonary ex‐ am findings (e.g., decreased breath sound, bronchophony, dullness on percussion, rhon‐ chus, crackles) on the probability of pneumonia depends on the cut-off value to define an abnormal result. For example, a respiratory rate greater than 20 breaths/min had a LR+

to 1.3 [26,29], whereas a respiratory rate greater than 25 breaths/min had a LR+ of 1.5 to 3.4 [25,28] (Table 2). When the estimated prevalence of pneumonia in primary care services is

tive predictive value ranging between 3.2% and 8.1% [25-29]. In contrast, two studies have

around 2-3%, the presence of crackles on pulmonary examination (LR+

of 1.2

=1.6-2.7) had a posi‐

1.3 NS ━ NS 1.2

Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with...

http://dx.doi.org/10.5772/53475

103

NS 2.2 ━ 4.3 3.8

**Sputum production**

**Normal**

**Dullness to percussion**

**Normal lung**

result not significant.

acquired pneumonia in adults (LR+)[25-29].

**2.2. Physical examination**


**Table 1.** Differential diagnosis of adult patients with community-acquired pneumonia in the primary health care system.


LR+: positive likelihood ratio for pneumonia (sensitivity/1-specificity). HR: heart rate, RR: respiratory rate, T: tempera‐ ture. Normal vital signs: heart rate < 100 beats/min, respiratory rate < 20 breaths/min and temperature < 37.8 °C. NS: result not significant.

**Table 2.** Predictive value of clinical manifestations (symptoms and signs) associated with the diagnosis of communityacquired pneumonia in adults (LR+)[25-29].

#### **2.2. Physical examination**

There are no individual items from the clinical history whose presence or absence would re‐ duce or increase the odds of disease sufficiently to exclude or confirm the diagnosis of pneu‐ monia without obtain a chest radiograph [18,20]. Though the presence of fever, comorbidities, history of dementia or immunosuppression may be helpful, they are not con‐ firmatory, particularly given the typically low prevalence of pneumonia in the primary health services (2-5%) [1-3]. For example, when the estimated prevalence of pneumonia in

positive predictive value (PPV) ranging between 2.6% and 6.2% [25,28,29], reflecting the low prevalence of pneumonia in the ambulatory care setting. Meanwhile the presence of odyno‐

Collagen vascular disease: Systemic lupus erythematosus, rheumatoid arthritis, Wegener granulomatosis, Churg-

**Table 1.** Differential diagnosis of adult patients with community-acquired pneumonia in the primary health care

=0.5-0.7) in the same context had a positive predictive value rang‐

=1.3-2.1) had a

primary care services is around 2-3%, the presence of subjective fever (LR+

phagia or rhinorrhea (LR+

Upper respiratory tract infections Acute bronchitis and bronchiolitis

Exacerbations of asthma and COPD

Primary neoplastic disease and metastatic lung disease

*Common causes*

Influenza - Flu

Pulmonary tuberculosis Congestive heart failure Pulmonary embolism

Pulmonary atelectasis

Strauss syndrome.

Eosinophilic pneumonia

Cryptogenic organizing pneumonia (COP)

Sarcoidosis

system.

Hypersensitivity pneumonitis Drug-induced lung diseases

Radiation-induced lung disease Carcinomatous lymphangiosis

*Rare causes*

ing between 1.0% and 2.1% [25,29].

102 Respiratory Disease and Infection - A New Insight

The effect of vital sign abnormalities (e.g., tachycardia, tachypnea, fever) or pulmonary ex‐ am findings (e.g., decreased breath sound, bronchophony, dullness on percussion, rhon‐ chus, crackles) on the probability of pneumonia depends on the cut-off value to define an abnormal result. For example, a respiratory rate greater than 20 breaths/min had a LR+ of 1.2 to 1.3 [26,29], whereas a respiratory rate greater than 25 breaths/min had a LR+ of 1.5 to 3.4 [25,28] (Table 2). When the estimated prevalence of pneumonia in primary care services is around 2-3%, the presence of crackles on pulmonary examination (LR+ =1.6-2.7) had a posi‐ tive predictive value ranging between 3.2% and 8.1% [25-29]. In contrast, two studies have shown that having a normal heart rate (below 100 beats/min) without fever (temperature < 37.8 °C) and tachypnea (respiratory rate < 20 breaths/min) reduces significantly the probabil‐ ity of community-acquired pneumonia (LR- = 0.18), thereby reducing the pretest odds of pneumonia by more than fivefold [26,29].

clinical features [18,20]. In the literature we find several protocols or decision rules that are designed specifically to help primary care physicians in the assessment of adult patients with lower respiratory tract infections (Table 3) [25-29,32-34]. Thus, in the clinical practice guidelines is often recommended to primary care physicians request of chest radiography in patients presenting respiratory symptoms based on some of these decision rules, in order to

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105

optimize patient`s care [16,17,35].

**Heckerling et al.** Each variable scores a point.

**Singal et al.** Estimating the probability of pneumonia.

Where Y: -3.095 + 1.214 x Cough + 1.007 x Fever + 0.823 x Crackles

throat; + 5.0 for dyspnea; + 8.2 for lateral chest pain; + 0.9 for crackles.

**Diehr et al.** Rhinorrhea Sore throat Night sweats Mialgyas

Crackles

mg/L.

setting[25-28,32-34].

Absence of asthma

Sputum production RR > 25 breaths/min Tº ≥ 37.8 °C or 100 °F

Heart rate > 100 beats/min Temperature > 37.8 °C or 100 °F Decreased breath sounds

Heart rate > 100 beats/min Respiratory rate > 20 breaths/min

Temperature > 37.8 °C

Probability = 1/(1 + e-Y)

Each variable = 1 if present

**Clinical prediction rules for pneumonia diagnosis in adults**

**Score** -2 points -1 point 1 point 1 point 1 point 2 points 2 points

**Gennis et al.** If one or more variables are present requesting chest radiograph.

**Melbye et al.** A logistic regression model is proposed for the diagnosis of pneumonia.

**González Ortiz et al.** A logistic regression model is proposed for the diagnosis of pneumonia.

**Hopstaken et al.** A logistic regression model is proposed for the diagnosis of pneumonia.

Y= + 4.7 for fever (reported by patient) with duration of illness of one week or more; – 4.5 for coryza; – 2.1 for sore

Y= –1.87; + 1.3 for pathologic auscultation; + 1.64 for neutrophilia; + 1.70 for pleural pain; + 1.21 for dyspnea.

**Table 3.** Clinical predictive rules for pneumonia diagnosed by chest radiography in the ambulatory care

Y= –4.15; + 0.91 for dry cough; + 1.01 for diarrhea; + 0.64 for temperature ≥ 38 °C; + 2.78 for C-reactive protein ≥ 20

In the Chilean study [29], the major clinical predictors of pneumonia were fever (≥38 °C), tachypnea (≥20 breaths/min), mental confusion, orthopnea, cyanosis, dullness on percus‐ sion, bronchophony, crackles and oxygen saturation less than 90% breathing room air (LR + : 2.0 to 9.5). In contrast, sore throat, runny nose, normal vital signs and lung ausculta‐ tion without clinical abnormalities were less frequent in patients with final diagnosis of pneumonia (LR+ : 0.3-0.5). Unlike other studies, Saldías et al. examined some combinations of symptoms and signs showing that significantly increases the pretest probability. The combination of clinical variables increase the probability of pneumonia, such as the pres‐ ence of fever and tachypnea associated with orthopnea, dullness on percussion, crackles or oxygen saturation below 90% (LR+ : 4.9 to 14.7). At the same time, the probability of pneumonia is very low in patients presenting with respiratory symptoms and normal vi‐ tal signs and lung auscultation (LR+ : 0.1).

In summary, individual symptoms and signs at presentation distinguish poorly between community-acquired pneumonia and other causes of respiratory illnesses [13,18,20]. Thus, in 10-20% of ambulatory patients with a suspected lower respiratory tract infection CXR is requested [19]. CXR can diagnose pneumonia in cases with the presence of pulmonary infil‐ trates and differentiate pneumonia from other conditions that may present with similar symptoms (e.g., acute bronchitis or influenza) [2,3]. In addition, the results may suggest spe‐ cific aetiologies (e.g., lung abscess, TB infection), identify coexisting conditions (e.g., bron‐ chial obstruction, pleural effusion, neoplasms) and evaluate the severity of illness (e.g., multilobar or bilateral infiltrates, rapid progression of infiltrates, pleural effusion) [6,8]. Al‐ though chest radiography is frequently used for diagnosing pneumonia, little is known about the influence of CXR on the probability estimation of pneumonia by general practi‐ tioners and on change in patient management [18,20]. Chest radiography is considered the gold standard for pneumonia diagnosis; however, we do not know its sensitivity and specif‐ icity, and we have limited data on the clinical implications of false-positive and false-nega‐ tive results [18]. In the absence of empirical evidence, the decision to order a chest radiograph needs to rely on expert opinion in seeking strategies to optimize the balance be‐ tween harms and benefits [16,17].

#### **2.3. Clinical judgment and decision guidelines**

Although physicians often planning the diagnostic and laboratory exams considering the prevalence of the disease and its estimate of the probability in the population being evaluat‐ ed, the diagnostic threshold of professionals varies considerably even when faced with simi‐ lar clinical situations [30,31]. As the predictive value of individual signs and symptoms to the diagnosis of pneumonia is relatively low, to resolve this problem, it has been designed some predictive rules or decision guidelines incorporating the presence or absence of specif‐ ic clinical findings intended to guide clinicians in the management of patients with similar clinical features [18,20]. In the literature we find several protocols or decision rules that are designed specifically to help primary care physicians in the assessment of adult patients with lower respiratory tract infections (Table 3) [25-29,32-34]. Thus, in the clinical practice guidelines is often recommended to primary care physicians request of chest radiography in patients presenting respiratory symptoms based on some of these decision rules, in order to optimize patient`s care [16,17,35].

shown that having a normal heart rate (below 100 beats/min) without fever (temperature < 37.8 °C) and tachypnea (respiratory rate < 20 breaths/min) reduces significantly the probabil‐

In the Chilean study [29], the major clinical predictors of pneumonia were fever (≥38 °C), tachypnea (≥20 breaths/min), mental confusion, orthopnea, cyanosis, dullness on percus‐ sion, bronchophony, crackles and oxygen saturation less than 90% breathing room air (LR

: 2.0 to 9.5). In contrast, sore throat, runny nose, normal vital signs and lung ausculta‐ tion without clinical abnormalities were less frequent in patients with final diagnosis of

of symptoms and signs showing that significantly increases the pretest probability. The combination of clinical variables increase the probability of pneumonia, such as the pres‐ ence of fever and tachypnea associated with orthopnea, dullness on percussion, crackles

pneumonia is very low in patients presenting with respiratory symptoms and normal vi‐

In summary, individual symptoms and signs at presentation distinguish poorly between community-acquired pneumonia and other causes of respiratory illnesses [13,18,20]. Thus, in 10-20% of ambulatory patients with a suspected lower respiratory tract infection CXR is requested [19]. CXR can diagnose pneumonia in cases with the presence of pulmonary infil‐ trates and differentiate pneumonia from other conditions that may present with similar symptoms (e.g., acute bronchitis or influenza) [2,3]. In addition, the results may suggest spe‐ cific aetiologies (e.g., lung abscess, TB infection), identify coexisting conditions (e.g., bron‐ chial obstruction, pleural effusion, neoplasms) and evaluate the severity of illness (e.g., multilobar or bilateral infiltrates, rapid progression of infiltrates, pleural effusion) [6,8]. Al‐ though chest radiography is frequently used for diagnosing pneumonia, little is known about the influence of CXR on the probability estimation of pneumonia by general practi‐ tioners and on change in patient management [18,20]. Chest radiography is considered the gold standard for pneumonia diagnosis; however, we do not know its sensitivity and specif‐ icity, and we have limited data on the clinical implications of false-positive and false-nega‐ tive results [18]. In the absence of empirical evidence, the decision to order a chest radiograph needs to rely on expert opinion in seeking strategies to optimize the balance be‐

Although physicians often planning the diagnostic and laboratory exams considering the prevalence of the disease and its estimate of the probability in the population being evaluat‐ ed, the diagnostic threshold of professionals varies considerably even when faced with simi‐ lar clinical situations [30,31]. As the predictive value of individual signs and symptoms to the diagnosis of pneumonia is relatively low, to resolve this problem, it has been designed some predictive rules or decision guidelines incorporating the presence or absence of specif‐ ic clinical findings intended to guide clinicians in the management of patients with similar

: 0.1).

: 0.3-0.5). Unlike other studies, Saldías et al. examined some combinations

= 0.18), thereby reducing the pretest odds of

: 4.9 to 14.7). At the same time, the probability of

ity of community-acquired pneumonia (LR-

pneumonia by more than fivefold [26,29].

104 Respiratory Disease and Infection - A New Insight

or oxygen saturation below 90% (LR+

tal signs and lung auscultation (LR+

tween harms and benefits [16,17].

**2.3. Clinical judgment and decision guidelines**

+

pneumonia (LR+


**Table 3.** Clinical predictive rules for pneumonia diagnosed by chest radiography in the ambulatory care setting[25-28,32-34].

Prediction rules based on clinical information have been developed to support the diagnosis of pneumonia and help limit the use of expensive diagnostic tests [36,37]. However, these prediction rules need to be validated in the primary care setting. Several clinical prediction rules have been developed to predict pneumonia in adults but they were not superior to clinical judgment to predict pneumonia in the ambulatory setting [20,36,37]. In summary, combination of history and physical examination findings at presentation only moderately increase the probability of pneumonia. Thus, the clinical guidelines endorse the need for chest radiography to confirm all diagnoses of community-acquired pneumonia [16,17,35].

emergency physicians ranged between 1.5 and 4.8. Similar findings were described by Wipf et al. [39], who determined the accuracy of various physical examination maneuvers in diag‐ nosing pneumonia and compared the interobserver reliability of the maneuvers among three examiners. The authors concluded that the clinical findings investigated in chest ex‐ amination does not confirm or exclude with certainty the diagnosis of pneumonia, and the degree of interobserver agreement was highly variable for different physical examination

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**Variables Clinical diagnosis Diehr et al Singal et al Heckerling et al Gennis et al**

**Sensitivity** 0.79 (0.72-0.84) 0.77 (0.70-0.83) 0.76 (0.69-0.82) 0.84 (0.78-0.90) 0.92 (0.87-0.96) **Specificity** 0.66 (0.63-0.69) 0.64 (0.61-0.68) 0.54 (0.51-0.58) 0.41\*(0.38-0.44) 0.31\*(0.28-0.33) **PPV** 0.55 (0.50-0.59) 0.54 (0.49-0.58) 0.46 (0.42-0.50) 0.43 (0.39-0,45) 0.42 (0.39-0.44) **NPV** 0.85 (0.81-0.89) 0.84 (0.79-0.88) 0.81 (0.76-0.86) 0.84 (0.77-0.89) 0.88 (0.80-0.94) **Accuracy** 0.70 (0.65-0.75) 0.69 (0.67-0.74) 0.62 (0.57-0.67) 0.56\*(0.50-0.62) 0.53\*(0.47-0.59) **LR+** 2.3 (1.9-2.7) 2.2 (1.8-2.6) 1.7 (1.4-1.9) 1.4\* (1.2-1.6) 1.4\* (1.2-1.4)

Note: 95% CI: confidence interval of 95%, PPV: positive predictive value; NPV: negative predictive value. \* p <0.05

**Table 4.** Predictive value of clinical judgment and four predictive rules in the diagnosis of community-acquired

**Clinical diagnosis Sensitivity Specificity PPV NPV LR+**

**Physician A 83% 83% 70% 91% 4.8**

**Physician B 75% 73% 56% 86% 2.8**

**Physician C 77% 47% 36% 85% 1.5**

**Average 79% 66% 55% 85% 2.3**

practice. Physician C was an emergency medicine specialist with less than three years of clinical practice.

ratio (LR+) for clinical diagnosis of community-acquired pneumonia[29].

Note: Physicians A and B were specialists in internal medicine and respiratory disease over five years of professional

**Table 5.** Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and positive likelihood

Lieberman et al. [40] evaluated the reliability of physicians' judgement relating to the pres‐ ence of pneumonia in adult patients with acute respiratory symptoms by clinical assessment alone compared with chest X-ray. Physicians' judgements of pneumonia had a sensitivity of 74% (95% CI 49-90%), a specificity of 84% (95% CI 78-88%), a NPV of 97% (95% CI 94-99%)

%, (95% CI) %, (95% CI) %, (95% CI) %, (95% CI) %, (95% CI)

findings.

compared with clinical judgment.

pneumonia in adults[38].

In two predictive rules, described by Diehr et al. [25] and Heckerling et al. [28], the pretest probability of pneumonia is amended according to the presence or absence of certain specif‐ ic symptoms. While other rule, described by Singal et al. [27], was designed using a logistic regression analysis and provides a probability of pneumonia ranging from 4% (absence of symptoms and signs) to 49% (presence of cough, fever and crackles). On the other hand, Gennis et al. [26] suggested applying chest radiograph in patients with suspected of CAP and alteration in any vital signs (heart rate above 100 beats/min, respiratory rate above 20 breaths/min or temperature higher than 37.8 °C). Melbye et al [32], González Ortiz et al. [33] and Hospstaken et al. [34] proposed a logistic regression model for diagnosis of pneumonia based on clinical and laboratory variables.

Two prospective studies have examined the predictive value of clinical judgment and four clinical predictive rules in the diagnosis of community-acquired pneumonia in adults [37,38]. Emerman et al. [37] compared physician judgment in the use of chest radiographs for diagnosing pneumonia with decision rules developed by Diehr, Singal, Heckerling, and Gennis in the emergency department and medical outpatient clinic of a major urban teach‐ ing hospital. The prevalence of pneumonia in this study was 7%, they found that clinical judgment allowed to reduce the application of unnecessary chest radiographs better than the four predictive rules (LR- = 0.25, 95%CI: 0.09 to 0.61) while clinical judgment allowed to increase the likelihood of pneumonia to around 13% (LR+ = 2.0, 95%CI: 1.5 to 2.4), which would have led to demand many unnecessary radiographic examinations. Among the four examined predictive rules, the application of x-ray only to patients with abnormal vital signs recommended by Gennis et al. [26] had higher diagnostic yield with a LR+ of 2.6 (95%CI: 1.6 to 3.7), which would have reduced by 40% the application of unnecessary radio‐ graphic examinations but would not have detected 38% of pneumonias confirmed by radiol‐ ogy (LR- = 0.50, 95%CI: 0.27 to 0.78 compared with LR- = 0.18 of the original study of Gennis et al.) [34]. The sensitivity of physician judgment (86%) exceeded that of all four decision rules (62-76%), but the higher specificity and accuracy of two of the decision rules [26,28] suggested that they may have a role in patient evaluation.

Saldías et al. [29,38] have shown that clinical diagnosis of pneumonia made by physicians in the emergency department had better sensitivity (range: 75-83%) than specificity (range: 47-83%) and better negative predictive value (NPV) (range: 85-91%) than PPV (range: 36-70%) (Table 4). In fact, a less experienced emergency physician had lower PPV and spe‐ cificity compared to internal medicine and respiratory disease physicians (Table 5). The chance to change the initial diagnosis of pneumonia or positive likelihood ratio of three emergency physicians ranged between 1.5 and 4.8. Similar findings were described by Wipf et al. [39], who determined the accuracy of various physical examination maneuvers in diag‐ nosing pneumonia and compared the interobserver reliability of the maneuvers among three examiners. The authors concluded that the clinical findings investigated in chest ex‐ amination does not confirm or exclude with certainty the diagnosis of pneumonia, and the degree of interobserver agreement was highly variable for different physical examination findings.

Prediction rules based on clinical information have been developed to support the diagnosis of pneumonia and help limit the use of expensive diagnostic tests [36,37]. However, these prediction rules need to be validated in the primary care setting. Several clinical prediction rules have been developed to predict pneumonia in adults but they were not superior to clinical judgment to predict pneumonia in the ambulatory setting [20,36,37]. In summary, combination of history and physical examination findings at presentation only moderately increase the probability of pneumonia. Thus, the clinical guidelines endorse the need for chest radiography to confirm all diagnoses of community-acquired pneumonia [16,17,35].

In two predictive rules, described by Diehr et al. [25] and Heckerling et al. [28], the pretest probability of pneumonia is amended according to the presence or absence of certain specif‐ ic symptoms. While other rule, described by Singal et al. [27], was designed using a logistic regression analysis and provides a probability of pneumonia ranging from 4% (absence of symptoms and signs) to 49% (presence of cough, fever and crackles). On the other hand, Gennis et al. [26] suggested applying chest radiograph in patients with suspected of CAP and alteration in any vital signs (heart rate above 100 beats/min, respiratory rate above 20 breaths/min or temperature higher than 37.8 °C). Melbye et al [32], González Ortiz et al. [33] and Hospstaken et al. [34] proposed a logistic regression model for diagnosis of pneumonia

Two prospective studies have examined the predictive value of clinical judgment and four clinical predictive rules in the diagnosis of community-acquired pneumonia in adults [37,38]. Emerman et al. [37] compared physician judgment in the use of chest radiographs for diagnosing pneumonia with decision rules developed by Diehr, Singal, Heckerling, and Gennis in the emergency department and medical outpatient clinic of a major urban teach‐ ing hospital. The prevalence of pneumonia in this study was 7%, they found that clinical judgment allowed to reduce the application of unnecessary chest radiographs better than

would have led to demand many unnecessary radiographic examinations. Among the four examined predictive rules, the application of x-ray only to patients with abnormal vital signs recommended by Gennis et al. [26] had higher diagnostic yield with a LR+

(95%CI: 1.6 to 3.7), which would have reduced by 40% the application of unnecessary radio‐ graphic examinations but would not have detected 38% of pneumonias confirmed by radiol‐ ogy (LR- = 0.50, 95%CI: 0.27 to 0.78 compared with LR- = 0.18 of the original study of Gennis et al.) [34]. The sensitivity of physician judgment (86%) exceeded that of all four decision rules (62-76%), but the higher specificity and accuracy of two of the decision rules [26,28]

Saldías et al. [29,38] have shown that clinical diagnosis of pneumonia made by physicians in the emergency department had better sensitivity (range: 75-83%) than specificity (range: 47-83%) and better negative predictive value (NPV) (range: 85-91%) than PPV (range: 36-70%) (Table 4). In fact, a less experienced emergency physician had lower PPV and spe‐ cificity compared to internal medicine and respiratory disease physicians (Table 5). The chance to change the initial diagnosis of pneumonia or positive likelihood ratio of three

= 0.25, 95%CI: 0.09 to 0.61) while clinical judgment allowed to

= 2.0, 95%CI: 1.5 to 2.4), which

of 2.6

based on clinical and laboratory variables.

106 Respiratory Disease and Infection - A New Insight

increase the likelihood of pneumonia to around 13% (LR+

suggested that they may have a role in patient evaluation.

the four predictive rules (LR-


Note: 95% CI: confidence interval of 95%, PPV: positive predictive value; NPV: negative predictive value. \* p <0.05 compared with clinical judgment.

**Table 4.** Predictive value of clinical judgment and four predictive rules in the diagnosis of community-acquired pneumonia in adults[38].


Note: Physicians A and B were specialists in internal medicine and respiratory disease over five years of professional practice. Physician C was an emergency medicine specialist with less than three years of clinical practice.

**Table 5.** Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and positive likelihood ratio (LR+) for clinical diagnosis of community-acquired pneumonia[29].

Lieberman et al. [40] evaluated the reliability of physicians' judgement relating to the pres‐ ence of pneumonia in adult patients with acute respiratory symptoms by clinical assessment alone compared with chest X-ray. Physicians' judgements of pneumonia had a sensitivity of 74% (95% CI 49-90%), a specificity of 84% (95% CI 78-88%), a NPV of 97% (95% CI 94-99%) and a PPV of only 27% (95% CI 16-42%). They concluded that the ability of physicians to negate radiologically confirmed pneumonia by clinical assessment in febrile adult respirato‐ ry tract infection patients was good, but that their ability to successfully predict this condi‐ tion was poor.

hours. A negative sputum or blood culture in a patient suspected of having community-ac‐ quired pneumonia does not rule out the possibility of a severe lower respiratory tract infec‐ tion [41]. Given these areas of uncertainty in clinical decision-making, a concerted effort has been undertaken to develop reliable and practical biomarkers for the diagnosis, risk predic‐ tion and management of CAP. To be helpful in routine clinical practice, a biomarker should provide additional actionable information – not already available by standard methods – that accomplishes at least one or more of the following: a) Assists in establishing a rapid and reliable diagnosis; b) Provides an indication of prognosis; c) Selects those patients most like‐ ly to benefit from a specific intervention; d) Reflects the efficacy or lack of efficacy of specific

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109

The usefulness of biomarkers for diagnosing lower respiratory tract infections (LRTI), and identifying particular disease entities amongst LRTIs (e.g., acute bronchitis, influenza,

Some observational studies indicate that C-reactive protein (CRP) may have some role in identifying patients with community-acquired pneumonia. Almirall et al. found significant‐ ly higher CRP values in adult patients with confirmed CAP compared to healthy controls and suspected CAP [42]. Flanders et al. evaluated CRP as a possible tool in the differential diagnosis of 168 adults with acute cough less than three weeks [43]. CRP levels correlated with the presence of pneumonia but not with its severity. A serum CRP level over 40 mg/L had a sensitivity of 70% and specificity of 90% to identify pneumonia. Holm et al. confirmed the low sensitivity (73%) and specificity (65%) of serum CRP in the differential diagnosis of LRTIs [44]. The authors concluded that only very high CRP levels (>100 mg/L) can be used as indicator for the presence of CAP. Accordingly, Stolz et al. showed that the specificity of CRP at the cut-off value of 100 mg/L to predict radiological confirmed pneumonia reaches

Falk et al. [46] assessed the diagnostic value of CRP in primary care and emergency depart‐ ments in terms of ruling in or ruling out CAP. Eight studies incorporating 2,194 patients were included. The median prevalence of CAP was 14.6% (range 5%-89%). At a CRP cut-

negative LR– was 0.33 (95% CI 0.25-0.43). In conclusion, CRP may be of value in ruling out a diagnosis of CAP in situations where pneumonia probability exceeds 10%, typically in emer‐ gency departments. However, in primary care services, additional diagnostic testing with CRP is unlikely to alter the probability of CAP sufficiently to change subsequent manage‐

Procalcitonin (PCT) is a 116 amino acid protein, precursor of calcitonin, which is physiologi‐ cally produced by the C-cells of the thyroid after intracellular processing of the prohormone. The half-life of PCT is around 20-24 h and the plasma concentration in healthy individuals is

was 2.1 (95%CI 1.8-2.4] and the pooled

COPD exacerbation, pneumonia) is still a matter of controversy.

point of less than 20 mg/L, the pooled positive LR+

ment decisions such as antibiotic prescribing or referral to hospital.

interventions.

91.2% [45].

*2.4.2. Procalcitonin*

*2.4.1. C- reactive protein*

In other study developed in two emergency departments from Madrid, Spain; Gonzalez et al. [33] showed that the clinical judgment had low sensitivity for the diagnosis of pneumo‐ nia (45%) with a moderate PPV (80%). The sensitivity and specificity of clinical diagnosis of pneumonia established by emergency medicine physicians were similar or slightly higher compared with the four clinical predictive rules described in the literature [37,38]. The area under the curve (AUC) of clinical judgment and the clinical decision rule described by Diehr et al. for diagnosis of pneumonia were similar (AUC = 0.79 and 0.75, respectively), and both were higher than those described by Heckerling (AUC = 0.70), Singal (AUC = 0.70) and Gen‐ nis et al. (AUC = 0.67) [38].

In summary, the clinical findings (history and physical examination) have only moderate sensitivity and specificity for diagnosis of pneumonia in immunocompetent adult patients presenting with fever or respiratory symptoms in the ambulatory care setting (Table 6). None of the decision rules described in the literature have been superior to clinical judgment in the diagnosis of pneumonia, yet no studies have examined its real contribution in the evaluation and management of patients presenting with respiratory symptoms or fever in the primary care services.


Note: PPV: positive predictive value, NPV: negative predictive value, LR: likelihood ratio.

**Table 6.** Predictive value of clinical judgment in diagnosing community-acquired pneumonia in adults[29,37,39-41].

#### **2.4. Biomarkers and lower respiratory tract infection diagnosis**

Numerous non-infectious processes can produce respiratory symptoms and new pulmonary infiltrates with systemic inflammatory signs and symptoms that can be easily confused with bacterial pneumonia (Table 1). Typically, Gram stains of respiratory secretions are often un‐ available or are difficult to evaluate, and microbiological culture reports take at least 24 to 48 hours. A negative sputum or blood culture in a patient suspected of having community-ac‐ quired pneumonia does not rule out the possibility of a severe lower respiratory tract infec‐ tion [41]. Given these areas of uncertainty in clinical decision-making, a concerted effort has been undertaken to develop reliable and practical biomarkers for the diagnosis, risk predic‐ tion and management of CAP. To be helpful in routine clinical practice, a biomarker should provide additional actionable information – not already available by standard methods – that accomplishes at least one or more of the following: a) Assists in establishing a rapid and reliable diagnosis; b) Provides an indication of prognosis; c) Selects those patients most like‐ ly to benefit from a specific intervention; d) Reflects the efficacy or lack of efficacy of specific interventions.

The usefulness of biomarkers for diagnosing lower respiratory tract infections (LRTI), and identifying particular disease entities amongst LRTIs (e.g., acute bronchitis, influenza, COPD exacerbation, pneumonia) is still a matter of controversy.

#### *2.4.1. C- reactive protein*

and a PPV of only 27% (95% CI 16-42%). They concluded that the ability of physicians to negate radiologically confirmed pneumonia by clinical assessment in febrile adult respirato‐ ry tract infection patients was good, but that their ability to successfully predict this condi‐

In other study developed in two emergency departments from Madrid, Spain; Gonzalez et al. [33] showed that the clinical judgment had low sensitivity for the diagnosis of pneumo‐ nia (45%) with a moderate PPV (80%). The sensitivity and specificity of clinical diagnosis of pneumonia established by emergency medicine physicians were similar or slightly higher compared with the four clinical predictive rules described in the literature [37,38]. The area under the curve (AUC) of clinical judgment and the clinical decision rule described by Diehr et al. for diagnosis of pneumonia were similar (AUC = 0.79 and 0.75, respectively), and both were higher than those described by Heckerling (AUC = 0.70), Singal (AUC = 0.70) and Gen‐

In summary, the clinical findings (history and physical examination) have only moderate sensitivity and specificity for diagnosis of pneumonia in immunocompetent adult patients presenting with fever or respiratory symptoms in the ambulatory care setting (Table 6). None of the decision rules described in the literature have been superior to clinical judgment in the diagnosis of pneumonia, yet no studies have examined its real contribution in the evaluation and management of patients presenting with respiratory symptoms or fever in

**Clinical diagnosis Sensitivity Specificity PPV NPV LR+\_**

**González et al. 45% 93% 80% 74% 6.6**

**Wipf et al. 47-69% 58-75% 48-64% 57-72% 1.1-2.0**

**Emerman et al. 86% 58% 14% 98% 2.0**

**Lieberman et al. 49-90% 78-88% 16-42% 94-99% \_\_\_\_**

**Saldías et al. 75-83% 47-83% 36-70% 85-91% 1.5-4.8**

**Table 6.** Predictive value of clinical judgment in diagnosing community-acquired pneumonia in adults[29,37,39-41].

Numerous non-infectious processes can produce respiratory symptoms and new pulmonary infiltrates with systemic inflammatory signs and symptoms that can be easily confused with bacterial pneumonia (Table 1). Typically, Gram stains of respiratory secretions are often un‐ available or are difficult to evaluate, and microbiological culture reports take at least 24 to 48

Note: PPV: positive predictive value, NPV: negative predictive value, LR: likelihood ratio.

**2.4. Biomarkers and lower respiratory tract infection diagnosis**

tion was poor.

nis et al. (AUC = 0.67) [38].

108 Respiratory Disease and Infection - A New Insight

the primary care services.

Some observational studies indicate that C-reactive protein (CRP) may have some role in identifying patients with community-acquired pneumonia. Almirall et al. found significant‐ ly higher CRP values in adult patients with confirmed CAP compared to healthy controls and suspected CAP [42]. Flanders et al. evaluated CRP as a possible tool in the differential diagnosis of 168 adults with acute cough less than three weeks [43]. CRP levels correlated with the presence of pneumonia but not with its severity. A serum CRP level over 40 mg/L had a sensitivity of 70% and specificity of 90% to identify pneumonia. Holm et al. confirmed the low sensitivity (73%) and specificity (65%) of serum CRP in the differential diagnosis of LRTIs [44]. The authors concluded that only very high CRP levels (>100 mg/L) can be used as indicator for the presence of CAP. Accordingly, Stolz et al. showed that the specificity of CRP at the cut-off value of 100 mg/L to predict radiological confirmed pneumonia reaches 91.2% [45].

Falk et al. [46] assessed the diagnostic value of CRP in primary care and emergency depart‐ ments in terms of ruling in or ruling out CAP. Eight studies incorporating 2,194 patients were included. The median prevalence of CAP was 14.6% (range 5%-89%). At a CRP cutpoint of less than 20 mg/L, the pooled positive LR+ was 2.1 (95%CI 1.8-2.4] and the pooled negative LR– was 0.33 (95% CI 0.25-0.43). In conclusion, CRP may be of value in ruling out a diagnosis of CAP in situations where pneumonia probability exceeds 10%, typically in emer‐ gency departments. However, in primary care services, additional diagnostic testing with CRP is unlikely to alter the probability of CAP sufficiently to change subsequent manage‐ ment decisions such as antibiotic prescribing or referral to hospital.

#### *2.4.2. Procalcitonin*

Procalcitonin (PCT) is a 116 amino acid protein, precursor of calcitonin, which is physiologi‐ cally produced by the C-cells of the thyroid after intracellular processing of the prohormone. The half-life of PCT is around 20-24 h and the plasma concentration in healthy individuals is typically below 0.1 µg/L. In some studies procalcitonin does not appear to be a significant marker for CAP [47,48]. However, a more recent evaluation of the role of highly sensitive CRP and PCT measurements showed a better discriminatory value of these biomarkers compared to clinical signs [49]. The diagnostic accuracy of clinical signs and symptoms and a range of laboratory markers were assessed in 545 patients with suspected lower respirato‐ ry tract infection admitted to the emergency department. The area under the curve of a clini‐ cal model including fever, cough, sputum production, abnormal chest auscultation and dyspnea was 0.79 (95%CI, 0.75-0.83). Combining the values for procalcitonin and highly sen‐ sitive C-reactive protein together increased the AUC to 0.92 (95%CI, 0.89-0.94), which was significantly better than the AUC for PCT, CRP and clinical signs and symptoms alone. The contribution to diagnostic reliability made by PCT was substantially greater than that made by CRP, which in turn performed better than the total leukocyte count. Clinical criteria such as sputum production and physical examination with chest auscultation were poor predic‐ tors for the diagnosis of CAP. To predict bacteremia, PCT also had a higher AUC (0.85, 95%CI 0.80-0.91) as compared to CRP (0.71, 95%CI 0.62-0.80; p =0.01), leukocyte count (0.68, 95%CI 0.59-0.77; p = 0.002) and elevated body temperature (0.46, 95%CI 0.37-0.56; p < 0.001). The added value of the PCT biomarker as a clinical decision-making tool has been evi‐ denced in several studies involving PCT measurement [50-57].

setting, measurement of soluble triggering receptor expressed on myeloid cells-1 in plasma

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The first study on 148 patients suffering from suspected CAP or VAP and receiving mechan‐ ical ventilation, sTREM was assessed in the BAL fluid and its levels were a better predictor for bacterial infection than CPIS, TNF-alfa and IL-1 levels [58]. The authors also analyzed the behavior of PCT and did not find any role for this biomarker in identifying pneumonia. An‐ other group evaluated the presence of sTREM in exhaled ventilator condensate (EVC) and in BAL fluid from 23 patients clinically suspected of having VAP [60]. In contrast with the first report, sTREM-1 was detected in the BAL fluid of all 14 VAP subjects but also in 8 of 9 sub‐ jects without pneumonia, and sTREM levels did not differ in the VAP subjects compared to the non-pneumonia subjects. However, sTREM-1 was detected in the EVC from 11 of 14 subjects with VAP, but from only 1 of 9 subjects without VAP, and was significantly higher in the pneumonia patients. Another study tends to rule out the value of sTREM detection in BAL as a useful tool in VAP diagnosis [62]. In this study, 105 consecutive patients receiving mechanical ventilation and undergoing BAL were enrolled. Of those, 19 patients (18.1%) met definite microbiologic criteria for bacterial or fungal VAP. All the statistical analysis performed showed that measurement of sTREM-1 was inferior to clinical parameters for the

In patients with community-acquired pneumonia, traditional criteria of infection based on clinical signs and symptoms, clinical scoring systems, and general inflammatory indicators (for example, leukocytosis, fever, sputum and blood cultures) are often of limited clinical value and remain an unreliable guide to diagnosis lower respiratory tract infections. Procal‐ citonin is superior to other commonly used markers in its specificity for bacterial infection, allowing alternative diagnoses to be excluded, mainly as a guide to the necessity for antibi‐ otic therapy [63-65]. It can therefore be viewed as a diagnostic and prognostic test. It more closely matches the criteria for usefulness than other candidate biomarkers such as C-reac‐ tive protein, which is rather a nonspecific marker of acute phase inflammation, and proin‐ flammatory cytokines such as plasma IL-6 levels that are highly variable, cumbersome to

The clinical presentation of pneumonia in the elderly may be subtle, lacking the typical acute symptoms observed in younger adults. Riquelme et al. [66] reported the initial clinical presentation of 101 elderly patients with CAP (mean age, 78 years; 66.3% men) who were admitted to a 1000-bed teaching hospital in Barcelona, Spain. The most frequently observed symptoms were dyspnea (72.3%), cough (66.3%), fever (63.4%), asthenia (57.4%), purulent sputum (51.5%), anorexia (49.5%), altered mental status (44.6%), and pleuritic chest pain (33.7%). The classic triad of symptoms of pneumonia − cough, dyspnea, and fever − was ob‐ served in only 30.7% of these elderly patients. Nineteen patients (18.8%) did not have cough, purulent sputum, or pleuritic chest pain. In a prospective study [67] designed to assess the clinical characteristics of 503 elderly patients (mean age, 76.3 years; 63.4% men) admitted for CAP to 16 hospitals across Spain, the most frequently observed symptoms were cough

or serum has proved unhelpful as a guide to either etiology or outcome [61].

diagnosis of VAP.

**2.5. Pneumonia in elderly**

measure, and lack specificity for systemic infection.

Circulating levels of the precursor hormone PCT, derived primarily from nonthyroidal tis‐ sues, can rise several thousand times above normal in various inflammatory conditions, but most notably in bacterial infection [54]. In differentiating bacterial infection from non-infec‐ tive causes of inflammation in hospitalized patients, a meta-analysis concluded that PCT was both more sensitive (85% vs. 78%) and more specific (83% vs. 60%) compared with CRP. PCT was also more sensitive (92% vs. 86%) in differentiating between a bacterial etiology and a viral etiology [55].

*PCT and ventilator-associated pneumonia:* The utility of PCT levels to improve the early diag‐ nosis of ventilator-associated pneumonia (VAP) has been evaluated in different studies. Due to the use of dissimilar thresholds the results were not consistent [56-58]. Ramirez et al. re‐ port a cohort study with sequential measurement of PCT and CRP in well characterized pa‐ tients with VAP [59]. The results of this study showed that PCT and CRP plasma levels were statistically higher in patients with confirmed VAP, PCT being the more accurate marker. The combination of the simplified clinical pulmonary infection score (CPIS) and serum PCT was able to exclude all false-positive diagnosis of VAP thus resulting in 100% specificity.

#### *2.4.3. Soluble triggering receptor expressed by myeloid cell*

The soluble triggering receptor expressed by myeloid cells-1 (sTREM) has been proposed as another potentially useful diagnostic tool for CAP and VAP [58, 60-62]. The sTREM is upre‐ gulated by microbial products, it accurately identifies bacterial or fungal pneumonia in bronchoalveolar lavage fluid (BAL) from mechanically ventilated patients, and is superior in this regard to clinical findings or other laboratory values. Such intervention is not appropri‐ ate, however, in the routine care of patients with community-acquired pneumonia. In this setting, measurement of soluble triggering receptor expressed on myeloid cells-1 in plasma or serum has proved unhelpful as a guide to either etiology or outcome [61].

The first study on 148 patients suffering from suspected CAP or VAP and receiving mechan‐ ical ventilation, sTREM was assessed in the BAL fluid and its levels were a better predictor for bacterial infection than CPIS, TNF-alfa and IL-1 levels [58]. The authors also analyzed the behavior of PCT and did not find any role for this biomarker in identifying pneumonia. An‐ other group evaluated the presence of sTREM in exhaled ventilator condensate (EVC) and in BAL fluid from 23 patients clinically suspected of having VAP [60]. In contrast with the first report, sTREM-1 was detected in the BAL fluid of all 14 VAP subjects but also in 8 of 9 sub‐ jects without pneumonia, and sTREM levels did not differ in the VAP subjects compared to the non-pneumonia subjects. However, sTREM-1 was detected in the EVC from 11 of 14 subjects with VAP, but from only 1 of 9 subjects without VAP, and was significantly higher in the pneumonia patients. Another study tends to rule out the value of sTREM detection in BAL as a useful tool in VAP diagnosis [62]. In this study, 105 consecutive patients receiving mechanical ventilation and undergoing BAL were enrolled. Of those, 19 patients (18.1%) met definite microbiologic criteria for bacterial or fungal VAP. All the statistical analysis performed showed that measurement of sTREM-1 was inferior to clinical parameters for the diagnosis of VAP.

In patients with community-acquired pneumonia, traditional criteria of infection based on clinical signs and symptoms, clinical scoring systems, and general inflammatory indicators (for example, leukocytosis, fever, sputum and blood cultures) are often of limited clinical value and remain an unreliable guide to diagnosis lower respiratory tract infections. Procal‐ citonin is superior to other commonly used markers in its specificity for bacterial infection, allowing alternative diagnoses to be excluded, mainly as a guide to the necessity for antibi‐ otic therapy [63-65]. It can therefore be viewed as a diagnostic and prognostic test. It more closely matches the criteria for usefulness than other candidate biomarkers such as C-reac‐ tive protein, which is rather a nonspecific marker of acute phase inflammation, and proin‐ flammatory cytokines such as plasma IL-6 levels that are highly variable, cumbersome to measure, and lack specificity for systemic infection.

#### **2.5. Pneumonia in elderly**

typically below 0.1 µg/L. In some studies procalcitonin does not appear to be a significant marker for CAP [47,48]. However, a more recent evaluation of the role of highly sensitive CRP and PCT measurements showed a better discriminatory value of these biomarkers compared to clinical signs [49]. The diagnostic accuracy of clinical signs and symptoms and a range of laboratory markers were assessed in 545 patients with suspected lower respirato‐ ry tract infection admitted to the emergency department. The area under the curve of a clini‐ cal model including fever, cough, sputum production, abnormal chest auscultation and dyspnea was 0.79 (95%CI, 0.75-0.83). Combining the values for procalcitonin and highly sen‐ sitive C-reactive protein together increased the AUC to 0.92 (95%CI, 0.89-0.94), which was significantly better than the AUC for PCT, CRP and clinical signs and symptoms alone. The contribution to diagnostic reliability made by PCT was substantially greater than that made by CRP, which in turn performed better than the total leukocyte count. Clinical criteria such as sputum production and physical examination with chest auscultation were poor predic‐ tors for the diagnosis of CAP. To predict bacteremia, PCT also had a higher AUC (0.85, 95%CI 0.80-0.91) as compared to CRP (0.71, 95%CI 0.62-0.80; p =0.01), leukocyte count (0.68, 95%CI 0.59-0.77; p = 0.002) and elevated body temperature (0.46, 95%CI 0.37-0.56; p < 0.001). The added value of the PCT biomarker as a clinical decision-making tool has been evi‐

Circulating levels of the precursor hormone PCT, derived primarily from nonthyroidal tis‐ sues, can rise several thousand times above normal in various inflammatory conditions, but most notably in bacterial infection [54]. In differentiating bacterial infection from non-infec‐ tive causes of inflammation in hospitalized patients, a meta-analysis concluded that PCT was both more sensitive (85% vs. 78%) and more specific (83% vs. 60%) compared with CRP. PCT was also more sensitive (92% vs. 86%) in differentiating between a bacterial etiology

*PCT and ventilator-associated pneumonia:* The utility of PCT levels to improve the early diag‐ nosis of ventilator-associated pneumonia (VAP) has been evaluated in different studies. Due to the use of dissimilar thresholds the results were not consistent [56-58]. Ramirez et al. re‐ port a cohort study with sequential measurement of PCT and CRP in well characterized pa‐ tients with VAP [59]. The results of this study showed that PCT and CRP plasma levels were statistically higher in patients with confirmed VAP, PCT being the more accurate marker. The combination of the simplified clinical pulmonary infection score (CPIS) and serum PCT was able to exclude all false-positive diagnosis of VAP thus resulting in 100% specificity.

The soluble triggering receptor expressed by myeloid cells-1 (sTREM) has been proposed as another potentially useful diagnostic tool for CAP and VAP [58, 60-62]. The sTREM is upre‐ gulated by microbial products, it accurately identifies bacterial or fungal pneumonia in bronchoalveolar lavage fluid (BAL) from mechanically ventilated patients, and is superior in this regard to clinical findings or other laboratory values. Such intervention is not appropri‐ ate, however, in the routine care of patients with community-acquired pneumonia. In this

denced in several studies involving PCT measurement [50-57].

*2.4.3. Soluble triggering receptor expressed by myeloid cell*

and a viral etiology [55].

110 Respiratory Disease and Infection - A New Insight

The clinical presentation of pneumonia in the elderly may be subtle, lacking the typical acute symptoms observed in younger adults. Riquelme et al. [66] reported the initial clinical presentation of 101 elderly patients with CAP (mean age, 78 years; 66.3% men) who were admitted to a 1000-bed teaching hospital in Barcelona, Spain. The most frequently observed symptoms were dyspnea (72.3%), cough (66.3%), fever (63.4%), asthenia (57.4%), purulent sputum (51.5%), anorexia (49.5%), altered mental status (44.6%), and pleuritic chest pain (33.7%). The classic triad of symptoms of pneumonia − cough, dyspnea, and fever − was ob‐ served in only 30.7% of these elderly patients. Nineteen patients (18.8%) did not have cough, purulent sputum, or pleuritic chest pain. In a prospective study [67] designed to assess the clinical characteristics of 503 elderly patients (mean age, 76.3 years; 63.4% men) admitted for CAP to 16 hospitals across Spain, the most frequently observed symptoms were cough (80.9%), fever (75.5%), dyspnea (69.8%), sputum production (65.8%), chills (53.1%), pleuritic chest pain (43.3%), asthenia (38.6%), and altered mental status (25.8%). The typical constella‐ tion of symptoms of pneumonia (cough, purulent sputum, and pleural pain) was noted in only 152 patients (30.2%). Metlay et al. [68] studied the influence of age on symptoms at pre‐ sentation in 1,812 patients with CAP. Cough, dyspnea, and pleuritic chest pain were signifi‐ cantly less common among elderly patients than among younger patients (81.7% vs. 87.8%, 68.8% vs. 73.9%, and 31.6% vs. 53.3%, respectively). After controlling for patient demo‐ graphics, comorbidities, and severity of illness, elderly patients exhibited significantly fewer respiratory symptoms than did younger patients (respiratory symptom score, 7.21 vs. 9.79, respectively; p < 0.01). In other study, altered mental status and the absence of fever were observed more frequently in patients over 80 years of age than in those under 80 years of age (21.0% vs. 10.7%; p < 0.001 and 32.1% vs. 21.9%; p < 0.001, respectively). These findings are consistent with Saldías et al. study [69], which reported a higher incidence of dyspnea and confusion (71% vs. 53%; p < 0.001 and 28% vs. 8%; p< 0.001, respectively) but a lower incidence of fever, chills and pleuritic chest pain in the older patients (63% vs. 76%; p = 0.007, 21% vs. 41%; p < 0.001 and 12% vs. 32%; p< 0.001, respectively). Compared to adults below 65 years of age hospitalized for CAP during the same period, the following clinical findings were more prevalent among the elderly population: the presence of comorbidity, dyspnea, decreased level of consciousness, suspected aspiration, hypoxemia and high serum urea nitrogen on admission to hospital. In the elderly, admission to intermediate and inten‐ sive care units was more frequent (47.7% vs. 29.2%, p< 0.001), and the length of hospital stay was longer (10.6 vs. 8.6 days, p= 0.03). Multilobar radiographic involvement, pleural effu‐ sion, the hospital complication rate and the need for mechanical ventilation were similar in both groups, but mortality, both in-hospital and at 30-days follow-up, was higher in the eld‐ erly population (9.8% vs. 3.2%; p= 0.03 and 13.1% vs. 4.8%; p= 0.02, respectively). Further‐ more, it has been suggested that the local inflammatory response to infection of the lungs is decreased in the elderly, resulting in less cough and sputum production [70]. The systemic inflammatory response (e.g., fever, leukocytosis, high serum C-reactive protein) is also re‐ duced secondary to decreased production of cytokines. Nevertheless, the decrease in inter‐ leukin-6 (IL-6), the most prevalent mediator of fever, did not reach statistical significance in a study that measured IL-6 levels in 59 elderly patients and 21 younger patients with CAP (211.6 vs. 284.5 pg/mL, respectively) [71]. In contrast, tachypnea remains prevalent and ap‐ pears to be a sensitive indicator of lower respiratory tract infection in the elderly [69,72]. Al‐ tered mental status, confusion, a sudden decline in functional physical capacity, and comorbidity decompensation may be the only symptoms of an infection (including pneumo‐ nia) in the elderly [66]. Clinicians should be cognizant of those symptoms to avoid delay in establishing the diagnosis and initiating antibiotic therapy.

ly on clinical criteria is also hampered by the large variability in the ability to detect focal signs on chest examination between different examiners. Pneumonia remains foremost a clinical diagnosis. However, symptoms of lower respiratory infection, including fever, cough, purulent sputum, dyspnea, and pleuritic chest pain as well as the clinical findings of fever, tachypnea, tachycardia, hypoxemia, and auscultatory signs of consolidation, are not

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Although most patients with CAP can be managed successfully in the community by their general practitioner without additional investigations, distinguishing CAP from other caus‐ es of respiratory symptoms and signs can be difficult, particularly where the presence of co‐ morbidities such as congestive heart failure, chronic lung disease, or COPD complicate the clinical picture. The elderly can present a particularly difficult diagnostic challenge because they more frequently present with non-specific or atypical symptoms and signs. Chest ra‐ diographs are therefore routinely required to confirm the clinical suspicion of pneumonia: The clinical history and physical examination suggest the presence of a lower respiratory tract infection, but the diagnosis is established when demonstrating the presence of new on‐

**3.1. Severity assessment in adult patients with community acquired pneumonia**

Once community-acquired pneumonia is diagnosed, a combination of history, physical ex‐ amination, and laboratory exams can help estimate the short-term risk for torpid evolution or death and, along with the patient's psychosocial characteristics, determine the appropri‐ ate site of treatment [6,16,17,32]. These decisions, including the need for parenteral therapy and supportive care, ultimately relate to the decision on whether to hospitalize the patient. The wide spectrum of severity in patients presenting in the ambulatory care setting explains the wide variation in case fatality rates for pneumonia reported in the national and interna‐ tional literature in different clinical contexts [1,4,6,8]. Thus adult patients with pneumonia without risk factors treated in the ambulatory setting has a low mortality risk (1-2%), rising to 5-15% in patients with comorbidities or specific risk factors that are admitted to hospital ward and increases to 20-50% in those admitted to the intensive care unit. The severity as‐ sessment allow us to decide the site of care (outpatient or in-hospital: general ward, inter‐ mediate care unit or ICU), the extension of laboratory and microbiological examination, coverage of empiric antibiotic treatment, route and length of treatment and level of medical and nursing care that requires the particular case. Recognition of patients at low risk of com‐ plications that can be managed as outpatients would significantly reduce the costs of health care, minimizing risks, without compromising the evolution and prognosis of CAP patients [73]. Hospital admission rates of adult patients with community-acquired pneumonia re‐ ported in the literature vary considerably, suggesting that physicians do not use uniform cri‐ teria to assess the risk of morbidity and mortality of patients. It has been reported that primary care physicians often overestimate the risk of complications and death in patients

unique to pneumonia.

set pulmonary infiltrates on chest radiography.

**3. Prognosis of pneumonia**

#### **2.6. Recommendation**

The clinical diagnosis of pneumonia without radiological confirmation lacks specificity be‐ cause the clinical feature (history and physical examination) does not allow differentiating the patient with pneumonia from other acute respiratory diseases (e.g., upper respiratory tract infections, bronchitis, bronchiolitis, influenza). The diagnosis of pneumonia based sole‐ ly on clinical criteria is also hampered by the large variability in the ability to detect focal signs on chest examination between different examiners. Pneumonia remains foremost a clinical diagnosis. However, symptoms of lower respiratory infection, including fever, cough, purulent sputum, dyspnea, and pleuritic chest pain as well as the clinical findings of fever, tachypnea, tachycardia, hypoxemia, and auscultatory signs of consolidation, are not unique to pneumonia.

Although most patients with CAP can be managed successfully in the community by their general practitioner without additional investigations, distinguishing CAP from other caus‐ es of respiratory symptoms and signs can be difficult, particularly where the presence of co‐ morbidities such as congestive heart failure, chronic lung disease, or COPD complicate the clinical picture. The elderly can present a particularly difficult diagnostic challenge because they more frequently present with non-specific or atypical symptoms and signs. Chest ra‐ diographs are therefore routinely required to confirm the clinical suspicion of pneumonia: The clinical history and physical examination suggest the presence of a lower respiratory tract infection, but the diagnosis is established when demonstrating the presence of new on‐ set pulmonary infiltrates on chest radiography.

#### **3. Prognosis of pneumonia**

(80.9%), fever (75.5%), dyspnea (69.8%), sputum production (65.8%), chills (53.1%), pleuritic chest pain (43.3%), asthenia (38.6%), and altered mental status (25.8%). The typical constella‐ tion of symptoms of pneumonia (cough, purulent sputum, and pleural pain) was noted in only 152 patients (30.2%). Metlay et al. [68] studied the influence of age on symptoms at pre‐ sentation in 1,812 patients with CAP. Cough, dyspnea, and pleuritic chest pain were signifi‐ cantly less common among elderly patients than among younger patients (81.7% vs. 87.8%, 68.8% vs. 73.9%, and 31.6% vs. 53.3%, respectively). After controlling for patient demo‐ graphics, comorbidities, and severity of illness, elderly patients exhibited significantly fewer respiratory symptoms than did younger patients (respiratory symptom score, 7.21 vs. 9.79, respectively; p < 0.01). In other study, altered mental status and the absence of fever were observed more frequently in patients over 80 years of age than in those under 80 years of age (21.0% vs. 10.7%; p < 0.001 and 32.1% vs. 21.9%; p < 0.001, respectively). These findings are consistent with Saldías et al. study [69], which reported a higher incidence of dyspnea and confusion (71% vs. 53%; p < 0.001 and 28% vs. 8%; p< 0.001, respectively) but a lower incidence of fever, chills and pleuritic chest pain in the older patients (63% vs. 76%; p = 0.007, 21% vs. 41%; p < 0.001 and 12% vs. 32%; p< 0.001, respectively). Compared to adults below 65 years of age hospitalized for CAP during the same period, the following clinical findings were more prevalent among the elderly population: the presence of comorbidity, dyspnea, decreased level of consciousness, suspected aspiration, hypoxemia and high serum urea nitrogen on admission to hospital. In the elderly, admission to intermediate and inten‐ sive care units was more frequent (47.7% vs. 29.2%, p< 0.001), and the length of hospital stay was longer (10.6 vs. 8.6 days, p= 0.03). Multilobar radiographic involvement, pleural effu‐ sion, the hospital complication rate and the need for mechanical ventilation were similar in both groups, but mortality, both in-hospital and at 30-days follow-up, was higher in the eld‐ erly population (9.8% vs. 3.2%; p= 0.03 and 13.1% vs. 4.8%; p= 0.02, respectively). Further‐ more, it has been suggested that the local inflammatory response to infection of the lungs is decreased in the elderly, resulting in less cough and sputum production [70]. The systemic inflammatory response (e.g., fever, leukocytosis, high serum C-reactive protein) is also re‐ duced secondary to decreased production of cytokines. Nevertheless, the decrease in inter‐ leukin-6 (IL-6), the most prevalent mediator of fever, did not reach statistical significance in a study that measured IL-6 levels in 59 elderly patients and 21 younger patients with CAP (211.6 vs. 284.5 pg/mL, respectively) [71]. In contrast, tachypnea remains prevalent and ap‐ pears to be a sensitive indicator of lower respiratory tract infection in the elderly [69,72]. Al‐ tered mental status, confusion, a sudden decline in functional physical capacity, and comorbidity decompensation may be the only symptoms of an infection (including pneumo‐ nia) in the elderly [66]. Clinicians should be cognizant of those symptoms to avoid delay in

establishing the diagnosis and initiating antibiotic therapy.

The clinical diagnosis of pneumonia without radiological confirmation lacks specificity be‐ cause the clinical feature (history and physical examination) does not allow differentiating the patient with pneumonia from other acute respiratory diseases (e.g., upper respiratory tract infections, bronchitis, bronchiolitis, influenza). The diagnosis of pneumonia based sole‐

**2.6. Recommendation**

112 Respiratory Disease and Infection - A New Insight

#### **3.1. Severity assessment in adult patients with community acquired pneumonia**

Once community-acquired pneumonia is diagnosed, a combination of history, physical ex‐ amination, and laboratory exams can help estimate the short-term risk for torpid evolution or death and, along with the patient's psychosocial characteristics, determine the appropri‐ ate site of treatment [6,16,17,32]. These decisions, including the need for parenteral therapy and supportive care, ultimately relate to the decision on whether to hospitalize the patient.

The wide spectrum of severity in patients presenting in the ambulatory care setting explains the wide variation in case fatality rates for pneumonia reported in the national and interna‐ tional literature in different clinical contexts [1,4,6,8]. Thus adult patients with pneumonia without risk factors treated in the ambulatory setting has a low mortality risk (1-2%), rising to 5-15% in patients with comorbidities or specific risk factors that are admitted to hospital ward and increases to 20-50% in those admitted to the intensive care unit. The severity as‐ sessment allow us to decide the site of care (outpatient or in-hospital: general ward, inter‐ mediate care unit or ICU), the extension of laboratory and microbiological examination, coverage of empiric antibiotic treatment, route and length of treatment and level of medical and nursing care that requires the particular case. Recognition of patients at low risk of com‐ plications that can be managed as outpatients would significantly reduce the costs of health care, minimizing risks, without compromising the evolution and prognosis of CAP patients [73]. Hospital admission rates of adult patients with community-acquired pneumonia re‐ ported in the literature vary considerably, suggesting that physicians do not use uniform cri‐ teria to assess the risk of morbidity and mortality of patients. It has been reported that primary care physicians often overestimate the risk of complications and death in patients with CAP, hospitalizing consequently a significant number of patients at low risk [74]. The risk stratification of patients should help to reduce this variability and improve the decision of admission and cost effective management of the disease.

ate analysis, despite that large number of studies have shown its importance in the

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*Respiratory rate:* Regardless of age of the patient, the presence of tachypnea is one of the most reliable indicators of severity of pneumonia in univariate and multivariate analysis [6,69,75-77,79,81-83,86,87]. There is a linear relationship between respiratory rate and mor‐ tality in pneumonia patients, but in the clinical practice, it is recommended that the respira‐ tory rate above of 20 breaths/min should be considered a reliable sign of severity in patients

*Mental status:* Altered mental status has been identified as an independent risk factor of death in several studies, including elderly population [6,14,66,69,72,79,82]. However, the definition of altered mental status has varied in different studies, thus complicating their in‐ tegration as a prognostic factor. Despite this, the quantitative or qualitative impairment of consciousness are an excellent predictor of prognosis in patients with community-acquired

*Blood pressure:* Systolic hypotension (SBP <90 mmHg) or low diastolic blood pressure (DBP ≤ 60 mmHg) and the presence of septic shock on admission to hospital are independent fac‐ tors of poor prognosis in multivariate analysis of several studies [6,69,75-82,84]. In the ICU, the presence of septic shock or prolonged systolic hypotension for more than 12 hours which does not improve with adequate volume replacement and/or vasopressor drugs pre‐

*Oxygenation*: Hypoxemia and oxygen administration with a FiO2 ≥ 0.5 to maintain adequate tissue oxygenation or the application of PEEP are indicators of poor prognosis [6,78,79,83]. The severe acute respiratory failure and the need for mechanical ventilation in ICU admis‐ sion or during hospital stay are also predictors of mortality. The presence of hypoxemia or hypercapnia should be corrected immediately and is a determining factor in deciding hospi‐

*Chest radiography*: Bilateral or multilobar pulmonary opacities, rapid progression of pulmo‐ nary infiltrates over 72 hours, the presence of cavitation or pleural effusion are poor prog‐ nostic factors associated to hospital complications and short-term mortality [6,74-77,82-84]. The performance of serial chest radiographs to assess the clinical evolution of hospitalized patients with pneumonia is not recommended outside the ICU, unless there is clinical evi‐

with high mortality in univariate analysis. However, multivariate analysis results have been controversial and suggest that leukopenia may be a better predictor of mortality [6,79].

*Renal function:* The high blood urea nitrogen on admission to hospital has been recognized as a poor prognostic factor in patients with community acquired pneumonia in the univari‐ ate and multivariate analysis, probably reflecting the deterioration of tissue perfusion [6,69,75-77, 79,81,82]. It is important to emphasize, the main prognostic laboratory tests

) or leuko‐

) on admission to hospital was associated

*Leukocytes count:* The presence of leukopenia (less than 4,000 leukocytes per mm3

univariate analysis.

with pneumonia.

pneumonia.

scription is another sign of poor prognosis.

dence suggestive of treatment failure or a complication.

cytosis (greater than 20,000 leukocytes per mm3

talization of a particular case.

#### **3.2. Clinical, radiographic and laboratory prognostic factors in community-acquired pneumonia**

Numerous studies have examined hospital or ICU admission risk factors associated with complicated clinical course or poor prognosis in CAP patients, particularly related to hospi‐ tal or short-term mortality [6,16,17,69]. Univariate studies have described more than 40 clini‐ cal and laboratory parameters associated with mortality [6,36,69,75-77]. However, an independent association with short-term risk of death or hospital complication rate was found only for some clinical variables using multivariate analysis.

To facilitate handling of short-term prognostic factors in pneumonia, it is convenient to group them in different categories: a) Sociodemographic factors: age, gender, origin, ethnici‐ ty, social factors; b) Clinical history: preexisting disease, immunosuppression, altered men‐ tal status, fever, cough, sputum production, dyspnea, chills, chest pain; c) Physical examination: tachycardia, hypotension, tachypnea, hypothermia, hyperthermia, confusion, or pulmonary exam abnormalities; d) Chest radiograph: multilobar or bilateral pulmonary infiltrates, cavitation or pleural effusion; e) Laboratory tests: hypoxemia, hypercapnia, acidosis, high blood urea nitrogen, anemia, leukopenia, leukocytosis, hypoalbuminemia, hy‐ perglycemia, and raised biomarkers of inflammation; f) Microbiological exams: bacteremic pneumonia, lung infection by pneumococcus, anaerobic, atypical microorganisms, gramnegative bacilli or *S. aureus* [6].

*Age:* Several studies have demonstrated an association between extreme ages (below one year and over 65 years) and the risk of death in the hospital [4-6,14,23,78,79,82,83]. The com‐ munity-acquired pneumonia in the elderly usually manifests with atypical or nonspecific symptoms (e.g., mental confusion, anorexia, arrhythmias, congestive heart failure), difficult‐ ing the diagnostic process and delaying specific treatment adversely compromising the prognosis of patients [14,66,69,70]. The absence of fever, prostration, multiple comorbidities, nutritional disorders and institutionalization are poor prognostic factors in the elderly [14,66,72]. However, based on evidence from clinical studies, there are not reasons that sup‐ port the use of different clinical variables to assess the severity in elderly people.

*Comorbidity:* The presence of coronary heart disease, congestive heart failure, cerebrovascu‐ lar disease with motor dysfunction or dementia, diabetes mellitus, chronic respiratory dis‐ ease (COPD, bronchiectasis), cancer, immunosuppression, chronic renal failure, alcoholism, malnutrition and chronic hepatic disease are associated to increased hospital mortality in adult patients with pneumonia [6,69,75-77,83,85]. However, the contribution of different co‐ morbidities to severity of community-acquired pneumonia in adults has been difficult to es‐ tablish, due to lack of uniformity in the definition of chronic diseases in different studies and stratification problems with the severity of various comorbidities. This could partly ex‐ plain the low predictive power of specific comorbidities as risk factors of death in multivari‐ ate analysis, despite that large number of studies have shown its importance in the univariate analysis.

with CAP, hospitalizing consequently a significant number of patients at low risk [74]. The risk stratification of patients should help to reduce this variability and improve the decision

Numerous studies have examined hospital or ICU admission risk factors associated with complicated clinical course or poor prognosis in CAP patients, particularly related to hospi‐ tal or short-term mortality [6,16,17,69]. Univariate studies have described more than 40 clini‐ cal and laboratory parameters associated with mortality [6,36,69,75-77]. However, an independent association with short-term risk of death or hospital complication rate was

To facilitate handling of short-term prognostic factors in pneumonia, it is convenient to group them in different categories: a) Sociodemographic factors: age, gender, origin, ethnici‐ ty, social factors; b) Clinical history: preexisting disease, immunosuppression, altered men‐ tal status, fever, cough, sputum production, dyspnea, chills, chest pain; c) Physical examination: tachycardia, hypotension, tachypnea, hypothermia, hyperthermia, confusion, or pulmonary exam abnormalities; d) Chest radiograph: multilobar or bilateral pulmonary infiltrates, cavitation or pleural effusion; e) Laboratory tests: hypoxemia, hypercapnia, acidosis, high blood urea nitrogen, anemia, leukopenia, leukocytosis, hypoalbuminemia, hy‐ perglycemia, and raised biomarkers of inflammation; f) Microbiological exams: bacteremic pneumonia, lung infection by pneumococcus, anaerobic, atypical microorganisms, gram-

*Age:* Several studies have demonstrated an association between extreme ages (below one year and over 65 years) and the risk of death in the hospital [4-6,14,23,78,79,82,83]. The com‐ munity-acquired pneumonia in the elderly usually manifests with atypical or nonspecific symptoms (e.g., mental confusion, anorexia, arrhythmias, congestive heart failure), difficult‐ ing the diagnostic process and delaying specific treatment adversely compromising the prognosis of patients [14,66,69,70]. The absence of fever, prostration, multiple comorbidities, nutritional disorders and institutionalization are poor prognostic factors in the elderly [14,66,72]. However, based on evidence from clinical studies, there are not reasons that sup‐

*Comorbidity:* The presence of coronary heart disease, congestive heart failure, cerebrovascu‐ lar disease with motor dysfunction or dementia, diabetes mellitus, chronic respiratory dis‐ ease (COPD, bronchiectasis), cancer, immunosuppression, chronic renal failure, alcoholism, malnutrition and chronic hepatic disease are associated to increased hospital mortality in adult patients with pneumonia [6,69,75-77,83,85]. However, the contribution of different co‐ morbidities to severity of community-acquired pneumonia in adults has been difficult to es‐ tablish, due to lack of uniformity in the definition of chronic diseases in different studies and stratification problems with the severity of various comorbidities. This could partly ex‐ plain the low predictive power of specific comorbidities as risk factors of death in multivari‐

port the use of different clinical variables to assess the severity in elderly people.

**3.2. Clinical, radiographic and laboratory prognostic factors in community-acquired**

of admission and cost effective management of the disease.

found only for some clinical variables using multivariate analysis.

**pneumonia**

114 Respiratory Disease and Infection - A New Insight

negative bacilli or *S. aureus* [6].

*Respiratory rate:* Regardless of age of the patient, the presence of tachypnea is one of the most reliable indicators of severity of pneumonia in univariate and multivariate analysis [6,69,75-77,79,81-83,86,87]. There is a linear relationship between respiratory rate and mor‐ tality in pneumonia patients, but in the clinical practice, it is recommended that the respira‐ tory rate above of 20 breaths/min should be considered a reliable sign of severity in patients with pneumonia.

*Mental status:* Altered mental status has been identified as an independent risk factor of death in several studies, including elderly population [6,14,66,69,72,79,82]. However, the definition of altered mental status has varied in different studies, thus complicating their in‐ tegration as a prognostic factor. Despite this, the quantitative or qualitative impairment of consciousness are an excellent predictor of prognosis in patients with community-acquired pneumonia.

*Blood pressure:* Systolic hypotension (SBP <90 mmHg) or low diastolic blood pressure (DBP ≤ 60 mmHg) and the presence of septic shock on admission to hospital are independent fac‐ tors of poor prognosis in multivariate analysis of several studies [6,69,75-82,84]. In the ICU, the presence of septic shock or prolonged systolic hypotension for more than 12 hours which does not improve with adequate volume replacement and/or vasopressor drugs pre‐ scription is another sign of poor prognosis.

*Oxygenation*: Hypoxemia and oxygen administration with a FiO2 ≥ 0.5 to maintain adequate tissue oxygenation or the application of PEEP are indicators of poor prognosis [6,78,79,83]. The severe acute respiratory failure and the need for mechanical ventilation in ICU admis‐ sion or during hospital stay are also predictors of mortality. The presence of hypoxemia or hypercapnia should be corrected immediately and is a determining factor in deciding hospi‐ talization of a particular case.

*Chest radiography*: Bilateral or multilobar pulmonary opacities, rapid progression of pulmo‐ nary infiltrates over 72 hours, the presence of cavitation or pleural effusion are poor prog‐ nostic factors associated to hospital complications and short-term mortality [6,74-77,82-84]. The performance of serial chest radiographs to assess the clinical evolution of hospitalized patients with pneumonia is not recommended outside the ICU, unless there is clinical evi‐ dence suggestive of treatment failure or a complication.

*Leukocytes count:* The presence of leukopenia (less than 4,000 leukocytes per mm3 ) or leuko‐ cytosis (greater than 20,000 leukocytes per mm3 ) on admission to hospital was associated with high mortality in univariate analysis. However, multivariate analysis results have been controversial and suggest that leukopenia may be a better predictor of mortality [6,79].

*Renal function:* The high blood urea nitrogen on admission to hospital has been recognized as a poor prognostic factor in patients with community acquired pneumonia in the univari‐ ate and multivariate analysis, probably reflecting the deterioration of tissue perfusion [6,69,75-77, 79,81,82]. It is important to emphasize, the main prognostic laboratory tests measured at admission in hospitalized adult patients with community-acquired pneumonia are the arterial blood gases and measurement of uremia.

**Patient characteristics Score (points)**

**Male Age Female Age - 10 Nursing home resident 10**

**Neoplastic disease 30 Liver disease 20 Congestive heart failure 10 Cerebrovascular disease 10 Renal disease 10**

**Altered mental status 20 Respiratory rate ≥30 breaths/min 20 Systolic blood pressure < 90 mmHg 20 Temperature < 35 °C or ≥40 °C 15 Heart rate ≥125 beats/min 10**

**pH < 7.35 30 BUN > 30 mg/dL 20 Sodium < 130 mEq/L 20 Glucose ≥250 mg/dL 10 Hematocrit < 30% l10 PaO2 < 60 mmHg or O2 sat < 90% 10 Pleural effusion 10**

**Risk class Score Mortality Site of care recommendation**

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**Risk categories Score Mortality Site of care recommendation**

**III** 3 22% Inpatient (ICU admission)

**I** 0 – 1 1.5% Outpatient **II** 2 9.2% Inpatient

**I** 50 0.1 – 0.4% Outpatient **II** 51 – 70 0.6 – 0.7% Outpatient **III** 71 – 90 0.9 – 2.8% Short stay inpatient **IV** 91 – 130 8.2 – 12.5 Inpatient **V** > 130 27.1 – 31.1% Inpatient

*Demographic factors* **Age (in years)**

*Coexisting conditions*

*Initial physical examination findings*

*Initial laboratory findings*

**British Thoracic Society criteria (CURB-65)**

**Systolic BP < 90 mmHg or Diastolic BP ≤60 mmHg**

**BUN > 7 mmol/L or 20 mg/dL**

**Respiratory rate ≥30 breaths/min**

**Confusion**

**Age ≥65 years**

*Microbiology:* Bacteremic pneumonia with positive blood cultures has two to three times greater risk of death [6,75-77,83]. Pneumonia caused by gram negative bacilli, *Staphylococ‐ cus aureus* and *Pseudomonas aeruginosa* tend to have more complications during the evolu‐ tion and increased lethality [6,83]. Pulmonary infection by *Legionella spp* is a common cause of severe pneumonia and admission to the intensive care unit, specially reported in Europe. However, the clinical-radiographic features has failed to differentiate between the different etiologic agents of pneumonia; in this way, the late microbiological information has not been useful for assessing the severity of the individual patient on admission to hospital or in the context of attention in primary health care services (outpatient clinics or emergency departments). Nevertheless, microbiological tests are useful for evaluating the severity and guide antimicrobial therapy in patients hospitalized with community-ac‐ quired pneumonia.

#### **3.3. Clinical predictive rules to assess the severity of the patient with pneumonia**

The evaluation of the severity of the patient with pneumonia depends on the experience of the clinician, who has been reported often underestimate the seriousness of the disease [78]. No prognostic factor is sufficiently sensitive and specific for predicting the evolution of the individual patient. Thus, in the medical literature have been described several prognostic in‐ dices that would help the clinician to identify patients with community acquired pneumonia that have low or high risk of complications and/or death during the evolution (Table 7) [76,79-82]. None of the developed predictive models has allowed stratifying patients into well-defined risk categories. The development and dissemination of clinical guidelines that examine the severity of the patient with pneumonia by objective criteria, have reduced the hospitalization of low risk patients, significantly reducing the cost of medical resources without affecting the evolution and prognosis of patients [16,17,35,88,89]. Severity predic‐ tive models based on clinical and laboratory exams are best viewed as adjunctive tools for the clinical evaluation of patients. In general, predictive models should be used with care and should never override clinical judgment. The periodic assessment of severity during the course of hospital stay is mandatory to allow adjustment of empirical antibiotic treatment to avoid adverse events associated to it.

Significant variation in admission rates among hospitals and among individual physicians has been well documented. Physicians often overestimate severity and hospitalize a signif‐ icant number of patients at low risk for death [90,91]. Because of these issues, interest in developing simple and objective clinical criterias available at primary health care has stimulated to develop such predictive rules by several research groups [76,79-82]. The rel‐ ative merits and limitations of various proposed criteria have been carefully evaluated [92]. The two most interesting predictive rules are the Pneumonia Severity Index (PSI) de‐ scribed by Fine et al. [81] for screening of patients at low risk for outpatient treatment, and the British Thoracic Society criteria (CURB-65) for screening of high risk patients with severe CAP [79,80].


measured at admission in hospitalized adult patients with community-acquired pneumonia

*Microbiology:* Bacteremic pneumonia with positive blood cultures has two to three times greater risk of death [6,75-77,83]. Pneumonia caused by gram negative bacilli, *Staphylococ‐ cus aureus* and *Pseudomonas aeruginosa* tend to have more complications during the evolu‐ tion and increased lethality [6,83]. Pulmonary infection by *Legionella spp* is a common cause of severe pneumonia and admission to the intensive care unit, specially reported in Europe. However, the clinical-radiographic features has failed to differentiate between the different etiologic agents of pneumonia; in this way, the late microbiological information has not been useful for assessing the severity of the individual patient on admission to hospital or in the context of attention in primary health care services (outpatient clinics or emergency departments). Nevertheless, microbiological tests are useful for evaluating the severity and guide antimicrobial therapy in patients hospitalized with community-ac‐

**3.3. Clinical predictive rules to assess the severity of the patient with pneumonia**

The evaluation of the severity of the patient with pneumonia depends on the experience of the clinician, who has been reported often underestimate the seriousness of the disease [78]. No prognostic factor is sufficiently sensitive and specific for predicting the evolution of the individual patient. Thus, in the medical literature have been described several prognostic in‐ dices that would help the clinician to identify patients with community acquired pneumonia that have low or high risk of complications and/or death during the evolution (Table 7) [76,79-82]. None of the developed predictive models has allowed stratifying patients into well-defined risk categories. The development and dissemination of clinical guidelines that examine the severity of the patient with pneumonia by objective criteria, have reduced the hospitalization of low risk patients, significantly reducing the cost of medical resources without affecting the evolution and prognosis of patients [16,17,35,88,89]. Severity predic‐ tive models based on clinical and laboratory exams are best viewed as adjunctive tools for the clinical evaluation of patients. In general, predictive models should be used with care and should never override clinical judgment. The periodic assessment of severity during the course of hospital stay is mandatory to allow adjustment of empirical antibiotic treatment to

Significant variation in admission rates among hospitals and among individual physicians has been well documented. Physicians often overestimate severity and hospitalize a signif‐ icant number of patients at low risk for death [90,91]. Because of these issues, interest in developing simple and objective clinical criterias available at primary health care has stimulated to develop such predictive rules by several research groups [76,79-82]. The rel‐ ative merits and limitations of various proposed criteria have been carefully evaluated [92]. The two most interesting predictive rules are the Pneumonia Severity Index (PSI) de‐ scribed by Fine et al. [81] for screening of patients at low risk for outpatient treatment, and the British Thoracic Society criteria (CURB-65) for screening of high risk patients with

are the arterial blood gases and measurement of uremia.

116 Respiratory Disease and Infection - A New Insight

quired pneumonia.

avoid adverse events associated to it.

severe CAP [79,80].


*3.3.2. British thoracic society rule*

*3.3.3. Severe CAP rule*

To identify high-risk patients has been useful the discriminant rule developed by the British Thoracic Society, confirming that advanced age, altered mental status or confusion, respira‐ tory rate above 30 breaths/min, diastolic blood pressure below 60 mmHg and blood urea ni‐ trogen greater than 20 mg/dL are associated with increased mortality [79,80]. The BTS original criteria of 1987 have subsequently been modified [78-80]. In the initial study, risk of death was increased 21-fold if a patient, at the time of admission, had at least 2 of the follow‐ ing 3 conditions: tachypnea, diastolic hypotension, and an elevated blood urea nitrogen lev‐ el. These criteria appear to function well except among patients with chronic renal failure and among elderly patients [94,95]. The most recent modification of the BTS criteria includes five easily measurable factors [80]. Multivariate analysis of 1,068 patients identified the fol‐ lowing factors as indicators of increased mortality: confusion (based on a specific mental test or disorientation to person, place, or time), BUN level above 17 mmol/L (20 mg/dL), respira‐ tory rate over 30 breaths/min, low blood pressure (systolic, below 90 mm Hg; or diastolic, below 60 mmHg), and age over 65 years; this gave rise to the acronym CURB-65. In the deri‐ vation and validation cohorts, the 30-day mortality among patients with 0, 1, or 2 factors was 0.7%, 2.1%, and 9.2%, respectively. Mortality was higher when 3, 4, or 5 factors were present and was reported as 14.5%, 40%, and 57%, respectively. The authors suggested that patients with a CURB-65 score of 0-1 be treated as outpatients, that those with a score of 2 be admitted to the wards, and that patients with a score of ≥ 3 often required ICU care. A sim‐ plified version (CRB-65), which does not require testing for BUN level, may be appropriate

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Severe CAP (SCAP) is a life-threatening condition that requires intensive care. Estimates of the frequency of severe CAP range from 5 to 35%, with mortality ranging from 20 to 50% [6]. These relatively wide ranges indicate disparities between definitions of SCAP. There is no universally accepted definition of SCAP. During the last decade, the term has been used for cases that ultimately result in death, and/or patients requiring admission to an intensive care unit. Such practical definitions seem to be insufficient because the risk of death from CAP is not the same as the need for inpatient care. On the other hand, the decision to admit a pa‐ tient to the ICU depends on the clinical judgment of individual clinicians and the local prac‐ tices of their hospitals, differences that could account for much of the variability regarding ICU admission. Studies focused on the evaluation of patients admitted to the ICU mix some variables evident at the time of admission with other potentially evolutionary criteria, which are not applicable to early hospital admission. Other criteria, such as mechanical ventilation and septic shock, are less subject to interpretive variability and better reflect illness severity [97]. España et al. [82] developed a clinical prediction rule for severe community-acquired pneumonia (SCAP) in 1,057 adult patients visiting the emergency department from one hos‐ pital, which was then validated in two different populations: 719 patients from the same center and 1,121 patients from four other hospitals. In the multivariate analyses, eight inde‐ pendent predictive factors were correlated with severe community-acquired pneumonia: ar‐

for decision making in the primary care practitioner's office [96].

*\* Severe CAP was defined by hospital* mortality, mechanical ventilation use and/or septic shock.

**Table 7.** Prognostic rules in adults patients with community-acquired pneumonia[79-81].

#### *3.3.1. Pneumonia severity index*

The PSI is based on derivation and validation cohorts of 14,199 and 38,039 hospitalized patients with CAP, respectively, plus an additional 2,287 combined inpatients and out‐ patients [81]. The Pneumonia Severity Index allows us stratify patients into five risk cat‐ egories. Patients with pneumonia risk class I have low risk of death and adverse events, with a mortality rate ranging between 0.1% and 0.4%. In an observational study, low-risk patients susceptible to ambulatory care had a 30-days hospitalization rate around 5.5% [93]. The model described by Fine et al. was developed as a two-stage predictive tool to identify low risk patients for ambulatory management. In a first step, we consider some epidemiological variables (age, gender, nursing home residence), the presence of certain specific comorbidities (congestive heart failure, malignancy, chronic liver disease, cerebrovascular disease and chronic kidney disease) and some physical ex‐ amination abnormalities (mental status, heart rate, blood pressure, respiratory rate and temperature). In a second step, we consider some laboratory and radiographic findings (for example, anemia, hypoxemia, azotemia, hyperglycemia and pleural effusion). On the basis of associated mortality rates, it has been suggested that risk class I and II pa‐ tients should be treated as outpatients, risk class III patients should be treated in an ob‐ servation unit or with a short hospitalization, and risk class IV and V patients should be treated as inpatients (Table 7). In general, patients younger than 60 years without co‐ morbidities and abnormalities in mental status and vital signs are classified into low risk categories, which could be treated as outpatients unless there are social factors that hinder their control or adherence to treatment (e.g., alcoholism, drug addiction, psychi‐ atric disorders or, rural origin).

#### *3.3.2. British thoracic society rule*

**Severe Community Acquired Pneumonia score (SCAP)**

118 Respiratory Disease and Infection - A New Insight

*3.3.1. Pneumonia severity index*

atric disorders or, rural origin).

*Variables Score* **Age ≥80 years** 5 **Systolic blood pressure < 90 mmHg** 11 **Respiratory rate > 30 breaths/min** 9 **Confusion** 5 **Blood urea nitrogen > 30 mg/dL** 5 **Multilobar or bilateral pulmonary infiltrates** 5 **PaO2 < 54 mmHg or PaO2/FiO2 < 250** 6 **pH < 7.30** 13

**Risk categories Score Severe CAP \* Low** 0 – 9 0.19 - 3.4% **Intermediate** 10 – 19 9.2 - 11.2% **High** 20 36.6 - 75.8%

*\* Severe CAP was defined by hospital* mortality, mechanical ventilation use and/or septic shock.

The PSI is based on derivation and validation cohorts of 14,199 and 38,039 hospitalized patients with CAP, respectively, plus an additional 2,287 combined inpatients and out‐ patients [81]. The Pneumonia Severity Index allows us stratify patients into five risk cat‐ egories. Patients with pneumonia risk class I have low risk of death and adverse events, with a mortality rate ranging between 0.1% and 0.4%. In an observational study, low-risk patients susceptible to ambulatory care had a 30-days hospitalization rate around 5.5% [93]. The model described by Fine et al. was developed as a two-stage predictive tool to identify low risk patients for ambulatory management. In a first step, we consider some epidemiological variables (age, gender, nursing home residence), the presence of certain specific comorbidities (congestive heart failure, malignancy, chronic liver disease, cerebrovascular disease and chronic kidney disease) and some physical ex‐ amination abnormalities (mental status, heart rate, blood pressure, respiratory rate and temperature). In a second step, we consider some laboratory and radiographic findings (for example, anemia, hypoxemia, azotemia, hyperglycemia and pleural effusion). On the basis of associated mortality rates, it has been suggested that risk class I and II pa‐ tients should be treated as outpatients, risk class III patients should be treated in an ob‐ servation unit or with a short hospitalization, and risk class IV and V patients should be treated as inpatients (Table 7). In general, patients younger than 60 years without co‐ morbidities and abnormalities in mental status and vital signs are classified into low risk categories, which could be treated as outpatients unless there are social factors that hinder their control or adherence to treatment (e.g., alcoholism, drug addiction, psychi‐

**Table 7.** Prognostic rules in adults patients with community-acquired pneumonia[79-81].

To identify high-risk patients has been useful the discriminant rule developed by the British Thoracic Society, confirming that advanced age, altered mental status or confusion, respira‐ tory rate above 30 breaths/min, diastolic blood pressure below 60 mmHg and blood urea ni‐ trogen greater than 20 mg/dL are associated with increased mortality [79,80]. The BTS original criteria of 1987 have subsequently been modified [78-80]. In the initial study, risk of death was increased 21-fold if a patient, at the time of admission, had at least 2 of the follow‐ ing 3 conditions: tachypnea, diastolic hypotension, and an elevated blood urea nitrogen lev‐ el. These criteria appear to function well except among patients with chronic renal failure and among elderly patients [94,95]. The most recent modification of the BTS criteria includes five easily measurable factors [80]. Multivariate analysis of 1,068 patients identified the fol‐ lowing factors as indicators of increased mortality: confusion (based on a specific mental test or disorientation to person, place, or time), BUN level above 17 mmol/L (20 mg/dL), respira‐ tory rate over 30 breaths/min, low blood pressure (systolic, below 90 mm Hg; or diastolic, below 60 mmHg), and age over 65 years; this gave rise to the acronym CURB-65. In the deri‐ vation and validation cohorts, the 30-day mortality among patients with 0, 1, or 2 factors was 0.7%, 2.1%, and 9.2%, respectively. Mortality was higher when 3, 4, or 5 factors were present and was reported as 14.5%, 40%, and 57%, respectively. The authors suggested that patients with a CURB-65 score of 0-1 be treated as outpatients, that those with a score of 2 be admitted to the wards, and that patients with a score of ≥ 3 often required ICU care. A sim‐ plified version (CRB-65), which does not require testing for BUN level, may be appropriate for decision making in the primary care practitioner's office [96].

#### *3.3.3. Severe CAP rule*

Severe CAP (SCAP) is a life-threatening condition that requires intensive care. Estimates of the frequency of severe CAP range from 5 to 35%, with mortality ranging from 20 to 50% [6]. These relatively wide ranges indicate disparities between definitions of SCAP. There is no universally accepted definition of SCAP. During the last decade, the term has been used for cases that ultimately result in death, and/or patients requiring admission to an intensive care unit. Such practical definitions seem to be insufficient because the risk of death from CAP is not the same as the need for inpatient care. On the other hand, the decision to admit a pa‐ tient to the ICU depends on the clinical judgment of individual clinicians and the local prac‐ tices of their hospitals, differences that could account for much of the variability regarding ICU admission. Studies focused on the evaluation of patients admitted to the ICU mix some variables evident at the time of admission with other potentially evolutionary criteria, which are not applicable to early hospital admission. Other criteria, such as mechanical ventilation and septic shock, are less subject to interpretive variability and better reflect illness severity [97]. España et al. [82] developed a clinical prediction rule for severe community-acquired pneumonia (SCAP) in 1,057 adult patients visiting the emergency department from one hos‐ pital, which was then validated in two different populations: 719 patients from the same center and 1,121 patients from four other hospitals. In the multivariate analyses, eight inde‐ pendent predictive factors were correlated with severe community-acquired pneumonia: ar‐ terial pH below 7.30, systolic blood pressure under 90 mmHg, respiratory rate above 30 breaths/min, altered mental status, blood urea nitrogen over 30 mg/dL, oxygen arterial pres‐ sure under 54 mmHg or ratio of arterial oxygen tension to fraction of inspired oxygen under 250 mmHg, age greater than or equal to 80 years, and multilobar or bilateral lung affecta‐ tion. The SCAP score was designed to identify high risk patients at admission, by predicting the hospital mortality, need for mechanical ventilation, and risk for septic shock.

and antibiotic therapies. The most popular severity scores, the pneumonia severity index and CURB-65 are accurate for predicting pneumonia-related mortality [108-114]. But clinical care should be based on a broader set of medical outcomes than just mortality, such as ICU admission, need for mechanical ventilation, progression to severe sepsis, or treatment fail‐ ure [115,116]. Unfortunately, there is no consensus surrounding serious complications that

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It has been reported that the SCAP score is slightly better than the PSI and CURB-65 in predict‐ ing adverse outcomes other than mortality in two independent cohorts [117]. In the external validation cohort, the rate of adverse outcomes increased steadily from low- to high-risk classes for the SCAP score as well as for the PSI and CURB-65 (p < 0.001). In the internal valida‐ tion cohort, there were no significant differences in outcomes such as ICU admission and me‐ chanical ventilation for the PSI and CURB-65. All three scores predicted treatment failure with low to moderate discrimination in the external validation cohort. It must be noted that the ini‐ tial severity of CAP is only one factor predicting treatment failure. Other factors, such as the causal microorganism and treatment-related factors, are not part of the three prediction tools. The SCAP score classified a significantly higher proportion of patients as low risk in both co‐ horts than the PSI and CURB-65, with lower rates of all adverse outcomes. Another goal of the tool is its negative predictive value. If the score is low, ICU admission and others adverse out‐ comes are very unlikely. In addition, patients identified as high risk by the SCAP score had somewhat higher rates of ICU admissions, need for mechanical ventilation, and severe sepsis compared with the PSI and CURB-65. Thus, applying the SCAP score would identify CAP pa‐ tients who should receive closer monitoring and more aggressive treatment. Given the some‐ what poorer predictive power of the PSI and CURB-65 in the internal validation cohort, the sensitivity, specificity, and AUC of the three scores were compared in the external validation cohort. Although the SCAP score had significantly better sensitivities and specificities than the

PSI and CURB-65, the differences were small and of uncertain clinical relevance.

**3.4. Biomarkers of inflammation for the severity assessment of CAP**

Saldías et al. [77] assessed the accuracy and discriminatory power of three validated rules (PSI, CURB-65 and SCAP) for predicting clinically relevant adverse outcomes (ICU admis‐ sion, need for mechanical ventilation and hospital complications rate) in patients hospital‐ ized with community-acquired pneumococcal pneumonia. The rate of all adverse outcomes and hospital length of stay increased directly with increasing PSI, CURB-65 and SCAP scores. The three severity scores allowed us to predict the risk of in-hospital complications and 30-day mortality. The PSI score was more sensitive and the SCAP was more specific to predict hospital complications and the risk of death. However, the SCAP was more sensitive and specific in predicting the use of mechanical ventilation. Thus, the severity scores vali‐ dated in the literature allow us to predict the risk of complications and death in adult pa‐ tients hospitalized with pneumococcal pneumonia. Nevertheless, the clinical indexes differ in their sensitivity, specificity and discriminatory power to predict different adverse events.

The clinical guidelines for the management of adult patients with CAP suggest the use of severity-based approach for the purpose of guiding therapeutic options, such as the need for hospital or ICU admission, suitability for ambulatory care, and choice and route of an‐

warrant hospitalization for patients with pneumonia.

The Severe Community Acquired Pneumonia score described by España et al. was validated to predict 30-day mortality in an internal validation cohort of consecutive adult patients ad‐ mitted to one hospital [98]. Consecutive inpatients from other three hospitals were used to externally validate the score and compare the SCAP with the PSI and CURB-65. The dis‐ criminatory power of these rules to predict 30-day mortality was tested by the area under curve (AUC), and their predictive accuracy with the sensitivity, specificity and predictive values. The 30-day mortality rate increased directly with increasing SCAP score (class 0: 0.5%, to class 4: 66.5% risk) in the internal validation cohort, and from 1.3% to 29.2% in ex‐ ternal cohort (p <0.001) with an AUC of 0.83 and 0.75, respectively (p= 0.024). The SCAP score identified 62.4% (95%CI 58.8-66.0) low-risk patients, 52.5% (95%CI 48.8-56.2) the PSI and 46.2% (95%CI 42.5-49.9) the CURB-65 in the external cohort. Patients classified as low risk by the three rules had similar 30-day mortality (SCAP: 2.5%, PSI: 1.6% and CURB-65: 2.7%). The SCAP score was valid to predict 30-day mortality among low-risk patients and to identify patients at low-risk was similar or greater than the other studied rules.

#### *3.3.4. Generic sepsis scores*

Generic scoring systems such as the systemic inflammatory response syndrome (SIRS) crite‐ ria and standardized early warning score (SEWS) have been extensively assessed in critical‐ ly ill patients and are relatively simple to calculate [99,100]. However, it has been reported that SIRS and SEWS perform less favourably than CURB-65 and PSI scores for severity as‐ sessment in CAP and prediction of progression to sepsis in severe CAP [101,102]. Consider‐ ing the limited number of studies to date does not support use of generic sepsis scores over pneumonia-specific scores in CAP.

The clinical pulmonary infection score (CPIS) - original or modified - has been proposed for the diagnosis and management of ventilator-associated pneumonia [103, 104]. The clinical pulmonary infection score has low diagnostic accuracy; however, incorporating gram stains results into the score may help clinical decision making in patients with clinically suspected pneumonia [105].

The use of APACHE II predictive model in the evaluation of patients with severe pneumonia in the ICU has demonstrated its usefulness as a predictor of mortality [106,107]. However, it has not been proved applicable in units of lower complexity of the hospital. The application of this prognostic tool out of the ICU is difficult, time consuming and impractical.

#### *3.3.5. Clinically relevant adverse outcome prediction*

Severity scores provide pivotal direction for the management of community-acquired pneu‐ monia, helping guide decisions such as the appropriate venue for care, diagnostic strategies, and antibiotic therapies. The most popular severity scores, the pneumonia severity index and CURB-65 are accurate for predicting pneumonia-related mortality [108-114]. But clinical care should be based on a broader set of medical outcomes than just mortality, such as ICU admission, need for mechanical ventilation, progression to severe sepsis, or treatment fail‐ ure [115,116]. Unfortunately, there is no consensus surrounding serious complications that warrant hospitalization for patients with pneumonia.

terial pH below 7.30, systolic blood pressure under 90 mmHg, respiratory rate above 30 breaths/min, altered mental status, blood urea nitrogen over 30 mg/dL, oxygen arterial pres‐ sure under 54 mmHg or ratio of arterial oxygen tension to fraction of inspired oxygen under 250 mmHg, age greater than or equal to 80 years, and multilobar or bilateral lung affecta‐ tion. The SCAP score was designed to identify high risk patients at admission, by predicting

The Severe Community Acquired Pneumonia score described by España et al. was validated to predict 30-day mortality in an internal validation cohort of consecutive adult patients ad‐ mitted to one hospital [98]. Consecutive inpatients from other three hospitals were used to externally validate the score and compare the SCAP with the PSI and CURB-65. The dis‐ criminatory power of these rules to predict 30-day mortality was tested by the area under curve (AUC), and their predictive accuracy with the sensitivity, specificity and predictive values. The 30-day mortality rate increased directly with increasing SCAP score (class 0: 0.5%, to class 4: 66.5% risk) in the internal validation cohort, and from 1.3% to 29.2% in ex‐ ternal cohort (p <0.001) with an AUC of 0.83 and 0.75, respectively (p= 0.024). The SCAP score identified 62.4% (95%CI 58.8-66.0) low-risk patients, 52.5% (95%CI 48.8-56.2) the PSI and 46.2% (95%CI 42.5-49.9) the CURB-65 in the external cohort. Patients classified as low risk by the three rules had similar 30-day mortality (SCAP: 2.5%, PSI: 1.6% and CURB-65: 2.7%). The SCAP score was valid to predict 30-day mortality among low-risk patients and to

the hospital mortality, need for mechanical ventilation, and risk for septic shock.

identify patients at low-risk was similar or greater than the other studied rules.

Generic scoring systems such as the systemic inflammatory response syndrome (SIRS) crite‐ ria and standardized early warning score (SEWS) have been extensively assessed in critical‐ ly ill patients and are relatively simple to calculate [99,100]. However, it has been reported that SIRS and SEWS perform less favourably than CURB-65 and PSI scores for severity as‐ sessment in CAP and prediction of progression to sepsis in severe CAP [101,102]. Consider‐ ing the limited number of studies to date does not support use of generic sepsis scores over

The clinical pulmonary infection score (CPIS) - original or modified - has been proposed for the diagnosis and management of ventilator-associated pneumonia [103, 104]. The clinical pulmonary infection score has low diagnostic accuracy; however, incorporating gram stains results into the score may help clinical decision making in patients with clinically suspected

The use of APACHE II predictive model in the evaluation of patients with severe pneumonia in the ICU has demonstrated its usefulness as a predictor of mortality [106,107]. However, it has not been proved applicable in units of lower complexity of the hospital. The application of this

Severity scores provide pivotal direction for the management of community-acquired pneu‐ monia, helping guide decisions such as the appropriate venue for care, diagnostic strategies,

prognostic tool out of the ICU is difficult, time consuming and impractical.

*3.3.5. Clinically relevant adverse outcome prediction*

*3.3.4. Generic sepsis scores*

120 Respiratory Disease and Infection - A New Insight

pneumonia [105].

pneumonia-specific scores in CAP.

It has been reported that the SCAP score is slightly better than the PSI and CURB-65 in predict‐ ing adverse outcomes other than mortality in two independent cohorts [117]. In the external validation cohort, the rate of adverse outcomes increased steadily from low- to high-risk classes for the SCAP score as well as for the PSI and CURB-65 (p < 0.001). In the internal valida‐ tion cohort, there were no significant differences in outcomes such as ICU admission and me‐ chanical ventilation for the PSI and CURB-65. All three scores predicted treatment failure with low to moderate discrimination in the external validation cohort. It must be noted that the ini‐ tial severity of CAP is only one factor predicting treatment failure. Other factors, such as the causal microorganism and treatment-related factors, are not part of the three prediction tools. The SCAP score classified a significantly higher proportion of patients as low risk in both co‐ horts than the PSI and CURB-65, with lower rates of all adverse outcomes. Another goal of the tool is its negative predictive value. If the score is low, ICU admission and others adverse out‐ comes are very unlikely. In addition, patients identified as high risk by the SCAP score had somewhat higher rates of ICU admissions, need for mechanical ventilation, and severe sepsis compared with the PSI and CURB-65. Thus, applying the SCAP score would identify CAP pa‐ tients who should receive closer monitoring and more aggressive treatment. Given the some‐ what poorer predictive power of the PSI and CURB-65 in the internal validation cohort, the sensitivity, specificity, and AUC of the three scores were compared in the external validation cohort. Although the SCAP score had significantly better sensitivities and specificities than the PSI and CURB-65, the differences were small and of uncertain clinical relevance.

Saldías et al. [77] assessed the accuracy and discriminatory power of three validated rules (PSI, CURB-65 and SCAP) for predicting clinically relevant adverse outcomes (ICU admis‐ sion, need for mechanical ventilation and hospital complications rate) in patients hospital‐ ized with community-acquired pneumococcal pneumonia. The rate of all adverse outcomes and hospital length of stay increased directly with increasing PSI, CURB-65 and SCAP scores. The three severity scores allowed us to predict the risk of in-hospital complications and 30-day mortality. The PSI score was more sensitive and the SCAP was more specific to predict hospital complications and the risk of death. However, the SCAP was more sensitive and specific in predicting the use of mechanical ventilation. Thus, the severity scores vali‐ dated in the literature allow us to predict the risk of complications and death in adult pa‐ tients hospitalized with pneumococcal pneumonia. Nevertheless, the clinical indexes differ in their sensitivity, specificity and discriminatory power to predict different adverse events.

#### **3.4. Biomarkers of inflammation for the severity assessment of CAP**

The clinical guidelines for the management of adult patients with CAP suggest the use of severity-based approach for the purpose of guiding therapeutic options, such as the need for hospital or ICU admission, suitability for ambulatory care, and choice and route of an‐ timicrobial agents. The use of prognostic scores, like CURB-65 and PSI, is recommended to support clinical judgment [16,17,35]. Despite their widespread use in clinical routine, traditional markers, such as severity of disease assessment by the patient`s fever, white blood cell count and also CRP level are not reliable for the assessment of disease severity and mortality risk in CAP. The pneumonia severity index (PSI) is a widely accepted and validated severity scoring system that assesses the risk of mortality for pneumonia pa‐ tients in a two step algorithm, combining clinical signs, demographic data and laboratory values [81]. However, its complexity is high, jeopardizing its dissemination and imple‐ mentation, especially in everyday clinical practice. Therefore, the CURB-65 score has been proposed as a simpler alternative [79,80].

**4. Conclusion**

20,000 cells/mm3

complications and death.

for site of care and treatment planning.

classify patients into three risk categories:

**4.1. Pneumonia severity assessment in the ambulatory setting**

In assessing the probability of death in adult patients with community-acquired pneumo‐ nia, the clinical history and physical examination abnormalities significantly influenced the prognosis [4,6,49,69,75-87]. In medical history, advanced age (over 65-70 years), specif‐ ic comorbidities, symptoms of dyspnea and confusion were associated with increased risk of death. Among comorbid conditions, the strongest predictors of death were neurologic disease, cancer, immunosuppression, alcoholism, malnutrition, renal disease, liver disease and congestive heart failure. In physical examination, altered mental status, hypotension, tachypnea and hypothermia were associated with increased odds of death. Laboratory ab‐ normalities significantly associated to bad prognosis are azotemia (blood urea nitrogen

bar or bilateral infiltrates on chest radiograph were also associated to increased risk of

Individual clinical and laboratory abnormalities are associated with only moderate in‐ creases in the odds of death [6,49,75-87]. Thus, combinations of factors are necessary to ac‐ curately assess short-term risk for death and guide site-of-care decisions. These prognosis rules include demographic factors (age, gender and nursing home residence), comorbid conditions (for example, neoplastic disease, diabetes, pulmonary disease, and heart dis‐ ease), symptoms and signs (for example, altered mental status, lack of pleuritic chest pain or fever, tachypnea, and hypotension), and laboratory and radiographic findings (for ex‐ ample, hypoxemia, azotemia, leukopenia, acidosis, hypoalbuminemia and multilobar infil‐ trates). The Pneumonia Patient Outcomes Research Team (PORT) Severity Index and CURB-65 are well validated prognostic rules recommended in the clinical guidelines [16,17,35] to assess severity of pneumonia patients in the ambulatory care setting. In eval‐ uating clinical findings as a guide to initial site of treatment, most studies on prognosis have focused on mortality as the sole outcome, which is problematic because a high risk for death may not be the only reason for hospitalization. Increased risk for other serious adverse events (e.g., ICU admission, mechanical ventilation support, progression to septic shock), reliability in adhering to therapy, returning for follow-up, and availability of sup‐ portive care at home are also important determinants for hospitalization. Therefore, clini‐ cal judgment should always prevail over the severity index calculation in clinical decision

Clinicians are advised to implement a simple and practical strategy for assessing the severi‐ ty and risk of complications in patients with community-acquired pneumonia assisted in the ambulatory care setting (outpatient clinics and emergency departments). It is suggested to

). Pleural effusion, rapid progression of pulmonary infiltrates and multilo‐

Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with...

) and leukocytosis ( ≥

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123

level above 20 mg/dL), hypoxemia, leukopenia ( ≤ 4,000 cells/mm<sup>3</sup>

A number of studies have explored the prognostic value of biomarkers in patients with CAP. Muller et al. [49] reported a significant relationship between procalcitonin levels and PSI categories, with PCT being markedly elevated in the highest PSI class. However, it must be taken into account that many PSI class V patients had low PCT values. Huang et al. [118], report a multicenter, prospective, observational cohort study in a large population of 1,651 patients admitted to 28 community or teaching emergency departments for CAP to deter‐ mine whether procalcitonin can provide prognostic information beyond the Pneumonia Se‐ verity Index and CURB-65. In this study procalcitonin levels did not add relevant prognostic information for most pneumonia patients. Used alone, procalcitonin had modest test charac‐ teristics: specificity (35%), sensitivity (92%), positive likelihood ratio (1.41), and negative likelihood ratio (0.22). However, among higher-risk groups as assessed by the Pneumonia Severity Index score, low procalcitonin level reliably predicted lower mortality.

The predictive value of procalcitonin was compared to CRP, leukocyte count and CRB-65 score in a large study of the CAPNETZ competence network [119] involving 1,671 patients with proven CAP, clinical and laboratory variables were determined at admission and pa‐ tients were followed-up for 28 days for outcome. The PCT levels at admission were a better predictor of CAP severity and outcome than leukocyte count and CRP levels, with a similar prognostic accuracy as the CRB-65 score. The area under the curve for PCT and CRB-65 was comparable (0.80, 95%CI 0.75-0.84 versus 0.79, 95%CI 0.74-0.84), but each significantly high‐ er compared with CRP (0.62, 95%CI 0.54-0.68) and leukocyte count (0.61, 95%CI 0.54-0.68). Another finding from this study, a PCT threshold of ≤ 0.228 ng/mL identified low-risk pa‐ tients within all CRB-65 risk groups.

Another study from the CAPNETZ network explored the role of pro-atrial natriuretic pep‐ tide (MR-proANP), pro-vasopressin (CT-proAVP), PCT and CRP for severity assessment and outcome prediction in 589 adult patients with CAP [120]. MR-proANP, CT-proAVP and PCT levels, but not CRP, increased with increasing severity of CAP, classified according to the CRB-65 score. The area under the curve values for CT-proAVP (0.86, 95%CI 0.83-0.89) and MR-proANP (0.76, 95%CI 0.72-0.80) were similar to the AUC of CRB-65 (0.73, 95%CI 0.70-0.77). In multivariate Cox proportional hazards regression analyses high levels of MRproANP and CT-proAVP were the strongest predictors of mortality. Thus, the authors con‐ cluded that MR-proANP and CT-proAVP are predictors of CAP severity and 28-day mortality comparable to the clinical CRB-65 score.

#### **4. Conclusion**

timicrobial agents. The use of prognostic scores, like CURB-65 and PSI, is recommended to support clinical judgment [16,17,35]. Despite their widespread use in clinical routine, traditional markers, such as severity of disease assessment by the patient`s fever, white blood cell count and also CRP level are not reliable for the assessment of disease severity and mortality risk in CAP. The pneumonia severity index (PSI) is a widely accepted and validated severity scoring system that assesses the risk of mortality for pneumonia pa‐ tients in a two step algorithm, combining clinical signs, demographic data and laboratory values [81]. However, its complexity is high, jeopardizing its dissemination and imple‐ mentation, especially in everyday clinical practice. Therefore, the CURB-65 score has been

A number of studies have explored the prognostic value of biomarkers in patients with CAP. Muller et al. [49] reported a significant relationship between procalcitonin levels and PSI categories, with PCT being markedly elevated in the highest PSI class. However, it must be taken into account that many PSI class V patients had low PCT values. Huang et al. [118], report a multicenter, prospective, observational cohort study in a large population of 1,651 patients admitted to 28 community or teaching emergency departments for CAP to deter‐ mine whether procalcitonin can provide prognostic information beyond the Pneumonia Se‐ verity Index and CURB-65. In this study procalcitonin levels did not add relevant prognostic information for most pneumonia patients. Used alone, procalcitonin had modest test charac‐ teristics: specificity (35%), sensitivity (92%), positive likelihood ratio (1.41), and negative likelihood ratio (0.22). However, among higher-risk groups as assessed by the Pneumonia

Severity Index score, low procalcitonin level reliably predicted lower mortality.

The predictive value of procalcitonin was compared to CRP, leukocyte count and CRB-65 score in a large study of the CAPNETZ competence network [119] involving 1,671 patients with proven CAP, clinical and laboratory variables were determined at admission and pa‐ tients were followed-up for 28 days for outcome. The PCT levels at admission were a better predictor of CAP severity and outcome than leukocyte count and CRP levels, with a similar prognostic accuracy as the CRB-65 score. The area under the curve for PCT and CRB-65 was comparable (0.80, 95%CI 0.75-0.84 versus 0.79, 95%CI 0.74-0.84), but each significantly high‐ er compared with CRP (0.62, 95%CI 0.54-0.68) and leukocyte count (0.61, 95%CI 0.54-0.68). Another finding from this study, a PCT threshold of ≤ 0.228 ng/mL identified low-risk pa‐

Another study from the CAPNETZ network explored the role of pro-atrial natriuretic pep‐ tide (MR-proANP), pro-vasopressin (CT-proAVP), PCT and CRP for severity assessment and outcome prediction in 589 adult patients with CAP [120]. MR-proANP, CT-proAVP and PCT levels, but not CRP, increased with increasing severity of CAP, classified according to the CRB-65 score. The area under the curve values for CT-proAVP (0.86, 95%CI 0.83-0.89) and MR-proANP (0.76, 95%CI 0.72-0.80) were similar to the AUC of CRB-65 (0.73, 95%CI 0.70-0.77). In multivariate Cox proportional hazards regression analyses high levels of MRproANP and CT-proAVP were the strongest predictors of mortality. Thus, the authors con‐ cluded that MR-proANP and CT-proAVP are predictors of CAP severity and 28-day

proposed as a simpler alternative [79,80].

122 Respiratory Disease and Infection - A New Insight

tients within all CRB-65 risk groups.

mortality comparable to the clinical CRB-65 score.

In assessing the probability of death in adult patients with community-acquired pneumo‐ nia, the clinical history and physical examination abnormalities significantly influenced the prognosis [4,6,49,69,75-87]. In medical history, advanced age (over 65-70 years), specif‐ ic comorbidities, symptoms of dyspnea and confusion were associated with increased risk of death. Among comorbid conditions, the strongest predictors of death were neurologic disease, cancer, immunosuppression, alcoholism, malnutrition, renal disease, liver disease and congestive heart failure. In physical examination, altered mental status, hypotension, tachypnea and hypothermia were associated with increased odds of death. Laboratory ab‐ normalities significantly associated to bad prognosis are azotemia (blood urea nitrogen level above 20 mg/dL), hypoxemia, leukopenia ( ≤ 4,000 cells/mm<sup>3</sup> ) and leukocytosis ( ≥ 20,000 cells/mm3 ). Pleural effusion, rapid progression of pulmonary infiltrates and multilo‐ bar or bilateral infiltrates on chest radiograph were also associated to increased risk of complications and death.

Individual clinical and laboratory abnormalities are associated with only moderate in‐ creases in the odds of death [6,49,75-87]. Thus, combinations of factors are necessary to ac‐ curately assess short-term risk for death and guide site-of-care decisions. These prognosis rules include demographic factors (age, gender and nursing home residence), comorbid conditions (for example, neoplastic disease, diabetes, pulmonary disease, and heart dis‐ ease), symptoms and signs (for example, altered mental status, lack of pleuritic chest pain or fever, tachypnea, and hypotension), and laboratory and radiographic findings (for ex‐ ample, hypoxemia, azotemia, leukopenia, acidosis, hypoalbuminemia and multilobar infil‐ trates). The Pneumonia Patient Outcomes Research Team (PORT) Severity Index and CURB-65 are well validated prognostic rules recommended in the clinical guidelines [16,17,35] to assess severity of pneumonia patients in the ambulatory care setting. In eval‐ uating clinical findings as a guide to initial site of treatment, most studies on prognosis have focused on mortality as the sole outcome, which is problematic because a high risk for death may not be the only reason for hospitalization. Increased risk for other serious adverse events (e.g., ICU admission, mechanical ventilation support, progression to septic shock), reliability in adhering to therapy, returning for follow-up, and availability of sup‐ portive care at home are also important determinants for hospitalization. Therefore, clini‐ cal judgment should always prevail over the severity index calculation in clinical decision for site of care and treatment planning.

#### **4.1. Pneumonia severity assessment in the ambulatory setting**

Clinicians are advised to implement a simple and practical strategy for assessing the severi‐ ty and risk of complications in patients with community-acquired pneumonia assisted in the ambulatory care setting (outpatient clinics and emergency departments). It is suggested to classify patients into three risk categories:


Clinical judgment is essential to decide the setting of care and treatment of patients with community-acquired pneumonia, especially those located in the intermediate risk catego‐ ry. In general, patients younger than 65 years without preexisting diseases, abnormal vital signs or altered mental status at admission, could be managed as outpatients considering its low risk of death and complications. Elderly patients (aged above 65 years) with specif‐ ic comorbidities and two or more risk factors from the British Thoracic Society rule (CURB-65), it is recommended to handle them in the hospital with severe pneumonia cri‐ teria.

In primary health care services, we recommend to assess the severity of adult patients with community acquired pneumonia considering only clinical variables available in primary care services:


In general terms, in young adults without risk factors it is recommended outpatient man‐ agement, in presence of one risk factor it is recommended ambulatory or short-term hospital care depending on previous experience and clinical judgment, in presence of two or more risk factors it is recommended to refer the patient to the hospital (Figure 1).

**Figure 1. Severity assessment in adult patients with community-acquired pneumonia attended in primary**

Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with...

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125

**health care services (outpatient clinics and emergency departments).**

**a.** Low-risk patients (short-term mortality less than 1-2%) susceptible to ambulatory treat‐

**b.** High-risk patients (short-term mortality around 20-30%) that must be managed in the hospital and probably in specialized units (Intermediate Care Unit or ICU) with severe

**c.** Intermediate-risk patients with advanced age, comorbidities or independent risk factors

Clinical judgment is essential to decide the setting of care and treatment of patients with community-acquired pneumonia, especially those located in the intermediate risk catego‐ ry. In general, patients younger than 65 years without preexisting diseases, abnormal vital signs or altered mental status at admission, could be managed as outpatients considering its low risk of death and complications. Elderly patients (aged above 65 years) with specif‐ ic comorbidities and two or more risk factors from the British Thoracic Society rule (CURB-65), it is recommended to handle them in the hospital with severe pneumonia cri‐

In primary health care services, we recommend to assess the severity of adult patients with community acquired pneumonia considering only clinical variables available in primary

**•** Comorbidity: coronary heart disease, congestive heart failure, chronic pulmonary disease (COPD, bronchiectasis), diabetes mellitus, cerebrovascular disease with motor dysfunc‐ tion or dementia, chronic renal failure, chronic liver disease, alcoholism, malignancies,

**•** Chest X-ray: bilateral or multilobar pulmonary infiltrates, cavitation or pleural effusion.

**•** Presence of decompensated comorbidity (e.g., COPD exacerbation, congestive heart fail‐

In general terms, in young adults without risk factors it is recommended outpatient man‐ agement, in presence of one risk factor it is recommended ambulatory or short-term hospital care depending on previous experience and clinical judgment, in presence of two or more

of death, but they cannot be classified into a specific category.

**•** Altered mental status: drowsiness, stupor, coma or mental confusion.

**•** Tachycardia or heart rate ≥ 120 beats/min.

**•** Tachypnea or respiratory rate ≥ 20 breaths/min.

**•** Low blood pressure or hypotension (BP < 90/60 mmHg).

**•** Pulse oximetry: SpO2 saturation less than 90% on room air.

ure, myocardial infarction, hyperglycemia, arrhythmias).

risk factors it is recommended to refer the patient to the hospital (Figure 1).

ment or brief hospitalization.

124 Respiratory Disease and Infection - A New Insight

pneumonia criteria.

teria.

care services:

**•** Age over 65 years.

malnutrition.

**Figure 1. Severity assessment in adult patients with community-acquired pneumonia attended in primary health care services (outpatient clinics and emergency departments).**

Nevertheless, after evaluating the severity of the case, when the clinician needs to decide the site of care (outpatient or hospital admission), it is important to consider the clinical and so‐ cial variables involved in each particular case. We should especially avoid that high-risk pa‐ tients receive outpatient treatment, but it is also important to minimize the number of lowrisk patients that are unnecessarily admitted to hospital. Different studies have allowed developing a list of risk factors that determine the need for hospital admission and aid the clinicians in estimating the severity of CAP patients. Clinical judgment and experience of the physician must predominate over predictive models, which are not infallible, and should always consider the aspirations and concerns of patients in making decisions about the site of care and treatment prescribed.

[5] The top ten causes of death – World Health Organization. Updated June 2011. http://

Clinical Diagnosis and Severity Assessment in Immunocompetent Adult Patients with...

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127

[6] Fine MJ, Smith MA, Carson CA, Mutha SS, Sankey SS, Weissfeld LA, Kapoor WN. Prognosis and outcomes of patients with community-acquired pneumonia: a meta-

[7] Marrie TJ. Community-acquired pneumonia in the elderly. Clin Infect Dis

[8] File TM. The epidemiology of respiratory tract infections. Semin Respir Infect

[9] Gonzalez R, Steiner JF, Sande MA. Antibiotic prescribing for adults with colds, upper respiratory tract infections, and bronchitis by ambulatory care physicians. JAMA

[10] Metlay JP, Stafford RS, Singer DE. National trends in the use of antibiotics by pri‐ mary care physicians for adult patients with cough. Arch Intern Med

[11] Dalhoff K. Worldwide guidelines for respiratory tract infections: community-ac‐

[12] Hoare Z, Lim WS. Pneumonia: update on diagnosis and management. BMJ 2006; 332:

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[14] Janssens JP, Krause KH. Pneumonia in the very old. Lancet Infect Dis 2004;4:112-24.

[15] Goossens H, Little P. Community acquired pneumonia in primary care. BMJ 2006;

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1077-9.

#### **Author details**

Fernando Peñafiel Saldías1\*, Orlando Díaz Patiño2 and Pablo Aguilera Fuenzalida3

\*Address all correspondence to: fsaldias@med.puc.cl

1 Departamento de Enfermedades Respiratorias y Programa de Medicina de Urgencia, Facultad de Medicina, Pontificia Universidad Católica de Chile, Chile

2 Departamento de Enfermedades Respiratorias y Medicina Intensiva, Facultad de Medicina, Pontificia Universidad Católica de Chile, Chile

3 Programa de Medicina de Urgencia, Facultad de Medicina, Pontificia Universidad Católica de Chile, Chile

#### **References**


[5] The top ten causes of death – World Health Organization. Updated June 2011. http:// www.who.int/mediacentre/factsheets/fs310/en/index.html

Nevertheless, after evaluating the severity of the case, when the clinician needs to decide the site of care (outpatient or hospital admission), it is important to consider the clinical and so‐ cial variables involved in each particular case. We should especially avoid that high-risk pa‐ tients receive outpatient treatment, but it is also important to minimize the number of lowrisk patients that are unnecessarily admitted to hospital. Different studies have allowed developing a list of risk factors that determine the need for hospital admission and aid the clinicians in estimating the severity of CAP patients. Clinical judgment and experience of the physician must predominate over predictive models, which are not infallible, and should always consider the aspirations and concerns of patients in making decisions about

1 Departamento de Enfermedades Respiratorias y Programa de Medicina de Urgencia,

2 Departamento de Enfermedades Respiratorias y Medicina Intensiva, Facultad de Medicina,

3 Programa de Medicina de Urgencia, Facultad de Medicina, Pontificia Universidad Católica

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Facultad de Medicina, Pontificia Universidad Católica de Chile, Chile

and Pablo Aguilera Fuenzalida3

the site of care and treatment prescribed.

126 Respiratory Disease and Infection - A New Insight

Fernando Peñafiel Saldías1\*, Orlando Díaz Patiño2

Pontificia Universidad Católica de Chile, Chile

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\*Address all correspondence to: fsaldias@med.puc.cl

**Author details**

de Chile, Chile

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**Chapter 6**

**Pneumonia in Children**

http://dx.doi.org/10.5772/54052

pneumonia seems to be increasing.

**1. Introduction**

Irena Wojsyk-Banaszak and Anna Bręborowicz

Pneumonia causes substantial morbidity in children worldwide and is a leading cause of death in children in the developing world. The incidence of pneumonia is the highest in children under 5 years of age and in recent years the incidence of complicated and severe

Etiological factors vary with age, source of infection (community vs. hospital acquired pneu‐ monia) and underlying host defects (e.g immunodeficiency). Viruses are the most common etiological factors in preschool children, although in many cases more than one causative agents can be identified. There are several emerging pathogens in community acquired pneumonia in children: virulent strains of *Streptococcus pneumoniae* that are not present in currently available vaccines, Panton-Velentine leucocidin producing *Staphylococcus aureus*,

Diagnosis in most of milder cases of community acquired pneumonia is based on clinical judgement alone, since laboratory tests and radiologic examination do not provide clues concerning etiology. Children with severe pneumonia, hospital acquired pneumonia and

Treatment of mild and moderate cases consists in supportive care and antibiotic treatment. First-line recommended therapy in previously healthy children regardless of age is amoxicil‐ lin, as it provides sufficient coverage against the most common invasive bacterial pathogen, namely *Streptococcus pneumoniae*. For hospital acquired pneumonia initial empiric treatment should be based on local antimicrobial susceptibility patterns, and modified adequately as

Despite the fact, that if properly diagnosed and treated pneumonia resolves with no residual changes, in some cases due to pathogen virulence and/or host susceptibility its course might

and reproduction in any medium, provided the original work is properly cited.

© 2013 Wojsyk-Banaszak and Bręborowicz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

human Bocaviruses and metapneumoviruses being the most important.

immunocompromised children require invasive diagnostic approach.

soon as the results of microbiological tests are available.

properly cited.

Additional information is available at the end of the chapter

**Chapter 6**

### **Pneumonia in Children**

Irena Wojsyk-Banaszak and Anna Bręborowicz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54052

#### **1. Introduction**

Pneumonia causes substantial morbidity in children worldwide and is a leading cause of death in children in the developing world. The incidence of pneumonia is the highest in children under 5 years of age and in recent years the incidence of complicated and severe pneumonia seems to be increasing.

Etiological factors vary with age, source of infection (community vs. hospital acquired pneu‐ monia) and underlying host defects (e.g immunodeficiency). Viruses are the most common etiological factors in preschool children, although in many cases more than one causative agents can be identified. There are several emerging pathogens in community acquired pneumonia in children: virulent strains of *Streptococcus pneumoniae* that are not present in currently available vaccines, Panton-Velentine leucocidin producing *Staphylococcus aureus*, human Bocaviruses and metapneumoviruses being the most important.

Diagnosis in most of milder cases of community acquired pneumonia is based on clinical judgement alone, since laboratory tests and radiologic examination do not provide clues concerning etiology. Children with severe pneumonia, hospital acquired pneumonia and immunocompromised children require invasive diagnostic approach.

Treatment of mild and moderate cases consists in supportive care and antibiotic treatment. First-line recommended therapy in previously healthy children regardless of age is amoxicil‐ lin, as it provides sufficient coverage against the most common invasive bacterial pathogen, namely *Streptococcus pneumoniae*. For hospital acquired pneumonia initial empiric treatment should be based on local antimicrobial susceptibility patterns, and modified adequately as soon as the results of microbiological tests are available.

Despite the fact, that if properly diagnosed and treated pneumonia resolves with no residual changes, in some cases due to pathogen virulence and/or host susceptibility its course might

properly cited.

© 2013 Wojsyk-Banaszak and Bręborowicz; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

be complicated with pleural effusion and empyema, pneumoatocele, lung abscess or necrot‐ izing pneumonia. A recognized complication of severe pneumonia is hyponatremia and SIADH (Syndrome of Inappropriate Antidiuretic Hormone Secretion).

substantial morbidity. Incidence of radiologically confirmed pneumonia in previously healthy children in Europe is 144-147/100,000 children/year and decreases with age, being the highest in children <5 years (328-338/100,000/year and 421/100,000/year in those aged 0-2 years) [3,4]. The rates of hospitalization due to pneumonia in this age group were 122/100,00/year in children ≤ 16 and 287/100,000/year in those ≤ 5 [4]. British studies show the rate of CAP presenting to General Practitioners in children < 5 to be 191/100, 000 person years [5], probably due to the fact that more severely sick children would present directly to the hospital. In a German study incidence of hospitalized pneumonia was 300/100,000/year in children 0-16 and 658/100,000/year in those aged 0-5. In 23% of those cases underlying conditions were present, and it is possible that many children with bronchiolitis were classi‐

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Since introduction of conjugate pneumococcal vaccine (PCV7) to national immunization programs in the USA and Europe the incidence of pneumococcal pneumonia has decreased (by 65% in the USA) and rates of CAP hospitalizations have decreased for children <1 but seem to be increasing for children > 5 [7-9]. At the same time the incidence of severe pneu‐ monia requiring hospital management as well as complicated pneumonia seems to be in‐ creasing. Between 1997 and 2006, the rate of local complications of CAP increased by 77.8% (5.4 and 9.6 cases per 100 000 population, respectively). Empyema accounted for >97% of all

Organisms causing pneumonia are varied and include bacteria, viruses, fungi and protozo‐ ans. Most cases of pneumonia are preceded by acute viral bronchitis. Viruses facilitate infec‐ tions with pathogenic microorganisms colonizing nasopharynx. These pathogens include *Streptococcus pneumoniae, Haemophilus influenzae* and *Moraxella catarrhalis*. Previous colonisa‐ tion with *Streptococcus mitis* and anaerobic cocci *Peptostreptococcus anaerobius* may have pro‐

Etiological factor of pneumonia can be identified in no more than 65-86% patients combin‐ ing multiple diagnostic tools including culture, serology and PCR [7,11]. In everyday clinical practice these methods are rarely used and treatment remains empiric based on national and

Viruses are responsible for 30-67% cases of CAP, and are the most common in children <2. The most frequently identified are respiratory syncytial virus (RSV) isolated in 13-29% and rhinovirus (3-45%) either in combination with bacteria or alone. Other viruses responsible for pneumonia comprise adenovirus (1-13%), influenza (4-22%) and parainfluenza virus (3-10%), rhinovirus (3-45%), human metapneumovirus (5-12%), human bocaviruses (5-15%). The less common are enterovirus, varicella-, herpes- and cytomegalovirus [7,11-13]. In older children bacterial infections are more frequent: *Streptococcus pneumoniae* being the leader (30-44% of CAP) followed by *Mycoplasma pneumoniae* (22-36%) and *Chlamydophila pneumoniae* (5-27%) [7,11,13-16]. *Streptococcus pneumoniae* remains the leading cause of severe pneumo‐

fied as having pneumonia [6].

local complications [8,9].

international guidelines.

tective effect against pathogenic strains.

**5. Etiology**

Burden of pneumonia can be diminished using preventive measures ranging from the sim‐ plest infection control methods like hand washing, limiting exposure to infectious cases, lim‐ iting exposure to tobacco smoke, vaccinations to passive immunization in selected cases.

#### **2. Definition**

Pneumonia is defined as an inflammation of lung tissue due to an infectious agent. Com‐ monly used clinical World Health Organization operational definition is based solely on clinical symptoms (cough or difficulties in breathing and tachypnoea) [1]. In the developing world the term Lower Respiratory Tract Infection (LRTI) is widely used instead of pneumo‐ nia, because of poor access to x-ray and difficulties in radiological confirmation of diagnosis.

Depending on the place of acquisition pneumonia can be divided into:


Recently a third type - Health Care Associated Pneumonia (HCAP) has been distinguished in adult patients.

The significance of this classification is based on its clinical utility since in most cases patho‐ gens responsible for CAP and HAP are different, warranting varying approach and empiric treatment.

#### **3. Community acquired pneumonia**

CAP can be defined as pneumonia in previously healthy children caused by an infectious agent contracted outside the hospital. The common clinical practice is to confirm the diagno‐ sis by radiological findings of consolidations.

#### **4. Epidemiology**

Globally the incidence of pneumonia in children < 5 years in developing countries is 0.28 ep‐ isodes per child - year (150 mln/year), compared to 0.05 episodes per child - year in devel‐ oped countries [2]. Pneumonia is responsible for 18% of death (2 mln/year) in young children worldwide, mostly occurring in impoverished countries with limited access to healthcare system. In more affluent societies pneumonia is rarely fatal, it leads however to substantial morbidity. Incidence of radiologically confirmed pneumonia in previously healthy children in Europe is 144-147/100,000 children/year and decreases with age, being the highest in children <5 years (328-338/100,000/year and 421/100,000/year in those aged 0-2 years) [3,4]. The rates of hospitalization due to pneumonia in this age group were 122/100,00/year in children ≤ 16 and 287/100,000/year in those ≤ 5 [4]. British studies show the rate of CAP presenting to General Practitioners in children < 5 to be 191/100, 000 person years [5], probably due to the fact that more severely sick children would present directly to the hospital. In a German study incidence of hospitalized pneumonia was 300/100,000/year in children 0-16 and 658/100,000/year in those aged 0-5. In 23% of those cases underlying conditions were present, and it is possible that many children with bronchiolitis were classi‐ fied as having pneumonia [6].

Since introduction of conjugate pneumococcal vaccine (PCV7) to national immunization programs in the USA and Europe the incidence of pneumococcal pneumonia has decreased (by 65% in the USA) and rates of CAP hospitalizations have decreased for children <1 but seem to be increasing for children > 5 [7-9]. At the same time the incidence of severe pneu‐ monia requiring hospital management as well as complicated pneumonia seems to be in‐ creasing. Between 1997 and 2006, the rate of local complications of CAP increased by 77.8% (5.4 and 9.6 cases per 100 000 population, respectively). Empyema accounted for >97% of all local complications [8,9].

#### **5. Etiology**

be complicated with pleural effusion and empyema, pneumoatocele, lung abscess or necrot‐ izing pneumonia. A recognized complication of severe pneumonia is hyponatremia and

Burden of pneumonia can be diminished using preventive measures ranging from the sim‐ plest infection control methods like hand washing, limiting exposure to infectious cases, lim‐ iting exposure to tobacco smoke, vaccinations to passive immunization in selected cases.

Pneumonia is defined as an inflammation of lung tissue due to an infectious agent. Com‐ monly used clinical World Health Organization operational definition is based solely on clinical symptoms (cough or difficulties in breathing and tachypnoea) [1]. In the developing world the term Lower Respiratory Tract Infection (LRTI) is widely used instead of pneumo‐ nia, because of poor access to x-ray and difficulties in radiological confirmation of diagnosis.

Recently a third type - Health Care Associated Pneumonia (HCAP) has been distinguished

The significance of this classification is based on its clinical utility since in most cases patho‐ gens responsible for CAP and HAP are different, warranting varying approach and empiric

CAP can be defined as pneumonia in previously healthy children caused by an infectious agent contracted outside the hospital. The common clinical practice is to confirm the diagno‐

Globally the incidence of pneumonia in children < 5 years in developing countries is 0.28 ep‐ isodes per child - year (150 mln/year), compared to 0.05 episodes per child - year in devel‐ oped countries [2]. Pneumonia is responsible for 18% of death (2 mln/year) in young children worldwide, mostly occurring in impoverished countries with limited access to healthcare system. In more affluent societies pneumonia is rarely fatal, it leads however to

SIADH (Syndrome of Inappropriate Antidiuretic Hormone Secretion).

Depending on the place of acquisition pneumonia can be divided into:

**a.** Community Acqiured Pneumonia (CAP)

**b.** Hospital Acquired Pneumonia (HAP).

**3. Community acquired pneumonia**

sis by radiological findings of consolidations.

**2. Definition**

138 Respiratory Disease and Infection - A New Insight

in adult patients.

**4. Epidemiology**

treatment.

Organisms causing pneumonia are varied and include bacteria, viruses, fungi and protozo‐ ans. Most cases of pneumonia are preceded by acute viral bronchitis. Viruses facilitate infec‐ tions with pathogenic microorganisms colonizing nasopharynx. These pathogens include *Streptococcus pneumoniae, Haemophilus influenzae* and *Moraxella catarrhalis*. Previous colonisa‐ tion with *Streptococcus mitis* and anaerobic cocci *Peptostreptococcus anaerobius* may have pro‐ tective effect against pathogenic strains.

Etiological factor of pneumonia can be identified in no more than 65-86% patients combin‐ ing multiple diagnostic tools including culture, serology and PCR [7,11]. In everyday clinical practice these methods are rarely used and treatment remains empiric based on national and international guidelines.

Viruses are responsible for 30-67% cases of CAP, and are the most common in children <2. The most frequently identified are respiratory syncytial virus (RSV) isolated in 13-29% and rhinovirus (3-45%) either in combination with bacteria or alone. Other viruses responsible for pneumonia comprise adenovirus (1-13%), influenza (4-22%) and parainfluenza virus (3-10%), rhinovirus (3-45%), human metapneumovirus (5-12%), human bocaviruses (5-15%). The less common are enterovirus, varicella-, herpes- and cytomegalovirus [7,11-13]. In older children bacterial infections are more frequent: *Streptococcus pneumoniae* being the leader (30-44% of CAP) followed by *Mycoplasma pneumoniae* (22-36%) and *Chlamydophila pneumoniae* (5-27%) [7,11,13-16]. *Streptococcus pneumoniae* remains the leading cause of severe pneumo‐ nia requiring hospitalization even in countries with reduced rates of invasive pneumococcal disease [17]. Since introduction of PCV7 the most common pneumococcal isolates are 1 (pre‐ dominantly responsible for empyema), 19A, 3, 6A and 7F (all included in 13-valent vaccine) [18]. Contrary to previous reports *Mycoplasma pneumoniae* seems to be equally frequent in school and preschool children [11,13]. Less common bacterial causes of CAP in children in‐ clude *Haemophilus influenzae* type B (5-9%), *Staphylococcus aureus*, *Moraxella catarrhalis* (1.5-4%), *Bordatella pertussis, Streptococcus pyogenes* (1-7%), *Chlamydia trachomatis* and a new pathogen identified in the 1990ies – *Simkania negevensis* [7,12]. Unlike in adults *Legionella pneumophila* is a rare cause of CAP in children [19].

**7. Clinical manifestations**

**•** toxic appearance

**•** chest pain,

**•** headache

Typical clinical symptoms of pneumonia consist of:

dren is rare, because they tend to swallow it.

**Table 1.** Tachypnoe defined according to WHO criteria [1]

pneumonia in little children) and/or vomiting

age and according to BTS comprise of: temperature 38,5 0

world to help field healthcare workers assess the need for hospital referral.

**•** cough (30% of children presenting to outpatient clinic with cough, after excluding those with wheeze, have radiographic signs of pneumonia, and cough was reported in 76% of children with CAP) [13,23]. It should be noted that sputum production in preschool chil‐

**•** signs of respiratory distress: tachypnoe (table 1), history of breathlessness or difficulty in breathing – chest retractions, nasal flaring, grunting, use of accessory muscles of respira‐ tion. Tachypnoe is a very sensitive marker of pneumonia. 50-80% of children with WHO defined tachypnoe had radiological signs of pneumonia, and the absence of tachypnoe is the best single finding for ruling out the disease [13,23]. In children <5 tachypnoe had sen‐ sitivity of 74% and specificity of 67% for radiologically confirmed pneumonia, but its clin‐ ical value was lower in the first 3 days of illness. In infants < 12 months respiratory rate of

**•** fever (present in 88-96% of children with radiologically confirmed pneumonia) [13]

70 breaths/min had a sensitivity of 63% and specificity of 89% for hypoxemia [7].

0-2 months "/>60 2-12 months "/>50 1-4 years "/>40 ≥ 5 years "/>30

**•** abdominal pain (referred pain from the diaphragmatic pleura might be the first sign of

Based on clinical symptoms pneumonia can be divided into severe pneumonia that warrants hospitalization and mild, moderate or non-severe. Signs of severe pneumonia differ with

minute in infants and > 50 breaths/minute in older children, moderate to severe recessions in infants and severe difficulty in breathing in older children, nasal flaring, cyanosis, intermit‐ tent apnoea, grunting, not feeding in infants and signs of dehydration in older children, ta‐ chycardia, capillary refill time ≥2s [7]. Agitation may be the sign of hypoxemia. Table 2 presents a simplified approach recommended by WHO to be implemented in developing

C, respiratory rate > 70 breaths/

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**Age Respiratory rate/minute**

In malaria–endemic regions of tropical Africa a challenging etiological factor of pneumonia is multidrug–resistant non typhoidal Salmonella, and in regions where tuberculosis is en‐ demic it is increasingly being recognized as a cause of acute pneumonia [20].

8-40% of cases represent a mixed viral - bacterial or bacterial - bacterial infection [3,4,7,12,16,19]. Primary viral infection predisposes to bacterial pneumonia: influenza epi‐ demics in developed countries coincide with epidemics of *Streptococcus pneumoniae* and *Staphylococcus aureus* pneumonias and measles or RSV infections contribute to increased pneumonia mortality in developing countries [2].

#### **6. Risk factors for cap**

There are several known risk factors for CAP to consider in addition to immunization status, epidemiological data and exposure to other children, especially preschoolers. Un‐ derlying comorbidities like diabetes mellitus, asplenia or splenic dysfunction, chronic car‐ diac disease, nephrotic syndrome, severe liver disease are risk factors for invasive pneumococcal disease including pneumonia. Other risk factors for CAP include: asthma, history of wheezing episodes, otitis media treated by tympanocentesis in the first 2 years of life (risk factor for children <5), tobacco smoke exposure, malnutrition, immunological deficits (primary or secondary), mucocilliary dysfunction (cystic fibrosis, cilliary dyskine‐ sia), congenital malformation of airways, impaired swallowing, microaspiration, gastroe‐ sophageal reflux, neuromuscular disorders, treatment with gastric acid inhibitors (risk factor in adults, in children its role was confirmed in one study). Environmental factors like indoor air pollution caused by cooking and heating with biomass fuels (like wood or dung), living in crowded conditions and parental smoking also increase a child's sus‐ ceptibility to pneumonia [1,7]. Tobacco smoke exposure has been found to increase risk of hospitalization for pneumonia in children < 5 [21]. Conditions predisposing to severe pneumonia include age <5 and prematurity (24-28 GA) [11]. Viral infections, especially influenza and prior antibiotic exposure additionally predispose to pneumococcal and staphylococcal pneumonia. Antibiotics alter bacterial microflora in the airways destroy‐ ing commensal bacteria like alfa-hemolytic Streptococci while viruses release neuramini‐ dase and other enzymes promoting adherence and expression of pneumococcal receptors on host cells like platelet activating factor receptor or CD14 [17,22].

#### **7. Clinical manifestations**

Typical clinical symptoms of pneumonia consist of:


nia requiring hospitalization even in countries with reduced rates of invasive pneumococcal disease [17]. Since introduction of PCV7 the most common pneumococcal isolates are 1 (pre‐ dominantly responsible for empyema), 19A, 3, 6A and 7F (all included in 13-valent vaccine) [18]. Contrary to previous reports *Mycoplasma pneumoniae* seems to be equally frequent in school and preschool children [11,13]. Less common bacterial causes of CAP in children in‐ clude *Haemophilus influenzae* type B (5-9%), *Staphylococcus aureus*, *Moraxella catarrhalis* (1.5-4%), *Bordatella pertussis, Streptococcus pyogenes* (1-7%), *Chlamydia trachomatis* and a new pathogen identified in the 1990ies – *Simkania negevensis* [7,12]. Unlike in adults *Legionella*

In malaria–endemic regions of tropical Africa a challenging etiological factor of pneumonia is multidrug–resistant non typhoidal Salmonella, and in regions where tuberculosis is en‐

8-40% of cases represent a mixed viral - bacterial or bacterial - bacterial infection [3,4,7,12,16,19]. Primary viral infection predisposes to bacterial pneumonia: influenza epi‐ demics in developed countries coincide with epidemics of *Streptococcus pneumoniae* and *Staphylococcus aureus* pneumonias and measles or RSV infections contribute to increased

There are several known risk factors for CAP to consider in addition to immunization status, epidemiological data and exposure to other children, especially preschoolers. Un‐ derlying comorbidities like diabetes mellitus, asplenia or splenic dysfunction, chronic car‐ diac disease, nephrotic syndrome, severe liver disease are risk factors for invasive pneumococcal disease including pneumonia. Other risk factors for CAP include: asthma, history of wheezing episodes, otitis media treated by tympanocentesis in the first 2 years of life (risk factor for children <5), tobacco smoke exposure, malnutrition, immunological deficits (primary or secondary), mucocilliary dysfunction (cystic fibrosis, cilliary dyskine‐ sia), congenital malformation of airways, impaired swallowing, microaspiration, gastroe‐ sophageal reflux, neuromuscular disorders, treatment with gastric acid inhibitors (risk factor in adults, in children its role was confirmed in one study). Environmental factors like indoor air pollution caused by cooking and heating with biomass fuels (like wood or dung), living in crowded conditions and parental smoking also increase a child's sus‐ ceptibility to pneumonia [1,7]. Tobacco smoke exposure has been found to increase risk of hospitalization for pneumonia in children < 5 [21]. Conditions predisposing to severe pneumonia include age <5 and prematurity (24-28 GA) [11]. Viral infections, especially influenza and prior antibiotic exposure additionally predispose to pneumococcal and staphylococcal pneumonia. Antibiotics alter bacterial microflora in the airways destroy‐ ing commensal bacteria like alfa-hemolytic Streptococci while viruses release neuramini‐ dase and other enzymes promoting adherence and expression of pneumococcal receptors

demic it is increasingly being recognized as a cause of acute pneumonia [20].

on host cells like platelet activating factor receptor or CD14 [17,22].

*pneumophila* is a rare cause of CAP in children [19].

140 Respiratory Disease and Infection - A New Insight

pneumonia mortality in developing countries [2].

**6. Risk factors for cap**

**•** signs of respiratory distress: tachypnoe (table 1), history of breathlessness or difficulty in breathing – chest retractions, nasal flaring, grunting, use of accessory muscles of respira‐ tion. Tachypnoe is a very sensitive marker of pneumonia. 50-80% of children with WHO defined tachypnoe had radiological signs of pneumonia, and the absence of tachypnoe is the best single finding for ruling out the disease [13,23]. In children <5 tachypnoe had sen‐ sitivity of 74% and specificity of 67% for radiologically confirmed pneumonia, but its clin‐ ical value was lower in the first 3 days of illness. In infants < 12 months respiratory rate of 70 breaths/min had a sensitivity of 63% and specificity of 89% for hypoxemia [7].


**Table 1.** Tachypnoe defined according to WHO criteria [1]


Based on clinical symptoms pneumonia can be divided into severe pneumonia that warrants hospitalization and mild, moderate or non-severe. Signs of severe pneumonia differ with age and according to BTS comprise of: temperature 38,5 0 C, respiratory rate > 70 breaths/ minute in infants and > 50 breaths/minute in older children, moderate to severe recessions in infants and severe difficulty in breathing in older children, nasal flaring, cyanosis, intermit‐ tent apnoea, grunting, not feeding in infants and signs of dehydration in older children, ta‐ chycardia, capillary refill time ≥2s [7]. Agitation may be the sign of hypoxemia. Table 2 presents a simplified approach recommended by WHO to be implemented in developing world to help field healthcare workers assess the need for hospital referral.

Physical examination:

**•** crackles (present in 33-90% of children with pneumonia), diminished breath sounds over affected site, bronchial breath sounds specific for lobar consolidation, absent breath sounds and dullness to percussion suggestive of effusion. A pleural rub may be heard if pneumonia is accompanied by pleuritis. Crackles and bronchial breath sounds have sensi‐ tivity of 75% and specificity of 57% in pneumonia diagnosis [7].

**8. Additional tests**

**2.** Laboratory studies

with parapneumonic effusion [19].

**1.** Pulsoximetry should be performed in all children with pneumonia since its results facil‐ itate assessment of severity and therefore the need for hospital referral. Pulsoximetry

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Determining the specific pathogen in children with CAP is difficult. Little children do not expectorate sputum, nasopharyngeal swabs are not reliable since bacteria present in the up‐ per airways are not necessarily the same as those causing pneumonia. Invasive diagnostic tools, though efficacious are hardly acceptable in otherwise healthy children most of whom improve with empiric treatment. British Thoracic Society (BTS) standards, Pediatric Infec‐ tious Diseases Society guidelines as well as American Academy of Pediatrics Policy state‐ ments do not recommend microbiological investigation of the child with pneumonia treated as an outpatient. For patients admitted to the hospital, especially those admitted to ICU and

**•** blood cultures are positive in <10% of patients with pneumonia and < 2% of patients treat‐ ed in the outpatient setting. They should nevertheless be performed since if positive, they provide information on CAP etiology and antibiotic resistance [7,19]. In children with complicated pneumonia prevalence of bacteremia vary from 7.8% to 26.5% in pneumonia

**•** nasopharyngeal aspirates or nasal lavage samples may be helpful in identifying respirato‐ ry viruses including respiratory syncytial virus, parainfluenza virus, influenza virus and adenovirus by immunofluorescence method. The results of these tests are particularly useful for cohorting infected children during outbreaks and for epidemic purposes [7]. **•** sputum is difficult to obtain in small children. A reliable sputum sample, as opposed to saliva, contains <10 epithelial cells per low-powered field [35]. Sputum induced by inhala‐ tion with 5% hypertonic saline has much higher bacterial yield and seems to be a valuable

**•** children who require mechanical ventilation should have tracheal aspirates taken for

**•** aspirated pleural fluid should be sent for microscopy, culture and antigen detection. Cul‐ tures are positive in 9% - 18% of cases (sensitivity 23%, specificity 100%) [7, 37]. Pneumo‐ coccal antigen detection in pleural fluid has sensitivity of 90% and specificity of 95% [38].

**•** pneumococcal antigens detection in urine is not specific, as it is often positive in young

**•** Serological testing: fourfold rise in antibody titers in complement fixation test is a golden diagnostics standard, unfortunately not useful for treatment guidance. In many laborato‐

should be definitely performed in all children admitted to hospital [7].

those with complications of CAP, microbiological diagnosis should be attempted.

**3.** Determination of etiology – microbiological investigations

tool in microbiological diagnosis in children with CAP [36].

Pleural fluid should be checked for *Mycobacteria*.

children with nasopharyngeal colonization

Gram stain and culture at the time of endotracheal tube placement [19].


It is important to note that no clinical or radiological sign either alone or in combination, is sensitive and specific enough to differentiate between viral, atypical or typical bacterial eti‐ ology of pneumonia.


\*Look table 1

**Table 2.** Severity of pneumonia – WHO classification [2,27]

#### **8. Additional tests**

Physical examination:

142 Respiratory Disease and Infection - A New Insight

ology of pneumonia.

\*Look table 1

**Table 2.** Severity of pneumonia – WHO classification [2,27]

*pneumonia* (up to 30%) infection [7].

**•** crackles (present in 33-90% of children with pneumonia), diminished breath sounds over affected site, bronchial breath sounds specific for lobar consolidation, absent breath sounds and dullness to percussion suggestive of effusion. A pleural rub may be heard if pneumonia is accompanied by pleuritis. Crackles and bronchial breath sounds have sensi‐

**•** presence of wheeze, especially in the absence of fever, makes the diagnosis of typical bac‐ terial pneumonia unlikely [24]. It is however a common sign in viral and *Mycoplasma*

**•** combining several clinical symptoms into diagnostic algorithm improves sensitivity and specificity of diagnosis. WHO criteria for defining pneumonia (cough or difficulties in breathing and tachypnoea) investigated in a Brasilian study of 390 children have sensitivi‐ ty of 94% for children <2, and 62% for children ≥2 and specificities of 20% and 16% respec‐ tively. Adding fever improved specificity to 44% and 50% [25]. In an Australian study of febrile children < 5 presenting to tertiary emergency department, clinical indicators of pneumonia confirmed radiologically and microbiologically comprised an unwell appear‐ ance, fever ≥ 39 0 C, breathing difficulties, chronic disease, prolonged capillary refill time,

tachypnoe, crackles on auscultation and lack of antipneumococcal vaccination [26].

**Pneumonia/non severe pneumonia** Cough

It is important to note that no clinical or radiological sign either alone or in combination, is sensitive and specific enough to differentiate between viral, atypical or typical bacterial eti‐

**Severe pneumonia** Signs of pneumonia & ≥1

**Very severe pneumonia** Signs of severe pneumonia & ≥1

Problems with breathing Tachypnoe \* No signs of severe pneumonia present



tivity of 75% and specificity of 57% in pneumonia diagnosis [7].


Determining the specific pathogen in children with CAP is difficult. Little children do not expectorate sputum, nasopharyngeal swabs are not reliable since bacteria present in the up‐ per airways are not necessarily the same as those causing pneumonia. Invasive diagnostic tools, though efficacious are hardly acceptable in otherwise healthy children most of whom improve with empiric treatment. British Thoracic Society (BTS) standards, Pediatric Infec‐ tious Diseases Society guidelines as well as American Academy of Pediatrics Policy state‐ ments do not recommend microbiological investigation of the child with pneumonia treated as an outpatient. For patients admitted to the hospital, especially those admitted to ICU and those with complications of CAP, microbiological diagnosis should be attempted.


ries enzyme linked immunosorbent assays (ELISA) has replaced complement fixation tests as less time consuming. Positive anti *Mycoplasma* IgM antibody titer 9-11 days from the onset of illness is also suggestive of recent infection. Cross reactions with adenovirus and *Legionella pneumophila* have been described [23]. Cold agglutinins measurement value is limited – in school children the positive predictive value for *Mycoplasma* of a rapid cold agglutinin test was 70% [7]. In *Chlamydophila pneumoniae* infection IgM rise is observed af‐ ter 3 weeks and IgG rise after 6-8 weeks.


**Figure 2.** Round focus of consolidation in the left upper lobe. Pneumonia.

**•** clinical signs of severe pneumonia (listed above),

**•** parents' inability to manage the illness at home

PIDS), PaO2 <60 mmHg and PaCO2 >50 mmHg, central cyanosis

**•** diffuse radiological changes: multilobar pneumonia, pleural effusion

**•** CAP caused by pathogen with increased virulence eg. MRSA(PIDS)

Most children with CAP can be safely managed on outpatient basis. Indications for hospital

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**•** hypoxemia – oxygen saturation < 92% (according to BTS) or <90% (according to AAP and

**•** underlying conditions eg. congenital heart defect, cystic fibrosis, bronchopulmonary dys‐

**9. Management**

referral comprise:

**•** signs of sepsis or septic shock

**•** young age – < 6 months of life

plasia, immune deficiencies

**•** outpatient treatment failure

**4.** Chest radiography

**Figure 1.** Alveolar consolidations in the left lower lobe and in the right lower lobe*. Mycoplasma pneumoniae* pneumonia

**Figure 2.** Round focus of consolidation in the left upper lobe. Pneumonia.

#### **9. Management**

ries enzyme linked immunosorbent assays (ELISA) has replaced complement fixation tests as less time consuming. Positive anti *Mycoplasma* IgM antibody titer 9-11 days from the onset of illness is also suggestive of recent infection. Cross reactions with adenovirus and *Legionella pneumophila* have been described [23]. Cold agglutinins measurement value is limited – in school children the positive predictive value for *Mycoplasma* of a rapid cold agglutinin test was 70% [7]. In *Chlamydophila pneumoniae* infection IgM rise is observed af‐

**•** *Legionella pneumophila* urinary antigen detection remains golden standard for the diagno‐ sis of legionellosis. The test remains positive for weeks after acute infection. Urinary anti‐ gen is positive only in case of infection with serogroup 1. Antigens are excreted in urine at the beginning of the second week of illness. Quick diagnostic tests are commercially avail‐ able with sensitivity of 80% and specificity of 99-100%. Infection with serogroup 1 can be

**•** real time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) can be used for in‐ vestigating the etiology of pneumonia. The advantage of this method is the availability of results on the same day. It does not however provide information on bacterial sensitivity nor is readily available outside research settings. Pneumolysin based PCR has been in‐ creasingly used to detect *Streptococcus pneumoniae* in blood and pleural fluid with sensitiv‐ ity of 100% and specificity of 95% [7]. Measuring bacterial load with RT-PCR may help predict the outcome of CAP as adult patients with bacterial load > 1000 copies/mL were at

**Figure 1.** Alveolar consolidations in the left lower lobe and in the right lower lobe*. Mycoplasma pneumoniae* pneumonia

excluded when the results of 3 consecutive urine samples are negative.

higher risk for sepsis, respiratory insufficiency and death [39].

ter 3 weeks and IgG rise after 6-8 weeks.

144 Respiratory Disease and Infection - A New Insight

**4.** Chest radiography

Most children with CAP can be safely managed on outpatient basis. Indications for hospital referral comprise:


Children who are not improving despite treatment and present with impeding respirato‐ ry failure or shock should be admitted to Intensive Care Unit. Criteria for ICU admis‐ sion comprise:

sessed in 24-48 hours. The reason for this change, despite the obvious concerns of increasing antibiotic resistance among bacteria as well as possible adverse reactions in children unnec‐ essarily treated with antibiotics, is the fact that based on clinical, laboratory and radiological markers, either alone or in combination, a reliable distinction between viral and bacterial in‐ fection is impossible. Children <2 especially with a history of conjugate pneumococcal vacci‐ nation and with mild symptoms of lower respiratory tract infection are unlikely to have pneumonia. In these children antibiotics might be withheld provided reassessment of the child is made if the symptoms persist or deterioration occurs [7]. PIDS guidelines, on the contrary, state that preschool children with CAP do not routinely require antibiotic therapy

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Antibiotic of choice for CAP treated in community is amoxicillin 90 mg/kg/day applied in two doses for 5-10 days. Results from two randomized trials on short course (3 days) oral antibiotics performed on infants in developing countries are difficult to interpret since many of these children had bronchiolitis with wheeze or upper respiratory tract in‐ fection and did not need antibiotics at all [43,44]. Amoxicillin is effective against the ma‐ jority pathogens responsible for CAP in children. It is well tolerated and affordable. Alternatives are co-amoxiclav, cefaclor and macrolides. For *Streptococcus pneumoniae* re‐ sistant to penicillin with MICs up to 4.0 µg/mL preferred treatment consists in ceftriax‐ one, and for MICs >4.0 µg/mL in vancomycin, linezolid or clindamycin though resistance to clindamycin seems to be increasing amounting to 15-40% in certain geographic re‐ gions [19]. It should be noted that interpretation of *in vitro* susceptibility tests to penicil‐ lin depends on the route of administration. Intravenously administered penicillin can achieve tissue concentrations effective against organisms with minimal inhibitory concen‐ tration (MICs) ≤2.0 µg/mL, possibly effective for strains with MICS of 4 µg/mL and not likely to be effective for strains with MICS ≥ 8 µg/mL. For orally administered penicillin corresponding values are <0.06 µg/mL, 0.12 – 1.0 µg/mL, and ≥2.0 µg/mL for resistant strains [19]. Clinical laboratory standards vary depending on the region. Those given above, were issued jointly by Infectious Diseases Society of America (IDSA) and Ameri‐ can Thoracic Society (ATS). BTS recommends cut off values for intravenously adminis‐ tered penicillin of <0.1 mg/L; 0.1-1.0 mg/L and > 1.0-4.0 mg/L and European Respiratory Society (ERS) recommends MIC breakpoints <0.5 mg/L; 0.5 – 2.0 mg/L and >2.0 mg/L re‐ spectively [17,19]. PIDS recommends levofloxacin for children from 6 months of age as preferred choice for oral therapy [19]. Macrolide antibiotics may be added if *Mycoplasma pneumoniae* or *Chlamydophila pneumonia* are suspected when the child is not improving af‐ ter 24 - 48 hours or in very severe cases. They are not recommended as first choice anti‐ biotics because up to 40% of currently isolated in USA strains of *S. pneumoniae* are

As is the case with indications for antibiotics in CAP there is no consensus as to how they should be administered. According to BTS guidelines if the child is feeding well and not vomiting, antibiotics should be given orally. Children with moderate pneumonia admitted because of respiratory distress can be treated with oral antibiotics and discharged when fe‐ ver and respiratory distress subside [7,45]. Intravenous route of antibiotic administration is

since pneumonia in this age group is predominantly of viral origin [19].

resistant to macrolides [19].


#### **10. General management**

All children treated for pneumonia should be reassessed in 48 hours if there is no clinical improvement or deterioration and persistence of fever. It is important that parents of chil‐ dren treated at home have clear written instructions on fever management, preventing de‐ hydration, recognizing signs of deterioration as well as further access to healthcare professionals [7].

Hospitalized hypoxemic children should be given oxygen to maintain oxygen saturation > 92%. Dehydrated children should be provided adequate amount of oral fluids and if unable to drink should receive intravenous fluids. Their electrolytes and creatinine serum levels should be measured on daily basis. Up to date there have been no studies proving beneficial effects of chest physiotherapy in children with pneumonia and therefore chest physiothera‐ py should not be performed.

A child should improve as evaluated by clinical symptoms and laboratory inflammatory markers in 48-72 hours after initiation of adequate treatment. Failure to improve war‐ rants further investigation for possible complications, resistant microorganisms or alter‐ native diagnosis.

#### **11. Antibiotic treatment**

There is no consensus between experts whether all children with CAP should receive antibi‐ otics. According to BTS guidelines issued in 2011 all children diagnosed with pneumonia should be treated with antibiotics. This is in contrast to previous guidelines stating that if viral etiology is suspected antibiotics might be withheld provided that the child is reas‐ sessed in 24-48 hours. The reason for this change, despite the obvious concerns of increasing antibiotic resistance among bacteria as well as possible adverse reactions in children unnec‐ essarily treated with antibiotics, is the fact that based on clinical, laboratory and radiological markers, either alone or in combination, a reliable distinction between viral and bacterial in‐ fection is impossible. Children <2 especially with a history of conjugate pneumococcal vacci‐ nation and with mild symptoms of lower respiratory tract infection are unlikely to have pneumonia. In these children antibiotics might be withheld provided reassessment of the child is made if the symptoms persist or deterioration occurs [7]. PIDS guidelines, on the contrary, state that preschool children with CAP do not routinely require antibiotic therapy since pneumonia in this age group is predominantly of viral origin [19].

Children who are not improving despite treatment and present with impeding respirato‐ ry failure or shock should be admitted to Intensive Care Unit. Criteria for ICU admis‐

**•** need for invasive mechanical ventilation or non-invasive positive pressure ventilation,

**•** altered mental status due to hypercarbia, hypoxemia or as a result of pneumonia

**•** hypoxemia requiring FiO2 greater than inspired concentration or flow feasible in general care area; pulsoximetry measurements ≤92% with inspired oxygen of ≥0.5 (according to

**•** rising respiratory rate and heart rate with clinical evidence of severe respiratory distress

All children treated for pneumonia should be reassessed in 48 hours if there is no clinical improvement or deterioration and persistence of fever. It is important that parents of chil‐ dren treated at home have clear written instructions on fever management, preventing de‐ hydration, recognizing signs of deterioration as well as further access to healthcare

Hospitalized hypoxemic children should be given oxygen to maintain oxygen saturation > 92%. Dehydrated children should be provided adequate amount of oral fluids and if unable to drink should receive intravenous fluids. Their electrolytes and creatinine serum levels should be measured on daily basis. Up to date there have been no studies proving beneficial effects of chest physiotherapy in children with pneumonia and therefore chest physiothera‐

A child should improve as evaluated by clinical symptoms and laboratory inflammatory markers in 48-72 hours after initiation of adequate treatment. Failure to improve war‐ rants further investigation for possible complications, resistant microorganisms or alter‐

There is no consensus between experts whether all children with CAP should receive antibi‐ otics. According to BTS guidelines issued in 2011 all children diagnosed with pneumonia should be treated with antibiotics. This is in contrast to previous guidelines stating that if viral etiology is suspected antibiotics might be withheld provided that the child is reas‐

sion comprise:

**•** fluid refractory shock

BTS) or ≥0.6 (according to PIDS)

146 Respiratory Disease and Infection - A New Insight

**10. General management**

py should not be performed.

**11. Antibiotic treatment**

professionals [7].

native diagnosis.

**•** recurrent apnea, grunting or slow irregular breathing

and exhaustion with or without hypercarbia [7,19]

Antibiotic of choice for CAP treated in community is amoxicillin 90 mg/kg/day applied in two doses for 5-10 days. Results from two randomized trials on short course (3 days) oral antibiotics performed on infants in developing countries are difficult to interpret since many of these children had bronchiolitis with wheeze or upper respiratory tract in‐ fection and did not need antibiotics at all [43,44]. Amoxicillin is effective against the ma‐ jority pathogens responsible for CAP in children. It is well tolerated and affordable. Alternatives are co-amoxiclav, cefaclor and macrolides. For *Streptococcus pneumoniae* re‐ sistant to penicillin with MICs up to 4.0 µg/mL preferred treatment consists in ceftriax‐ one, and for MICs >4.0 µg/mL in vancomycin, linezolid or clindamycin though resistance to clindamycin seems to be increasing amounting to 15-40% in certain geographic re‐ gions [19]. It should be noted that interpretation of *in vitro* susceptibility tests to penicil‐ lin depends on the route of administration. Intravenously administered penicillin can achieve tissue concentrations effective against organisms with minimal inhibitory concen‐ tration (MICs) ≤2.0 µg/mL, possibly effective for strains with MICS of 4 µg/mL and not likely to be effective for strains with MICS ≥ 8 µg/mL. For orally administered penicillin corresponding values are <0.06 µg/mL, 0.12 – 1.0 µg/mL, and ≥2.0 µg/mL for resistant strains [19]. Clinical laboratory standards vary depending on the region. Those given above, were issued jointly by Infectious Diseases Society of America (IDSA) and Ameri‐ can Thoracic Society (ATS). BTS recommends cut off values for intravenously adminis‐ tered penicillin of <0.1 mg/L; 0.1-1.0 mg/L and > 1.0-4.0 mg/L and European Respiratory Society (ERS) recommends MIC breakpoints <0.5 mg/L; 0.5 – 2.0 mg/L and >2.0 mg/L re‐ spectively [17,19]. PIDS recommends levofloxacin for children from 6 months of age as preferred choice for oral therapy [19]. Macrolide antibiotics may be added if *Mycoplasma pneumoniae* or *Chlamydophila pneumonia* are suspected when the child is not improving af‐ ter 24 - 48 hours or in very severe cases. They are not recommended as first choice anti‐ biotics because up to 40% of currently isolated in USA strains of *S. pneumoniae* are resistant to macrolides [19].

As is the case with indications for antibiotics in CAP there is no consensus as to how they should be administered. According to BTS guidelines if the child is feeding well and not vomiting, antibiotics should be given orally. Children with moderate pneumonia admitted because of respiratory distress can be treated with oral antibiotics and discharged when fe‐ ver and respiratory distress subside [7,45]. Intravenous route of antibiotic administration is reserved for children with severe, complicated pneumonia or sepsis for whom intravenous amoxicillin, co-amoxiclav, cefuroxime, cefotaxime or ceftriaxone are recommended. Accord‐ ing to PIDS guidelines however all children treated in hospital should receive antibiotics in‐ travenously to provide reliable blood and tissue concentrations [19]. In hospitalized children suspected of *S. aureus* infection vancomycin or clindamycin should be added to beta-lactam therapy. For children with penicillin allergy recommended drugs are cephalosporins and in case of type-I allergic reactions macrolides, vancomycin or clindamycin are suggested. In children who do not tolerate vancomycin or clindamycin, linezolid may be administered [19]. Antibiotic should be changed according to results of culture and sensitivity if these tests are positive. As soon as the child's condition improves, a switch to oral antibiotics should be considered [7].

Empyema should be suspected in every child with pneumonia with a history of prolonged fever, tachypnoe, pain on abdominal palpation, pleuritic chest pain, splinting of the affected side and persistence of high serum C-reactive protein levels [50]. On physical examination asymmetry of breath sounds, unilateral decreased chest wall expansion, dullness to percus‐ sion might be appreciated [27]. In some children with pneumonia empyema may develop during intravenous antibiotic treatment [50]. One of the identified risk factors for bacterial

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Chest radiographs show homogenous opacity over the entire lung (large effusion) (Figure 3). In smaller effusions an ascending rim of fluid along the lateral chest wall (meniscus sign) occurs. Costophrenical angle obliteration is the first sign of pleural effusion. Based on radio‐ graph it is not possible to distinguish effusion from empyema [47]. A method of choice for radiologic evaluation of patients with parapneumonic effusion and empyema is ultrasonog‐ raphy. It helps estimate the amount of fluid, its echogenicity, detects loculations and fibrin strands and is used to guide invasive procedures [28]. Chest CT should not be routinely per‐ formed, it may be useful however for diagnosis of underlying pathology eg. tumor in the

**Figure 3.** Opacification of left hemithorax with mediastinal shift to the opposite side. Alveolar consolidations in the

central field of the right lobe. Lobar pneumonia with pleural effusion caused by *Streptococcus pneumoniae*

empyema is precedent varicella [49].

mediastinum or lung abscess [46,53].

#### **12. Complications**

#### **12.1. Empyema and parapneumonic effusion**

Parapneumonic effusion is defined as pleural fluid collection in association with underlying pneumonia and empyema is defined as the accumulation of purulent fluid in the pleural cavity [46]. Incidence of parapneumonic effusion is increasing (by 70-100% between 1990s and the beginning of the present century), affecting 0.6% of all children with CAP, 2 - 10% of pneumonia hospitalizations and 1/3 of pneumococcal pneumonia hospitalizations [47-50]. Predominant etiological factors are *S. pneumonia* (serotype 1,3,14,19A) responsible for 10-66% of empyema cases, *S. aureus* including MRSA (4-30%) and *S. pyogenes.* The less com‐ mon include *Haemophilus influenzae*, *Mycobacterium* spp, *Pseudomonas aeruginosa*, anaerobes, *Mycoplasma pneumonia* and fungi [28,46,47,51,52]. Fluid collection is usually unilateral. Em‐ pyema classically exhibits three stages:


Some authors distinguish "pleuritis sicca stage" that precedes exudative stage, not necessa‐ rily leading to it [53].

Empyema should be suspected in every child with pneumonia with a history of prolonged fever, tachypnoe, pain on abdominal palpation, pleuritic chest pain, splinting of the affected side and persistence of high serum C-reactive protein levels [50]. On physical examination asymmetry of breath sounds, unilateral decreased chest wall expansion, dullness to percus‐ sion might be appreciated [27]. In some children with pneumonia empyema may develop during intravenous antibiotic treatment [50]. One of the identified risk factors for bacterial empyema is precedent varicella [49].

reserved for children with severe, complicated pneumonia or sepsis for whom intravenous amoxicillin, co-amoxiclav, cefuroxime, cefotaxime or ceftriaxone are recommended. Accord‐ ing to PIDS guidelines however all children treated in hospital should receive antibiotics in‐ travenously to provide reliable blood and tissue concentrations [19]. In hospitalized children suspected of *S. aureus* infection vancomycin or clindamycin should be added to beta-lactam therapy. For children with penicillin allergy recommended drugs are cephalosporins and in case of type-I allergic reactions macrolides, vancomycin or clindamycin are suggested. In children who do not tolerate vancomycin or clindamycin, linezolid may be administered [19]. Antibiotic should be changed according to results of culture and sensitivity if these tests are positive. As soon as the child's condition improves, a switch to oral antibiotics

Parapneumonic effusion is defined as pleural fluid collection in association with underlying pneumonia and empyema is defined as the accumulation of purulent fluid in the pleural cavity [46]. Incidence of parapneumonic effusion is increasing (by 70-100% between 1990s and the beginning of the present century), affecting 0.6% of all children with CAP, 2 - 10% of pneumonia hospitalizations and 1/3 of pneumococcal pneumonia hospitalizations [47-50]. Predominant etiological factors are *S. pneumonia* (serotype 1,3,14,19A) responsible for 10-66% of empyema cases, *S. aureus* including MRSA (4-30%) and *S. pyogenes.* The less com‐ mon include *Haemophilus influenzae*, *Mycobacterium* spp, *Pseudomonas aeruginosa*, anaerobes, *Mycoplasma pneumonia* and fungi [28,46,47,51,52]. Fluid collection is usually unilateral. Em‐

**•** Exudative – pleural space contains free flowing fluid with a low white cell count, so called parapneumonic effusion that results from increased vascular permeability and mi‐ gration of neutrophils, lymphocytes and eosinophils in the course of inflammatory proc‐

**•** Fibrinopurulent occurs 5-10 days from the onset of the disease and consists in the deposi‐ tion of fibrin in the pleural space that leads to septation and formation of loculations. The number of white cells increases (empyema) in response to bacterial invasion across the

**•** Organizing - includes infiltration of fibroblasts and evolution of thick elastic membrane in the pleural cavity (the "peel"). These membranes may impair lung function and prevent lung re-expansion. Empyema at this stage may heal spontaneously or a chronic empyema

Some authors distinguish "pleuritis sicca stage" that precedes exudative stage, not necessa‐

damaged epithelium and if left untreated it progresses into

should be considered [7].

148 Respiratory Disease and Infection - A New Insight

**12. Complications**

ess.

may develop [46].

rily leading to it [53].

**12.1. Empyema and parapneumonic effusion**

pyema classically exhibits three stages:

Chest radiographs show homogenous opacity over the entire lung (large effusion) (Figure 3). In smaller effusions an ascending rim of fluid along the lateral chest wall (meniscus sign) occurs. Costophrenical angle obliteration is the first sign of pleural effusion. Based on radio‐ graph it is not possible to distinguish effusion from empyema [47]. A method of choice for radiologic evaluation of patients with parapneumonic effusion and empyema is ultrasonog‐ raphy. It helps estimate the amount of fluid, its echogenicity, detects loculations and fibrin strands and is used to guide invasive procedures [28]. Chest CT should not be routinely per‐ formed, it may be useful however for diagnosis of underlying pathology eg. tumor in the mediastinum or lung abscess [46,53].

**Figure 3.** Opacification of left hemithorax with mediastinal shift to the opposite side. Alveolar consolidations in the central field of the right lobe. Lobar pneumonia with pleural effusion caused by *Streptococcus pneumoniae*

**•** Small: < 10 mm on lateral decubitus radiograph or opacifies < ¼ hemithorax

Conservative treatment with antibiotics is recommended for small effusions. Antibiotic se‐ lection is based on blood or pleural fluid culture results, and if these are not available, on treatment guidelines. Many patients improve with conservative treatment alone. In an American study over 50% of all patients with moderate to severe effusions recovered with antibiotic treatment alone [54]. Management of moderate effusions depends on child's de‐ gree of respiratory compromise: if clinical condition is good, treatment with antibiotics is ap‐ propriate and if the child presents signs of respiratory distress, treatment is the same as for large effusions: fluid should be removed either by tube thoracocentesis (for not loculated fluid) or chest tube with fibrinolytics or VATS (both for loculated fluid) [19]. Once the chest tube is inserted, no more than 10 ml/kg of fluid in little children and 1.5 liters of fluid in old‐ er children and adolescents should be removed in order to avoid re-expansion pulmonary edema. When this volume is reached, the drain should be clamped for an hour [46]. There is no clear evidence on advantage in clinical outcome of children treated with fibrinolytic agents versus VATS [55,56]. The recommended doses for fibrinolytic agents are [19,57,58]:

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**•** Urokinase (not available in USA) 10,000 U every 12 hours for 3 days in children < 1 year

**•** Tissue plasminogen activator 0.1 mg/kg; maximum of 3 mg three times a day for 3 to 4

Children who fail to improve despite antibiotics, drainage and fibrinolytics, should undergo VATS in order to debride fibrinous adhesions and remove dense loculated fluid. It seems prudent to ask for surgical opinion if the patient is not improving after 7 days of treatment. Another indication for surgery is bronchopleural fistula with pyopneumothorax [46]. As an alternative to VATS, especially in organized empyema in a child with non-resolving signs of systemic infection with fever, formal thoracotomy with decortications should be considered [19]. According to BTS guidelines indications for surgery referral are clinical signs and symptoms and not aberrant radiologic picture in an asymptomatic child [46]. Risk factors for the failure of tube thoracostomy include duration of symptoms > 7 days before the proce‐ dure, complex multiloculated empyema, pneumatocele, pulmonary necrosis and an under‐ lying medical condition [59,60]. A chest tube can be removed if fluid output is < 1 ml/kg/day

A more aggressive approach is to perform VATS in the first 48 hours of treatment. That gives a chance for bacteriological diagnosis and shorter hospital stay, though not all studies

**•** Moderate: >10 mm rim of fluid and opacifies < ½ hemithorax

and 40,000 U every 12 hours for 3 days in children > 1 year

With streptokinase and urokinase there is risk of hypersensitivity reactions.

calculated over the last 12 hours or 50-60 ml/day and there is no air leak [19].

**•** Streptokinase 12,000 - 25,000 IU/kg/dose daily for 3-5 days

**•** Large: opacifies > ½ hemithorax

days or 4 mg every 24 hours

confirm the latter observation [54].

**Figure 4.** Multiple abscesses in the right upper lobe. Diffuse alveolar consolidations with thickening of intraalveolar spaces in the middle and lower right lobes. Fluid in the right pleural space. *Staphylococcus aureus* pneumonia with lung abscesses and pleural effusion.

Pleural fluid, if obtained during thoracocentesis or video-assisted thoracoscopic surgery (VATS), should be sent for culture, Gram stain, cytology and molecular techniques if availa‐ ble. Bacteriological investigations should always be undertaken even though they are posi‐ tive in ¼ of cases since they may provide useful information guiding antibiotic therapy. Stain for acid-fast bacilli, culture and PCR should also be performed [46]. In most cases of bacterial empyema polymorphonuclear leukocytes are the predominant cells. In case of ma‐ lignancy the fluid may be blood stained with lymphocytic predominance, although malig‐ nant cells may not be present. In tuberculosis there is also predominance of lymphocytes in pleural fluid although in 10% of cases effusion might be neutrophilic [46]. Light criteria, use‐ ful for treatment guidance in adult patients, have not been properly validated in children and their routine use is not recommended [46,53].

All children with empyema or pleural effusion should be treated as inpatients [46]. There is no consensus however as to what is optimal treatment of empyema: antibiotics alone for small to moderate effusions, chest tube insertion with or without fibrinolytics or VATS for moderate to severe cases. Differences in management result to some extent from personal experience and availability of different treatment modalities, including experienced inter‐ ventional radiologists and pediatric thoracic surgeons. Generally, two most important fac‐ tors determining the need for chest tube insertion are the size of effusion and the child's degree of respiratory compromise [19]. There is agreement that due to an invasive nature of the procedure and the need for general anesthesia in younger children, a drain should be inserted instead of repeated needle thoracocenteses [46]. According to PIDS, pleural effu‐ sions can be divided depending on their size into:


**Figure 4.** Multiple abscesses in the right upper lobe. Diffuse alveolar consolidations with thickening of intraalveolar spaces in the middle and lower right lobes. Fluid in the right pleural space. *Staphylococcus aureus* pneumonia with

Pleural fluid, if obtained during thoracocentesis or video-assisted thoracoscopic surgery (VATS), should be sent for culture, Gram stain, cytology and molecular techniques if availa‐ ble. Bacteriological investigations should always be undertaken even though they are posi‐ tive in ¼ of cases since they may provide useful information guiding antibiotic therapy. Stain for acid-fast bacilli, culture and PCR should also be performed [46]. In most cases of bacterial empyema polymorphonuclear leukocytes are the predominant cells. In case of ma‐ lignancy the fluid may be blood stained with lymphocytic predominance, although malig‐ nant cells may not be present. In tuberculosis there is also predominance of lymphocytes in pleural fluid although in 10% of cases effusion might be neutrophilic [46]. Light criteria, use‐ ful for treatment guidance in adult patients, have not been properly validated in children

All children with empyema or pleural effusion should be treated as inpatients [46]. There is no consensus however as to what is optimal treatment of empyema: antibiotics alone for small to moderate effusions, chest tube insertion with or without fibrinolytics or VATS for moderate to severe cases. Differences in management result to some extent from personal experience and availability of different treatment modalities, including experienced inter‐ ventional radiologists and pediatric thoracic surgeons. Generally, two most important fac‐ tors determining the need for chest tube insertion are the size of effusion and the child's degree of respiratory compromise [19]. There is agreement that due to an invasive nature of the procedure and the need for general anesthesia in younger children, a drain should be inserted instead of repeated needle thoracocenteses [46]. According to PIDS, pleural effu‐

lung abscesses and pleural effusion.

150 Respiratory Disease and Infection - A New Insight

and their routine use is not recommended [46,53].

sions can be divided depending on their size into:

Conservative treatment with antibiotics is recommended for small effusions. Antibiotic se‐ lection is based on blood or pleural fluid culture results, and if these are not available, on treatment guidelines. Many patients improve with conservative treatment alone. In an American study over 50% of all patients with moderate to severe effusions recovered with antibiotic treatment alone [54]. Management of moderate effusions depends on child's de‐ gree of respiratory compromise: if clinical condition is good, treatment with antibiotics is ap‐ propriate and if the child presents signs of respiratory distress, treatment is the same as for large effusions: fluid should be removed either by tube thoracocentesis (for not loculated fluid) or chest tube with fibrinolytics or VATS (both for loculated fluid) [19]. Once the chest tube is inserted, no more than 10 ml/kg of fluid in little children and 1.5 liters of fluid in old‐ er children and adolescents should be removed in order to avoid re-expansion pulmonary edema. When this volume is reached, the drain should be clamped for an hour [46]. There is no clear evidence on advantage in clinical outcome of children treated with fibrinolytic agents versus VATS [55,56]. The recommended doses for fibrinolytic agents are [19,57,58]:


With streptokinase and urokinase there is risk of hypersensitivity reactions.

Children who fail to improve despite antibiotics, drainage and fibrinolytics, should undergo VATS in order to debride fibrinous adhesions and remove dense loculated fluid. It seems prudent to ask for surgical opinion if the patient is not improving after 7 days of treatment. Another indication for surgery is bronchopleural fistula with pyopneumothorax [46]. As an alternative to VATS, especially in organized empyema in a child with non-resolving signs of systemic infection with fever, formal thoracotomy with decortications should be considered [19]. According to BTS guidelines indications for surgery referral are clinical signs and symptoms and not aberrant radiologic picture in an asymptomatic child [46]. Risk factors for the failure of tube thoracostomy include duration of symptoms > 7 days before the proce‐ dure, complex multiloculated empyema, pneumatocele, pulmonary necrosis and an under‐ lying medical condition [59,60]. A chest tube can be removed if fluid output is < 1 ml/kg/day calculated over the last 12 hours or 50-60 ml/day and there is no air leak [19].

A more aggressive approach is to perform VATS in the first 48 hours of treatment. That gives a chance for bacteriological diagnosis and shorter hospital stay, though not all studies confirm the latter observation [54].

The optimal duration of antibiotic treatment for parapneumonic effusion and empyema de‐ pends on clinical response. Recommended route of administration is intravenous until the chest tube is removed, and then can be switched to oral route for 1 to 4 weeks or longer if the child has not fully recovered. However, there are no randomized clinical trials to sup‐ port this approach [19,46]. Long-term outcome in children is favorable. Radiological evi‐ dence of pleural disease completely resolves within 3 months in up to 80% of children and by 18 months in all children. Lung function tests results as well as exercise tolerance in most patients are normal 12 months after discharge [46,52,53]. Conditions predisposing to severe pneumonia with pleural effusion and empyema include immunodeficiencies and cystic fib‐ rosis and they should be excluded during follow-up period [46].

#### **12.2. Lung abscess**

Lung abscess is a thick-walled cavity containing necrotic tissue 2 cm or greater in diame‐ ter caused by an infection [28]. It may be either primary – occurring in healthy children without lung abnormalities or secondary – occurring in children with underlying condi‐ tion predisposing to lung disease. The most important mechanism of lung abscess forma‐ tion is aspiration, especially in children with neuromuscular disorders. Other risk factors include immunodeficiencies, underlying lung disease like congenital malformations, cyst‐ ic fibrosis, swallowing problems, eg. achalasia, poor dental hygiene. Abscesses may also ensue by hematogenous spread from septicemia or right-sided bacterial endocarditis, ex‐ tension from foci in abdominal cavity or retropharyngeal space or from airway obstruc‐ tion by foreign body [61,62].

**Figure 5.** Infiltrate with air-fluid level in the upper field of the right lobe. Lung abscess.

improve despite medical treatment.

and localized bronchiectasis.

**12.3. Necrotising pneumonia**

The mainstay of treatment is conservative antibiotic therapy with spectrum covering *S. pneumoniae, S. aureus* and Gram-negative bacilli and anaerobes in case of secondary abscess. For immunocompromised patients antibiotics should cover fungal pathogens. Antibiotic of choice is penicillin with clindamycin or metronidazole. Other experts recommend third-gen‐ eration cephalosporin and flucloxacillin, ticarcillin, ampicillin/clavulanic acid and piperacil‐ lin/tazobactam. One should consider the possibility of MRSA infection, especially if the abscess complicates pneumonia or results from hematogenous spread from other organs [61,62]. A 2-3 week course of intravenous therapy followed by oral treatment for 4 to 8 weeks is usually recommended [28]. In experienced interventional radiology centers CTguided aspiration of the lung abscess and placement of pigtail catheter is performed for di‐ agnostic and therapeutic reasons. Surgical intervention is indicated for abscesses failing to

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The overall outcome is favorable, mortality being much lower than in adults: <5% and most‐ ly occurring in children with secondary lung abscesses or underlying medical problems. The complications include empyema or pyopneumothorax if abscess ruptures into pleural cavi‐ ty, bronchopleural fistula if connection between the abscess cavity and pleural space persists

Necrotizing pneumonia (NP), defined as multiple cavitary lesions in consolidated areas, is a rare, though increasingly detected complication in children. It is characterized by liquefac‐

The main causative organisms are usually streptococci, anaerobic bacteria, *S. aureus* and *Klebsiella pneumonia*, however there are rare reports of other causative organisms including *Mycoplasma pneumoniae* [63]. Mixed infections are common. The most frequent sites for lung abscess formation in recumbent position are: the right upper lobe, the left lower lobe and the apical segments of both lower lobes. When the patient aspirates in supine position the poste‐ rior segments of the upper lobes are usually involved.

Clinical symptoms include cough, purulent sputum production, fever, dyspnea, chest pain, tachypnoe, weight loss, hemoptysis, malaise/lethargy. Physical signs do not differ from un‐ complicated pneumonia, decreased breath sounds and dull note on percussion may be ap‐ preciated. Symptoms may persist for several weeks [61].

Diagnosis is usually made by chest radiograph showing an inflammatory infiltrate of the pulmonary parenchyma with a cavity containing an air-fluid level. Initially it may appear as a solid lesion surrounded by an alveolar infiltrate. Bulging fissure representing increased volume of the affected lobe may be present. CT is usually performed to exclude other com‐ plications like empyema, pneumatocoele, underlying congenital abnormality like sequestra‐ tion, bronchogenic cyst or adenomatoid malformation. Features distinguishing abscess from other entities include well-marginated walls, density greater than water, contrast enhance‐ ment in adjacent tissues (Figure 4, Figure 5) [61].

**Figure 5.** Infiltrate with air-fluid level in the upper field of the right lobe. Lung abscess.

The mainstay of treatment is conservative antibiotic therapy with spectrum covering *S. pneumoniae, S. aureus* and Gram-negative bacilli and anaerobes in case of secondary abscess. For immunocompromised patients antibiotics should cover fungal pathogens. Antibiotic of choice is penicillin with clindamycin or metronidazole. Other experts recommend third-gen‐ eration cephalosporin and flucloxacillin, ticarcillin, ampicillin/clavulanic acid and piperacil‐ lin/tazobactam. One should consider the possibility of MRSA infection, especially if the abscess complicates pneumonia or results from hematogenous spread from other organs [61,62]. A 2-3 week course of intravenous therapy followed by oral treatment for 4 to 8 weeks is usually recommended [28]. In experienced interventional radiology centers CTguided aspiration of the lung abscess and placement of pigtail catheter is performed for di‐ agnostic and therapeutic reasons. Surgical intervention is indicated for abscesses failing to improve despite medical treatment.

The overall outcome is favorable, mortality being much lower than in adults: <5% and most‐ ly occurring in children with secondary lung abscesses or underlying medical problems. The complications include empyema or pyopneumothorax if abscess ruptures into pleural cavi‐ ty, bronchopleural fistula if connection between the abscess cavity and pleural space persists and localized bronchiectasis.

#### **12.3. Necrotising pneumonia**

The optimal duration of antibiotic treatment for parapneumonic effusion and empyema de‐ pends on clinical response. Recommended route of administration is intravenous until the chest tube is removed, and then can be switched to oral route for 1 to 4 weeks or longer if the child has not fully recovered. However, there are no randomized clinical trials to sup‐ port this approach [19,46]. Long-term outcome in children is favorable. Radiological evi‐ dence of pleural disease completely resolves within 3 months in up to 80% of children and by 18 months in all children. Lung function tests results as well as exercise tolerance in most patients are normal 12 months after discharge [46,52,53]. Conditions predisposing to severe pneumonia with pleural effusion and empyema include immunodeficiencies and cystic fib‐

Lung abscess is a thick-walled cavity containing necrotic tissue 2 cm or greater in diame‐ ter caused by an infection [28]. It may be either primary – occurring in healthy children without lung abnormalities or secondary – occurring in children with underlying condi‐ tion predisposing to lung disease. The most important mechanism of lung abscess forma‐ tion is aspiration, especially in children with neuromuscular disorders. Other risk factors include immunodeficiencies, underlying lung disease like congenital malformations, cyst‐ ic fibrosis, swallowing problems, eg. achalasia, poor dental hygiene. Abscesses may also ensue by hematogenous spread from septicemia or right-sided bacterial endocarditis, ex‐ tension from foci in abdominal cavity or retropharyngeal space or from airway obstruc‐

The main causative organisms are usually streptococci, anaerobic bacteria, *S. aureus* and *Klebsiella pneumonia*, however there are rare reports of other causative organisms including *Mycoplasma pneumoniae* [63]. Mixed infections are common. The most frequent sites for lung abscess formation in recumbent position are: the right upper lobe, the left lower lobe and the apical segments of both lower lobes. When the patient aspirates in supine position the poste‐

Clinical symptoms include cough, purulent sputum production, fever, dyspnea, chest pain, tachypnoe, weight loss, hemoptysis, malaise/lethargy. Physical signs do not differ from un‐ complicated pneumonia, decreased breath sounds and dull note on percussion may be ap‐

Diagnosis is usually made by chest radiograph showing an inflammatory infiltrate of the pulmonary parenchyma with a cavity containing an air-fluid level. Initially it may appear as a solid lesion surrounded by an alveolar infiltrate. Bulging fissure representing increased volume of the affected lobe may be present. CT is usually performed to exclude other com‐ plications like empyema, pneumatocoele, underlying congenital abnormality like sequestra‐ tion, bronchogenic cyst or adenomatoid malformation. Features distinguishing abscess from other entities include well-marginated walls, density greater than water, contrast enhance‐

rosis and they should be excluded during follow-up period [46].

**12.2. Lung abscess**

152 Respiratory Disease and Infection - A New Insight

tion by foreign body [61,62].

rior segments of the upper lobes are usually involved.

preciated. Symptoms may persist for several weeks [61].

ment in adjacent tissues (Figure 4, Figure 5) [61].

Necrotizing pneumonia (NP), defined as multiple cavitary lesions in consolidated areas, is a rare, though increasingly detected complication in children. It is characterized by liquefac‐ tion and cavitation of pulmonary tissue [63]. The most frequently associated pathogen is *Streptococcus pneumoniae*, especially serotypes 3 and 14. Other pathogens involved include group A *Streptococci, Staphylococcus aureus* and *Mycoplasma pneumoniae* [64-68]. The majority of patients have no prior medical history. Necrotizing pneumonia should be suspected in patients with complicated pneumonia who do not improve despite optimal medical treat‐ ment. Diagnosis can be established by computed tomography. Radiographic criteria for ne‐ crotizing pneumonia include the loss of normal pulmonary parenchymal architecture and the presence of areas of liquefaction replaced within 1-2 days by multiple small cavities [64]. Necrotizing pneumonia often coexists with pleural effusion.

with an underlying medical condition that increases the risk of invasive pneumococcal dis‐ ease (IPD) [69]. Underlying medical conditions that indicate the need for pneumococcal im‐

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**•** chronic heart disease, in particular cyanotic congenital heart disease and cardiac failure

**•** chronic lung disease including asthma if treated with prolonged high-dose oral corticoste‐

**•** functional or anatomical asplenia including children with sickle cell disease and other he‐

**•** immunocompromising conditions: HIV infection, chronic renal failure and nephrotic syn‐ drome, diseases associated with treatment with immunosuppressive drugs or radiation therapy (including malignant neoplasms, leukemias, lymphomas, Hodgkin disease, solid organ transplantation) and congenital immunodeficiency (including B- (humoral) or Tlymphocyte deficiency; complement deficiencies, particularly C1, C2, C3 and C4 and

Healthy children <5 and children with underlying medical conditions <6, who are fully immunized with PCV7 should receive a single supplemental dose of PCV13. Children between 6 and 18 with medical conditions favoring IPD (listed above) should receive a single dose of PCV13 regardless of whether they have previously received PCV7 or PPSV23 (2 doses of PPSV23 recommended). PCV13 which in addition to the 7 serotypes included in PCV7 (4,6B,9V,14,18C,19F,23F) contains the 6 pneumococcal serotypes (1,3,5,6A,7F,19A) responsible for 63% of cases of invasive pneumococcal disease occur‐ ring in children <5 in the USA has been licensed by the US Food and Drug Administra‐ tion in 2010 for use in children between 2 and 71 months of age. Because of the

*Streptococcus pneumoniae* is the most common pathogen in CAP in children and the most common cause of pneumonia mortality in children worldwide. It is responsible for at least 1.2 million deaths in infants annually, mostly in sub-Saharan Africa and Asia [45]. There are 92 known pneumococcal serotypes that differ by polysaccharide capsule. It was found that serotypes are correlated with different pneumonia outcomes, study results are not however equivocal. In pediatric patients serotypes 7F, 23F and 3 were correlated with the highest risk of death in the course of invasive pneumococcal disease [70]. In another study serotypes

phagocytic disorders excluding chronic granulomatous disease).

expended coverage provided it is meant to replace PCV7 [69].

**14. Specific bacterial causes of pneumonia**

**14.1.** *Streptococcus pneumoniae*

munization comprise:

**•** diabetes mellitus

**•** cochlear implant

moglobinopathies

**•** cerebrospinal fluid leaks

roids

Treatment consists in prolonged course of intravenous antibiotics active against CAP patho‐ gens including resistant strains of *S. pneumoniae*. Interventional procedures are contraindi‐ cated in children with NP, as they may increase the risk of complications such as bronchopleural fistula formation [35]. Generally, despite prolonged hospital course and as‐ sociated morbidity, the long term outcome in most children is favorable. Mortality rates are 5.5-7% [28,65].

#### **13. Prevention**

In order to prevent pneumonia several measures can be taken, starting with general recom‐ mendations like improving nourishment, housing conditions, heating systems, reducing to‐ bacco smoke exposure, promoting breast-feeding for the first 6 months of age, to more specific infection control measures like hand-washing, avoiding individuals with signs of respiratory tract infections, and vaccinations. For prevention of pneumonia immunization against the following microorganisms is recommended:


High risk infants: prematurely born (<35 week of GA), with hemodynamically significant congenital heart disease, bronchopulmonary dysplasia, congenital abnormalities of the air‐ ways and neuromuscular diseases should receive immune prophylaxis with RSV specific monoclonal antibody (palivizumab) in RSV season [68].

AAP recommends the routine use of 13-valent pneumococcal conjugate vaccine (PCV13) for healthy children 2 through 59 months of age and for children 60 through 71 months of age with an underlying medical condition that increases the risk of invasive pneumococcal dis‐ ease (IPD) [69]. Underlying medical conditions that indicate the need for pneumococcal im‐ munization comprise:


tion and cavitation of pulmonary tissue [63]. The most frequently associated pathogen is *Streptococcus pneumoniae*, especially serotypes 3 and 14. Other pathogens involved include group A *Streptococci, Staphylococcus aureus* and *Mycoplasma pneumoniae* [64-68]. The majority of patients have no prior medical history. Necrotizing pneumonia should be suspected in patients with complicated pneumonia who do not improve despite optimal medical treat‐ ment. Diagnosis can be established by computed tomography. Radiographic criteria for ne‐ crotizing pneumonia include the loss of normal pulmonary parenchymal architecture and the presence of areas of liquefaction replaced within 1-2 days by multiple small cavities [64].

Treatment consists in prolonged course of intravenous antibiotics active against CAP patho‐ gens including resistant strains of *S. pneumoniae*. Interventional procedures are contraindi‐ cated in children with NP, as they may increase the risk of complications such as bronchopleural fistula formation [35]. Generally, despite prolonged hospital course and as‐ sociated morbidity, the long term outcome in most children is favorable. Mortality rates are

In order to prevent pneumonia several measures can be taken, starting with general recom‐ mendations like improving nourishment, housing conditions, heating systems, reducing to‐ bacco smoke exposure, promoting breast-feeding for the first 6 months of age, to more specific infection control measures like hand-washing, avoiding individuals with signs of respiratory tract infections, and vaccinations. For prevention of pneumonia immunization

High risk infants: prematurely born (<35 week of GA), with hemodynamically significant congenital heart disease, bronchopulmonary dysplasia, congenital abnormalities of the air‐ ways and neuromuscular diseases should receive immune prophylaxis with RSV specific

AAP recommends the routine use of 13-valent pneumococcal conjugate vaccine (PCV13) for healthy children 2 through 59 months of age and for children 60 through 71 months of age

Necrotizing pneumonia often coexists with pleural effusion.

154 Respiratory Disease and Infection - A New Insight

against the following microorganisms is recommended:

monoclonal antibody (palivizumab) in RSV season [68].

**•** *Haemophilus influenzae* (conjugate vaccine)

**•** *Streptococcus pneumoniae* (conjugate and non-conjugate vaccine)

5.5-7% [28,65].

**13. Prevention**

**•** influenza virus

**•** measles virus

**•** varicella virus

**•** *Bordatella pertussiss*

**•** *Mycobacterium tuberculosis*


Healthy children <5 and children with underlying medical conditions <6, who are fully immunized with PCV7 should receive a single supplemental dose of PCV13. Children between 6 and 18 with medical conditions favoring IPD (listed above) should receive a single dose of PCV13 regardless of whether they have previously received PCV7 or PPSV23 (2 doses of PPSV23 recommended). PCV13 which in addition to the 7 serotypes included in PCV7 (4,6B,9V,14,18C,19F,23F) contains the 6 pneumococcal serotypes (1,3,5,6A,7F,19A) responsible for 63% of cases of invasive pneumococcal disease occur‐ ring in children <5 in the USA has been licensed by the US Food and Drug Administra‐ tion in 2010 for use in children between 2 and 71 months of age. Because of the expended coverage provided it is meant to replace PCV7 [69].

#### **14. Specific bacterial causes of pneumonia**

#### **14.1.** *Streptococcus pneumoniae*

*Streptococcus pneumoniae* is the most common pathogen in CAP in children and the most common cause of pneumonia mortality in children worldwide. It is responsible for at least 1.2 million deaths in infants annually, mostly in sub-Saharan Africa and Asia [45]. There are 92 known pneumococcal serotypes that differ by polysaccharide capsule. It was found that serotypes are correlated with different pneumonia outcomes, study results are not however equivocal. In pediatric patients serotypes 7F, 23F and 3 were correlated with the highest risk of death in the course of invasive pneumococcal disease [70]. In another study serotypes 1,6,14,19 were the most prevalent among children with complicated pneumonia, with sero‐ type 1 causing 24.4% of the complicated cases versus 3.6% of the uncomplicated cases [71].

Since universal introduction of pneumococcal conjugated 7-valent vaccine in 2000, there has been a decrease in incidence of pneumococcal pneumonia in children <5 [7]. There is, how‐ ever, concern that the serotypes included in the vaccine can be replaced with previously rare, potentially more virulent serotypes like serotype 1 or 19A [73,74]. Fortunately, these se‐

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There is a problem of increasing antibacterial resistance – up to 10% of cultured pneu‐ mococcal isolates in 2008 in Europe are not susceptible to penicillin, though the impact of *in vitro* resistance on clinical outcome of patients is not that clear. Fortunately, there was no increase in mortality or complication rate reported in children infected with re‐

The incidence of *Staphylococcus aureus* pneumonia has increased significantly during the past 20 years. An increasing number (up to 76% in Texas) of community-associated *S. aureus* is methicillin resistant (CA-MRSA) and in some regions it has become the main cause of complicated CAP in children [75-77]. CA-MRSA were described for the first time in the 1990s as a cause of infection in previously healthy young children and ado‐ lescents with no prior hospitalization or record with chronic healthcare facilities [77]. There is also significant increase in nasal colonization of healthy children with MRSA – in another study from the USA 36.4% of healthy children were colonized with *S. aureus* and 9.2% with MRSA. CA-MRSA has its own genotype different from hospital acquired strains [77]. It is worth noticing that 22% of MRSA strains had gene locus for PVL, and in a British study 11% of all *S. aureus* isolates from pneumonia patients carried the gene [78,79]. Even if the impact of nasal colonization on the risk of pneumonia is not clear, since 1/3 of patients with staphylococcal infection had no prior colonization documented

Primary *S. aureus* pneumonia results from direct invasion of the lungs through the tracheo‐ bronchial tree, and secondary pneumonia results from hematogenous spread. *S. aureus* CAP typically occurs in very young infants: 30% of cases occur in children younger than 3 months of age and 70% in those <1 year of age, more often in boys [60].CA-MRSA CAP fre‐ quently occurs in previously healthy children and adolescents and in many cases is preced‐ ed by influenza or flu-like illness or skin and soft tissue infection. Severe respiratory symptoms and hypotension develop rapidly. The USA300 clone is associated with venous

*S. aureus* possesses a variety of virulence factors including surface proteins (eg. protein A) that promote adherence and hence colonization of host tissues, invasions (leukocidin, kinas‐ es and hyaluronidase) that promote bacterial spread in tissues and membrane damaging toxins (eg. mentioned above leukocidin). There have been several reports in previously healthy children and adolescents of pneumonia caused by Panton – Valentine leukocidin producing *Staphylococcus aureus*. The pore–forming toxin encoded by *luk-S-PV* and *luk-F-PV* genes lyses neutrophils causing exaggerated though ineffective inflammatory response. Pa‐ tients present with rapidly progressing necrotizing pneumonia manifested by fever, leuco‐

rotypes have been included in the newer conjugated 13-valent vaccine.

[80], these data show a wide distribution of MRSA in the community.

thrombosis and subsequent septic pulmonary emboli [77].

sistant strains [71,73,74].

**14.2.** *Staphylococcus aureus*

*S. pneumoniae* commonly colonizes epithelium of nasopharynx in 20 - 40% of healthy chil‐ dren and > 60% of infants and children in day-care settings. After colonization a new strain eliminates other competing pneumococcal serotypes and persists for months in a carrier state. Bacteria with so called "persistent colonization phenotype", with low risk of tissue in‐ vasion are responsible for perpetual transmission within human populations and induce ac‐ quired B-cell mediated immunity to reinfection. To facilitate their stay within nasopharynx and evade host defenses they use different mechanisms like surface adhesions, IgA1 pro‐ tease and inhibitors of antibacterial peptides. Defects in host defense mechanisms can de‐ stroy the balance and lead to infection in immunocompromised host. Another phenotype – so called "invasive pneumococcal disease phenotype" is able to spread efficiently from per‐ son to person by coughing and rapidly induce the disease. Its main virulence factor is poly‐ saccharide capsule that prevents mechanical clearance by mucous secretions, restricts autolysis, reduces exposure to antibiotics and facilitates invasion and dissemination. Other virulence factors include: pore-forming cytotoxin – pneumolysin that among other patho‐ logical effects is able to inhibit cilliary movement of epithelial cells and impairs respiratory burst of phagocytic cells. Recent acquisition of an invasive serotype is more important in terms of further infection than long-term colonization and is in fact recognized as one of def‐ inite risk factors for pneumococcal pneumonia [45].

Some of the host immune mechanisms essential for defense against pneumococcal pneumo‐ nia are toll-like receptors (TLRs). Children with genetic deficiency of the common TLRadaptor protein – myeloid differentiation primary-response protein 88 (MyD88) or interleukin-1 receptor – associated kinase 4 (IRAK4), a kinase acting directly downstream from MyD88, are especially susceptible to invasive pneumococcal disease. Another genetic factor predisposing to invasive pneumococcal disease is polymorphism of genes coding in‐ hibitors of nuclear factor κΒ and defects in the complement C3 pathway crucial for opsoni‐ zation and consequently clearance of pneumococci [45].

Pneumonia usually begins with viral upper respiratory tract infection. The pathogen most commonly associated with dual infections with *Streptococcus pneumonia* is influenza virus. Co-infection with influenza virus attenuates host immune response diminishing its ability to clear pneumococcus. Influenza virus possesses neuraminidase that by exposing certain re‐ ceptors facilitates pneumococcus' adherence to respiratory epithelium [72]. Local innate im‐ mune defense systems like mucociliary clearance, cough reflex, antimicrobial peptides usually succeed in eliminating the pathogen. Should they fail, pneumonia ensues. Clinical symptoms are characteristic for bacterial pneumonia with high fever, chills, malaise, cough and dyspnoea. Cough becomes productive in older children, with purulent, blood tinged sputum. Pleural involvement is quite common. Untreated pneumococcal pneumonia may progress to respiratory failure, septic shock and consequently death.

The usual radiological presentation of pneumococcal pneumonia is lobar pneumonia, fre‐ quently accompanied by small pleural effusion. Changes may be confined to a single seg‐ ment or involve several segments or lobes or present as bronchopneumonia [41].

Since universal introduction of pneumococcal conjugated 7-valent vaccine in 2000, there has been a decrease in incidence of pneumococcal pneumonia in children <5 [7]. There is, how‐ ever, concern that the serotypes included in the vaccine can be replaced with previously rare, potentially more virulent serotypes like serotype 1 or 19A [73,74]. Fortunately, these se‐ rotypes have been included in the newer conjugated 13-valent vaccine.

There is a problem of increasing antibacterial resistance – up to 10% of cultured pneu‐ mococcal isolates in 2008 in Europe are not susceptible to penicillin, though the impact of *in vitro* resistance on clinical outcome of patients is not that clear. Fortunately, there was no increase in mortality or complication rate reported in children infected with re‐ sistant strains [71,73,74].

#### **14.2.** *Staphylococcus aureus*

1,6,14,19 were the most prevalent among children with complicated pneumonia, with sero‐ type 1 causing 24.4% of the complicated cases versus 3.6% of the uncomplicated cases [71]. *S. pneumoniae* commonly colonizes epithelium of nasopharynx in 20 - 40% of healthy chil‐ dren and > 60% of infants and children in day-care settings. After colonization a new strain eliminates other competing pneumococcal serotypes and persists for months in a carrier state. Bacteria with so called "persistent colonization phenotype", with low risk of tissue in‐ vasion are responsible for perpetual transmission within human populations and induce ac‐ quired B-cell mediated immunity to reinfection. To facilitate their stay within nasopharynx and evade host defenses they use different mechanisms like surface adhesions, IgA1 pro‐ tease and inhibitors of antibacterial peptides. Defects in host defense mechanisms can de‐ stroy the balance and lead to infection in immunocompromised host. Another phenotype – so called "invasive pneumococcal disease phenotype" is able to spread efficiently from per‐ son to person by coughing and rapidly induce the disease. Its main virulence factor is poly‐ saccharide capsule that prevents mechanical clearance by mucous secretions, restricts autolysis, reduces exposure to antibiotics and facilitates invasion and dissemination. Other virulence factors include: pore-forming cytotoxin – pneumolysin that among other patho‐ logical effects is able to inhibit cilliary movement of epithelial cells and impairs respiratory burst of phagocytic cells. Recent acquisition of an invasive serotype is more important in terms of further infection than long-term colonization and is in fact recognized as one of def‐

Some of the host immune mechanisms essential for defense against pneumococcal pneumo‐ nia are toll-like receptors (TLRs). Children with genetic deficiency of the common TLRadaptor protein – myeloid differentiation primary-response protein 88 (MyD88) or interleukin-1 receptor – associated kinase 4 (IRAK4), a kinase acting directly downstream from MyD88, are especially susceptible to invasive pneumococcal disease. Another genetic factor predisposing to invasive pneumococcal disease is polymorphism of genes coding in‐ hibitors of nuclear factor κΒ and defects in the complement C3 pathway crucial for opsoni‐

Pneumonia usually begins with viral upper respiratory tract infection. The pathogen most commonly associated with dual infections with *Streptococcus pneumonia* is influenza virus. Co-infection with influenza virus attenuates host immune response diminishing its ability to clear pneumococcus. Influenza virus possesses neuraminidase that by exposing certain re‐ ceptors facilitates pneumococcus' adherence to respiratory epithelium [72]. Local innate im‐ mune defense systems like mucociliary clearance, cough reflex, antimicrobial peptides usually succeed in eliminating the pathogen. Should they fail, pneumonia ensues. Clinical symptoms are characteristic for bacterial pneumonia with high fever, chills, malaise, cough and dyspnoea. Cough becomes productive in older children, with purulent, blood tinged sputum. Pleural involvement is quite common. Untreated pneumococcal pneumonia may

The usual radiological presentation of pneumococcal pneumonia is lobar pneumonia, fre‐ quently accompanied by small pleural effusion. Changes may be confined to a single seg‐

ment or involve several segments or lobes or present as bronchopneumonia [41].

inite risk factors for pneumococcal pneumonia [45].

156 Respiratory Disease and Infection - A New Insight

zation and consequently clearance of pneumococci [45].

progress to respiratory failure, septic shock and consequently death.

The incidence of *Staphylococcus aureus* pneumonia has increased significantly during the past 20 years. An increasing number (up to 76% in Texas) of community-associated *S. aureus* is methicillin resistant (CA-MRSA) and in some regions it has become the main cause of complicated CAP in children [75-77]. CA-MRSA were described for the first time in the 1990s as a cause of infection in previously healthy young children and ado‐ lescents with no prior hospitalization or record with chronic healthcare facilities [77]. There is also significant increase in nasal colonization of healthy children with MRSA – in another study from the USA 36.4% of healthy children were colonized with *S. aureus* and 9.2% with MRSA. CA-MRSA has its own genotype different from hospital acquired strains [77]. It is worth noticing that 22% of MRSA strains had gene locus for PVL, and in a British study 11% of all *S. aureus* isolates from pneumonia patients carried the gene [78,79]. Even if the impact of nasal colonization on the risk of pneumonia is not clear, since 1/3 of patients with staphylococcal infection had no prior colonization documented [80], these data show a wide distribution of MRSA in the community.

Primary *S. aureus* pneumonia results from direct invasion of the lungs through the tracheo‐ bronchial tree, and secondary pneumonia results from hematogenous spread. *S. aureus* CAP typically occurs in very young infants: 30% of cases occur in children younger than 3 months of age and 70% in those <1 year of age, more often in boys [60].CA-MRSA CAP fre‐ quently occurs in previously healthy children and adolescents and in many cases is preced‐ ed by influenza or flu-like illness or skin and soft tissue infection. Severe respiratory symptoms and hypotension develop rapidly. The USA300 clone is associated with venous thrombosis and subsequent septic pulmonary emboli [77].

*S. aureus* possesses a variety of virulence factors including surface proteins (eg. protein A) that promote adherence and hence colonization of host tissues, invasions (leukocidin, kinas‐ es and hyaluronidase) that promote bacterial spread in tissues and membrane damaging toxins (eg. mentioned above leukocidin). There have been several reports in previously healthy children and adolescents of pneumonia caused by Panton – Valentine leukocidin producing *Staphylococcus aureus*. The pore–forming toxin encoded by *luk-S-PV* and *luk-F-PV* genes lyses neutrophils causing exaggerated though ineffective inflammatory response. Pa‐ tients present with rapidly progressing necrotizing pneumonia manifested by fever, leuco‐ penia and hemoptysis preceded by viral infection, most common influenza [75]. Leucopenia is characteristic for PVL and is thought to be secondary to leukocidin destroying white blood cells [77]. Radiographic appearance is multiple nodular infiltrates, usually unilateral that may transform into cavitary lesions and pneumatocele. Radiographic progression of in‐ filtrates may be very rapid and should raise possibility of *S. aureus* pneumonia. Another characteristic radiographic sign – pneumatoceles – occurs in over half of cases and both its size and number may change hourly [61]. Staphylococcal pneumonia is often complicated with empyema, formation of lung abscesses, pneumothorax and acute respiratory distress syndrome (ARDS).

Patients present with symptoms of upper respiratory tract infection, fever, malaise, head‐ ache. Symptoms ensue gradually. Cough, which is initially unproductive, appears 3 to 5 days from the onset of disease. Associated symptoms include hoarsness, chills, chest pain, vomiting, nausea, diarrhea and myalgia. Coryza is a rare finding, and pleural effusion oc‐ curs in 5-20% of patients. On auscultation crackles, wheezes and bronchial breathing may be present. *Mycoplasma pneumonie* may present with extrapulmonary involvement including: nonexudative pharyngitis, cervical lymphadenopathy, otitis media, conjunctivitis, arthritis and rash. Illness usually resolves within 3 - 4 weeks, but it might be more severe in children with sickle cell disease and in Down syndrome. *Mycoplasma* may cause exacerbations in

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Radiological findings vary from reticular and interstitial pattern to lobar consolidations. Hi‐ lar adenopathy is present in 1/3 of patients. Characteristic is poor correlation between clini‐

In rare cases *Mycoplasma pneumonia*e pneumonia may be severe with massive lobar consoli‐ dation, pleural effusion, lung abscess or pneumatocele formation. Occasionally, fever and radiological changes progress, despite standard macrolide therapy. There is one report of successful treatment of refractory pneumonia with methylprednisolone in children [82]. Rare complications – obliterative bronchiolitis and diffuse interstitial fibrosis – have been described. In 1/3 of children 1 - 2 years after *Mycoplasma pneumoniae* infection abnormal find‐ ings including mosaic perfusion, bronchiectasis, bronchial wall thickening, decreased vascu‐ larity and air trapping are observed [81]. Extrapulmonary complications of *Mycoplasma*

**•** neural system: meningoencephalitis, transverse myelitis, cranial neuropathy, myeloradi‐

**•** skin: erythematous maculopapular or vesicular exanthems, erythema multiforme, Stevens

**•** hematologic: hemolytic anemia, thrombocytopenia, disseminated intravascular coagula‐

Hospital acquired pneumonia (HAP) is defined as pneumonia that occurs 48 hours or more after admission in a patient who had no signs of disease at the time he or she was presenting

pneumonia provoke signs from a variety of organs and systems. These include:

**•** heart: pericarditis, myocarditis, congestive heart failure, heart block

**•** genitourinary: glomerulonephritis, interstitial tubulonephritis

culopathy, a poliomyelitis-like syndrome, psychosis, Guillain-Barré syndrome

**•** gastrointestinal tract: nausea, vomiting, diarrhea, hepatic dysfunction, jaundice

asthmatic patients [28].

– Johnson syndrome

**•** musculoskeletal: myalgias, arthralgias

**15. Hospital acquired pneumonia**

tion

cal symptoms and physical and radiological findings. Treatment of choice consists in macrolide antibiotics.

Given the severe and potentially fatal nature of the infection, prompt initiation of appropri‐ ate antibiotic therapy is crucial. For hospitalized patients with pneumonia caused by methi‐ cillin susceptible *S. aureus* PIDS recommends intravenous therapy with β-lactamase stable penicillin (oxacillin, cloxacillin, flucloxacillin or nafcillin) or the first generation cephalospor‐ in (cefazolin). For more severe infections some experts recommend combination therapy with an aminoglycoside or rifampin although data from controlled clinical studies support‐ ing these recommendations are lacking [19]. The first line treatment for hospitalized chil‐ dren suspected of CA-MRSA is vancomycin. There is a concern, however, of adequate concentration of the drug in lung epithelial lining fluid – in adults it has been shown to ach‐ ieve 18% of serum levels [77]. Alternative choices are linezolid and clindamycin that have an additional advantage of blocking the production of PVL toxin and staphylococcal exotoxins so they are the drugs of choice in treatment of PVL-CA infections. Clindamycin should be used with care considering local susceptibility data and it should not be used in high inocu‐ lum infections such as empyema since in that case there is high risk that the bacteria will constitutively produce methylase [19]. Linezolid achieves higher concentrations in epithelial lining of the lung [80].

#### **14.3.** *Mycoplasma pneumoniae*

*Mycoplsamas* are the smallest self-replicating organisms able to live outside the host cells. They do not have cell wall, but a cell membrane containing sterols and do not stain well with Gram stain and antibiotics disrupting bacterial cell wall like β-lactams are inactive against these organisms. *Mycoplasma pneumonia*e pneumonia is usually a mild disease. Transmission occurs via person to person contact and incubation period is usually 1 to 2 weeks. Epidemics occur in approximately 4 - 7 year cycles. Recurrent infections are usual.

Infections with *Mycoplasma pneumonia* are common. Antibodies are present in 1/3 of all infants between 7 to 12 month of life and over 90% of adolescents. Very few infections occur in infants in the first 6 months of life probably due to presence of maternal anti‐ bodies. Recent studies show that contrary to previous reports *M. pneumoniae* CAP is also quite common in children <4 years. These microorganism are responsible for ¼ of infec‐ tions in this age group [81].

Microorganisms are acquired via respiratory route. In the airways they attach to a receptor on respiratory epithelium via adhesions. Lung injury in *M. pneumoniae* pneumonia is associ‐ ated with cell-mediated immunity of the host and it is also accompanied by ciliostasis [81].

Patients present with symptoms of upper respiratory tract infection, fever, malaise, head‐ ache. Symptoms ensue gradually. Cough, which is initially unproductive, appears 3 to 5 days from the onset of disease. Associated symptoms include hoarsness, chills, chest pain, vomiting, nausea, diarrhea and myalgia. Coryza is a rare finding, and pleural effusion oc‐ curs in 5-20% of patients. On auscultation crackles, wheezes and bronchial breathing may be present. *Mycoplasma pneumonie* may present with extrapulmonary involvement including: nonexudative pharyngitis, cervical lymphadenopathy, otitis media, conjunctivitis, arthritis and rash. Illness usually resolves within 3 - 4 weeks, but it might be more severe in children with sickle cell disease and in Down syndrome. *Mycoplasma* may cause exacerbations in asthmatic patients [28].

Radiological findings vary from reticular and interstitial pattern to lobar consolidations. Hi‐ lar adenopathy is present in 1/3 of patients. Characteristic is poor correlation between clini‐ cal symptoms and physical and radiological findings.

Treatment of choice consists in macrolide antibiotics.

penia and hemoptysis preceded by viral infection, most common influenza [75]. Leucopenia is characteristic for PVL and is thought to be secondary to leukocidin destroying white blood cells [77]. Radiographic appearance is multiple nodular infiltrates, usually unilateral that may transform into cavitary lesions and pneumatocele. Radiographic progression of in‐ filtrates may be very rapid and should raise possibility of *S. aureus* pneumonia. Another characteristic radiographic sign – pneumatoceles – occurs in over half of cases and both its size and number may change hourly [61]. Staphylococcal pneumonia is often complicated with empyema, formation of lung abscesses, pneumothorax and acute respiratory distress

Given the severe and potentially fatal nature of the infection, prompt initiation of appropri‐ ate antibiotic therapy is crucial. For hospitalized patients with pneumonia caused by methi‐ cillin susceptible *S. aureus* PIDS recommends intravenous therapy with β-lactamase stable penicillin (oxacillin, cloxacillin, flucloxacillin or nafcillin) or the first generation cephalospor‐ in (cefazolin). For more severe infections some experts recommend combination therapy with an aminoglycoside or rifampin although data from controlled clinical studies support‐ ing these recommendations are lacking [19]. The first line treatment for hospitalized chil‐ dren suspected of CA-MRSA is vancomycin. There is a concern, however, of adequate concentration of the drug in lung epithelial lining fluid – in adults it has been shown to ach‐ ieve 18% of serum levels [77]. Alternative choices are linezolid and clindamycin that have an additional advantage of blocking the production of PVL toxin and staphylococcal exotoxins so they are the drugs of choice in treatment of PVL-CA infections. Clindamycin should be used with care considering local susceptibility data and it should not be used in high inocu‐ lum infections such as empyema since in that case there is high risk that the bacteria will constitutively produce methylase [19]. Linezolid achieves higher concentrations in epithelial

*Mycoplsamas* are the smallest self-replicating organisms able to live outside the host cells. They do not have cell wall, but a cell membrane containing sterols and do not stain well with Gram stain and antibiotics disrupting bacterial cell wall like β-lactams are inactive against these organisms. *Mycoplasma pneumonia*e pneumonia is usually a mild disease. Transmission occurs via person to person contact and incubation period is usually 1 to 2 weeks. Epidemics occur in approximately 4 - 7 year cycles. Recurrent infections are usual. Infections with *Mycoplasma pneumonia* are common. Antibodies are present in 1/3 of all infants between 7 to 12 month of life and over 90% of adolescents. Very few infections occur in infants in the first 6 months of life probably due to presence of maternal anti‐ bodies. Recent studies show that contrary to previous reports *M. pneumoniae* CAP is also quite common in children <4 years. These microorganism are responsible for ¼ of infec‐

Microorganisms are acquired via respiratory route. In the airways they attach to a receptor on respiratory epithelium via adhesions. Lung injury in *M. pneumoniae* pneumonia is associ‐ ated with cell-mediated immunity of the host and it is also accompanied by ciliostasis [81].

syndrome (ARDS).

158 Respiratory Disease and Infection - A New Insight

lining of the lung [80].

**14.3.** *Mycoplasma pneumoniae*

tions in this age group [81].

In rare cases *Mycoplasma pneumonia*e pneumonia may be severe with massive lobar consoli‐ dation, pleural effusion, lung abscess or pneumatocele formation. Occasionally, fever and radiological changes progress, despite standard macrolide therapy. There is one report of successful treatment of refractory pneumonia with methylprednisolone in children [82]. Rare complications – obliterative bronchiolitis and diffuse interstitial fibrosis – have been described. In 1/3 of children 1 - 2 years after *Mycoplasma pneumoniae* infection abnormal find‐ ings including mosaic perfusion, bronchiectasis, bronchial wall thickening, decreased vascu‐ larity and air trapping are observed [81]. Extrapulmonary complications of *Mycoplasma* pneumonia provoke signs from a variety of organs and systems. These include:


#### **15. Hospital acquired pneumonia**

Hospital acquired pneumonia (HAP) is defined as pneumonia that occurs 48 hours or more after admission in a patient who had no signs of disease at the time he or she was presenting to the hospital [83]. It may be further divided into early onset (48-96 hours after admission) usually caused by pathogens responsible for CAP and late onset (> 96 hours after admission) caused by multidrug resistant nosocomial pathogens [84].

teral feeds, use of narcotic medications were found to be independent risk factors of VAP in

Nosocomial pneumonia should be suspected in any child with new respiratory symptoms during hospital stay, hypoxemia, increased oxygen or ventilation requirements, increased amount or altered characteristic of respiratory secretions. Most definitions include clinical and radiological signs, some additional bacteriological and laboratory data [83,92]. Accord‐ ing to National Healthcare Safety Network of Centers for Disease Control (CDC), HAP can be diagnosed in patients with new radiological changes (infiltrate, consolidation, cavitation, pneumatocele in infants in the 1st year of life) and at least three clinical criteria (clinically de‐ fined pneumonia) or two clinical and one laboratory criteria. For children with underlying pulmonary or heart disease radiologic changes must be confirmed in at least two serial x-

) or leucocytosis (≥12,000/mm<sup>3</sup>

**•** new onset or worsening of respiratory symptoms: cough, tachypnea, dyspnea,

In children up to12 years of age clinical criteria are slightly different and at least three (two

) or leukocytosis (≥15,000/mm<sup>3</sup>

**•** new onset or worsening of respiratory symptoms: apnea, cough, tachypnea, dyspnea, **•** nasal flaring with chest wall retractions or grunting, wheezing - infants ≤1 year of age

**•** bradycardia (<100 beats/minute) or tachycardia (>170 beats per minute) - infants ≤1 year

) and at least one (or two for clini‐

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) and ≥10% immature forms (in‐

C (or temperature instability for infants ≤1 year of age)

children [88,91].

**•** fever > 380

**•** fever > 380

of age

with no other cause or **•** leucopenia (<4,000/mm3

fants ≤1 year of age)

**•** new onset of purulent sputum, **•** increase in respiratory secretions,

**•** leucopenia (<4,000/mm3

**•** new onset of purulent sputum, **•** increase in respiratory secretions,

rays. Clinical criteria, that must be fulfilled include:

C with no other cause or

cally defined pneumonia) of the following:

**•** auscultary findings: rales, bronchial sounds,

**•** increased oxygen requirements, PaO2/FiO2 ≤ 240.

C or hypothermia < 36.50

**•** auscultary findings: rales, bronchial sounds,

in infants ≤1 year of age) must be fulfilled. These include:

**•** change in the character of sputum or respiratory secretions,

**•** change in the character of sputum or respiratory secretions,

Ventilator associated pneumonia (VAP), a type of HAP affecting mechanically ventilated patients is defined as pneumonia that occurs more than 48 hours after intubation [83].

A third type, only recently described, represents health-care associated pneumonia (HCAP) that develops in patients who fulfill one of the following conditions:


Definition of HCAP has not been validated in children, although, due to a growing number of pediatric patients with chronic medical conditions (eg. cerebral palsy, congenital malfor‐ mation syndromes, chronic pulmonary, heart and renal disease) who frequently have con‐ tacts with healthcare personnel, HCAP poses a significant problem in this population.

HAP occurs in 16-29% of pediatric patients and accounts for 10-15% of all nosocomial infec‐ tions in children and up to 67% of nosocomial infections in children admitted to pediatric intensive care units [85-87]. VAP occurs in about 3 to 32% of ventilated pediatric ICU pa‐ tients [87,88]. In Europe it is the most common and in the USA second most common noso‐ comial infection in children treated in intensive care units [88-90]. Mortality in HAP is much higher than in CAP and ranges from 10 to 70% depending on the etiological factors and co‐ morbidities [85,86,88]. HAP and VAP in particular increase length of hospital stay and hos‐ pital costs [86,88,91].

The most common etiological factors of pediatric HAP are respiratory viruses including res‐ piratory syncytial virus, adenovirus, influenza and parainfluenza viruses. Bacteria responsi‐ ble for late onset HAP comprise Gram-negative bacilli: *Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Acinetobacter* spp.*, Serratia* spp.., Gram positive organisms esp. *Staphylococcus aureus* and coagulase - negative *Staphylococci. P. aeruginosa* is the most com‐ mon bacterial pathogen in pediatric intensive care units associated with mortality rates up to 80% [90]. Most bacteria responsible for nosocomial infections are multidrug resistant, among them methicillin resistant *Staphylococci* (MRSA) and extended spectrum β-lactamase (ESBL) producing Gram negative bacilli. Immunocompromised children are at particular risk of infection caused by fungi, esp. *Aspergillus*, *Candida* and *Pneumocystis jiroveci*. In 38% of cases etiology of VAP in children is polymicrobial [88].

Risk factors for hospital acquired pneumonia comprise intubation and mechanical ventila‐ tion (increases the risk 6 to 21-fold increased risk), neuromuscular blockade, length of hospi‐ tal stay, immunosuppression, recent treatment with antibiotics and H2 blockers as well as overcrowding and understaffing of hospital wards [85,86]. Genetic syndrome, female gen‐ der, reintubation, transport out of the intensive care unit, surgery before ICU admission, en‐ teral feeds, use of narcotic medications were found to be independent risk factors of VAP in children [88,91].

Nosocomial pneumonia should be suspected in any child with new respiratory symptoms during hospital stay, hypoxemia, increased oxygen or ventilation requirements, increased amount or altered characteristic of respiratory secretions. Most definitions include clinical and radiological signs, some additional bacteriological and laboratory data [83,92]. Accord‐ ing to National Healthcare Safety Network of Centers for Disease Control (CDC), HAP can be diagnosed in patients with new radiological changes (infiltrate, consolidation, cavitation, pneumatocele in infants in the 1st year of life) and at least three clinical criteria (clinically de‐ fined pneumonia) or two clinical and one laboratory criteria. For children with underlying pulmonary or heart disease radiologic changes must be confirmed in at least two serial xrays. Clinical criteria, that must be fulfilled include:

**•** fever > 380 C with no other cause or

to the hospital [83]. It may be further divided into early onset (48-96 hours after admission) usually caused by pathogens responsible for CAP and late onset (> 96 hours after admission)

Ventilator associated pneumonia (VAP), a type of HAP affecting mechanically ventilated patients is defined as pneumonia that occurs more than 48 hours after intubation [83].

A third type, only recently described, represents health-care associated pneumonia (HCAP)

Definition of HCAP has not been validated in children, although, due to a growing number of pediatric patients with chronic medical conditions (eg. cerebral palsy, congenital malfor‐ mation syndromes, chronic pulmonary, heart and renal disease) who frequently have con‐ tacts with healthcare personnel, HCAP poses a significant problem in this population.

HAP occurs in 16-29% of pediatric patients and accounts for 10-15% of all nosocomial infec‐ tions in children and up to 67% of nosocomial infections in children admitted to pediatric intensive care units [85-87]. VAP occurs in about 3 to 32% of ventilated pediatric ICU pa‐ tients [87,88]. In Europe it is the most common and in the USA second most common noso‐ comial infection in children treated in intensive care units [88-90]. Mortality in HAP is much higher than in CAP and ranges from 10 to 70% depending on the etiological factors and co‐ morbidities [85,86,88]. HAP and VAP in particular increase length of hospital stay and hos‐

The most common etiological factors of pediatric HAP are respiratory viruses including res‐ piratory syncytial virus, adenovirus, influenza and parainfluenza viruses. Bacteria responsi‐ ble for late onset HAP comprise Gram-negative bacilli: *Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Acinetobacter* spp.*, Serratia* spp.., Gram positive organisms esp. *Staphylococcus aureus* and coagulase - negative *Staphylococci. P. aeruginosa* is the most com‐ mon bacterial pathogen in pediatric intensive care units associated with mortality rates up to 80% [90]. Most bacteria responsible for nosocomial infections are multidrug resistant, among them methicillin resistant *Staphylococci* (MRSA) and extended spectrum β-lactamase (ESBL) producing Gram negative bacilli. Immunocompromised children are at particular risk of infection caused by fungi, esp. *Aspergillus*, *Candida* and *Pneumocystis jiroveci*. In 38% of

Risk factors for hospital acquired pneumonia comprise intubation and mechanical ventila‐ tion (increases the risk 6 to 21-fold increased risk), neuromuscular blockade, length of hospi‐ tal stay, immunosuppression, recent treatment with antibiotics and H2 blockers as well as overcrowding and understaffing of hospital wards [85,86]. Genetic syndrome, female gen‐ der, reintubation, transport out of the intensive care unit, surgery before ICU admission, en‐

**•** antibiotic therapy or chemotherapy or wound care within 30 days of the infection

caused by multidrug resistant nosocomial pathogens [84].

**•** residence in a nursing home or long-term care facility

cases etiology of VAP in children is polymicrobial [88].

**•** attending haemodialisis center [83].

160 Respiratory Disease and Infection - A New Insight

pital costs [86,88,91].

that develops in patients who fulfill one of the following conditions: **•** hospitalization for 2 or more days within 90 days of the infection


In children up to12 years of age clinical criteria are slightly different and at least three (two in infants ≤1 year of age) must be fulfilled. These include:


**•** increased ventilation requirements, hypoxemia (Sat <94%) – this condition is obligatory for infants ≤1 year of age.

agent of the narrowest spectrum available. Antibiotics should be administered intravenous‐

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Infection control measures that should be meticulously implemented in order to decrease

**•** personal protective equipment – face masks, especially in the periods of increased inci‐

**•** use of non-invasive ventilation instead of mechanical ventilation in appropriate patients **•** care of medical equipment: ventilator circuits, suction devices, pulmonary function test‐

The authors wish to thank Katarzyna Jończyk-Potoczna MD, PHD for her excellent assis‐

Department of Pulmonology, Pediatric Allergy and Clinical Immunology, Karol Marcin‐

[1] World Health Organization. Pneumonia. Fact sheet No. 331.2011. Available at

www.who.int/mediacentre/factsheets/fs331/en. Accessed 03.08.2012

ly and switched to oral route if possible once the patient improves.

**•** cohorting patients infected or colonized with resistant microorganisms

**•** nursing practices: semirecumbent position for ventilated patients, mouth care

the number of HAP include:

dence of respiratory viruses

ing equipment

**•** avoiding H2 antagonists **•** judicious use of antibiotics

**Acknowledgements**

**Author details**

**References**

tance with radiological examinations.

Irena Wojsyk-Banaszak and Anna Bręborowicz

\*Address all correspondence to: iwojsyk@ump.edu.pl

kowski University of Medical Sciences, Szpitalna, Poznań, Poland

**•** appropriate hand hygiene, use of gloves

**•** avoiding overcrowding and understaffing

**•** immunization practices (as described above for CAP).

Laboratory criteria include positive cultures of blood (not related to other infections), pleu‐ ral fluid or specimens from lower respiratory tract (bronchoalveolar lavage - BAL or protect‐ ed specimen brushing), intracellular bacteria seen in ≥5% of cells obtained from BAL on direct microscopic exam, histopathologic evidence of abscess formation, focal consolida‐ tions, intensive polymorphonuclear cell accumulation in the small airways, lung parenchy‐ ma invasion by hyphae or pseudohyphae. Diagnostic value of BAL in children with VAP is found to be 50-72% and specificity 80-88%, and of quantification of intracellular organisms in BAL samples 30-55% and 89-95% respectively [93-96]. Laboratory criteria for HAP caused by atypical bacteria (*Mycoplasma* spp.*, Chlamydophila* spp.*, Legionella* spp.) or viruses include: positive culture from respiratory secretions, positive detection of antigen or antibody in res‐ piratory secretions, 4-fold IgG rise in paired sera, positive PCR, detection of *Legionella pneu‐ mophila* serogroup 1 antigens in urine [92].

Bacteriological diagnosis is often difficult, since hospitalized patients are commonly colon‐ ized with pathogenic flora. Therefore a quantitative criterion of bacterial yield (≥10<sup>4</sup> cfu/mL, or ≥10<sup>4</sup> cfu/g tissue in case of lung parenchyma specimen) has been established to aid the etiological diagnosis of pneumonia [92]. Applicability of invasive diagnostic techniques (lung biopsy) remains controversial for the fear of complications in unstable or severely sick patients and lack of data confirming their influence on clinical course or mortality rates.

Patients with HAP are most often debilitated individuals with multiple underlying condi‐ tions. HAP is a severe disease with high mortality rates. Therefore diagnosis should be es‐ tablished as quickly as possible using a wide array of diagnostic tools and techniques and empiric treatment should be implemented promptly while cultures and other microbiologi‐ cal studies are pending. Antibiotics should have spectrum broad enough to cover gram neg‐ ative and gram positive bacteria considering previous antibiotic exposure, local flora, antimicrobial susceptibility patterns and guidelines from the infectious diseases specialist. Risk factors for acquisition of antibiotic-resistant gram negative bacteria in pediatric inten‐ sive care unit (PICU) patients include younger age, severe general condition (appreciated based on PRISM {pediatric risk of mortality} score), intravenous antibiotics administration in the previous 12 months, PICU admission in the past, contacts with chronic care facilities [87,94]. Inappropriate antibiotic treatment is associated with increased mortality in patients with HAP [90]. Children with early onset HAP might be treated as those with CAP provid‐ ed they do not have specific risk factors for HCAP, VAP or colonization with multi-drug re‐ sistant bacteria. PICU patients should receive coverage against *Pseudomonas aeruginosa* (aminoglycoside with an appropriate β-lactam: piperacillin, ceftazidime or cefepime) and in hospital wards where incidence of methicillin resistant staphylococci or ESBL producing gram negative bacilli exceeds 5% vancomycin and carbapenems or ureidopenicillin deriva‐ tive plus β-lactamase inhibitor respectively should be included in empiric treatment regimes [92]. In children with immunodeficiencies antifungals should be administered. As soon as the causative organism and its sensitivity is known, therapy should be tailored using the agent of the narrowest spectrum available. Antibiotics should be administered intravenous‐ ly and switched to oral route if possible once the patient improves.

Infection control measures that should be meticulously implemented in order to decrease the number of HAP include:


**•** increased ventilation requirements, hypoxemia (Sat <94%) – this condition is obligatory

Laboratory criteria include positive cultures of blood (not related to other infections), pleu‐ ral fluid or specimens from lower respiratory tract (bronchoalveolar lavage - BAL or protect‐ ed specimen brushing), intracellular bacteria seen in ≥5% of cells obtained from BAL on direct microscopic exam, histopathologic evidence of abscess formation, focal consolida‐ tions, intensive polymorphonuclear cell accumulation in the small airways, lung parenchy‐ ma invasion by hyphae or pseudohyphae. Diagnostic value of BAL in children with VAP is found to be 50-72% and specificity 80-88%, and of quantification of intracellular organisms in BAL samples 30-55% and 89-95% respectively [93-96]. Laboratory criteria for HAP caused by atypical bacteria (*Mycoplasma* spp.*, Chlamydophila* spp.*, Legionella* spp.) or viruses include: positive culture from respiratory secretions, positive detection of antigen or antibody in res‐ piratory secretions, 4-fold IgG rise in paired sera, positive PCR, detection of *Legionella pneu‐*

Bacteriological diagnosis is often difficult, since hospitalized patients are commonly colon‐ ized with pathogenic flora. Therefore a quantitative criterion of bacterial yield (≥10<sup>4</sup> cfu/mL,

etiological diagnosis of pneumonia [92]. Applicability of invasive diagnostic techniques (lung biopsy) remains controversial for the fear of complications in unstable or severely sick patients and lack of data confirming their influence on clinical course or mortality rates.

Patients with HAP are most often debilitated individuals with multiple underlying condi‐ tions. HAP is a severe disease with high mortality rates. Therefore diagnosis should be es‐ tablished as quickly as possible using a wide array of diagnostic tools and techniques and empiric treatment should be implemented promptly while cultures and other microbiologi‐ cal studies are pending. Antibiotics should have spectrum broad enough to cover gram neg‐ ative and gram positive bacteria considering previous antibiotic exposure, local flora, antimicrobial susceptibility patterns and guidelines from the infectious diseases specialist. Risk factors for acquisition of antibiotic-resistant gram negative bacteria in pediatric inten‐ sive care unit (PICU) patients include younger age, severe general condition (appreciated based on PRISM {pediatric risk of mortality} score), intravenous antibiotics administration in the previous 12 months, PICU admission in the past, contacts with chronic care facilities [87,94]. Inappropriate antibiotic treatment is associated with increased mortality in patients with HAP [90]. Children with early onset HAP might be treated as those with CAP provid‐ ed they do not have specific risk factors for HCAP, VAP or colonization with multi-drug re‐ sistant bacteria. PICU patients should receive coverage against *Pseudomonas aeruginosa* (aminoglycoside with an appropriate β-lactam: piperacillin, ceftazidime or cefepime) and in hospital wards where incidence of methicillin resistant staphylococci or ESBL producing gram negative bacilli exceeds 5% vancomycin and carbapenems or ureidopenicillin deriva‐ tive plus β-lactamase inhibitor respectively should be included in empiric treatment regimes [92]. In children with immunodeficiencies antifungals should be administered. As soon as the causative organism and its sensitivity is known, therapy should be tailored using the

cfu/g tissue in case of lung parenchyma specimen) has been established to aid the

for infants ≤1 year of age.

162 Respiratory Disease and Infection - A New Insight

*mophila* serogroup 1 antigens in urine [92].

or ≥10<sup>4</sup>


#### **Acknowledgements**

The authors wish to thank Katarzyna Jończyk-Potoczna MD, PHD for her excellent assis‐ tance with radiological examinations.

#### **Author details**

Irena Wojsyk-Banaszak and Anna Bręborowicz

\*Address all correspondence to: iwojsyk@ump.edu.pl

Department of Pulmonology, Pediatric Allergy and Clinical Immunology, Karol Marcin‐ kowski University of Medical Sciences, Szpitalna, Poznań, Poland

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**Chapter 7**

**Cystic Fibrosis Pulmonary Exacerbation – Natural**

Cystic Fibrosis (CF) is the most common life-threatening inherited disease in the United Kingdom, affecting over 9,000 people, 56% of which are 16 years or older (UK CF Registry Annual Data Report 2009). Life expectancy currently remains well below the general popu‐ lation, but with improvement of care most CF patients born after 1980 are expected to reach adulthood. Much of the morbidity and mortality associated with CF are related to the respi‐

Recurrent acute infective pulmonary exacerbations are one of the most important features of CF. Pulmonary exacerbations are common events throughout the lifetime of a patient with CF. Frequent exacerbations are associated with impaired quality of life and accelerated de‐ cline in lung function (Marshall, 2004; Britto et al., 2002; Liou et al., 2001; Ramsey, 1996). Statistical models have demonstrated that frequent pulmonary exacerbations are also associ‐

Preventing and treating pulmonary exacerbations promptly and appropriately are therefore key therapeutic goals of the CF community that clinicians endeavour to employ in the effort to positively impact on long term outcomes, survival and quality of life of CF patients.

Despite CF being one of the most studied and understood genetic diseases, the exact patho‐ physiology of recurrent CF pulmonary exacerbations is still not completely understood. The most accepted hypothesis to explain how the primary defect in CF, the mutations in the

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sequeiros and Jarad; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**History, Causative Factors and Management**

Iara Maria Sequeiros and Nabil Jarad

http://dx.doi.org/10.5772/54336

ratory system (Goss & Burns, 2007).

ated with premature mortality (Liou et al., 2001).

**2. The pathophysiology of CF lung disease**

**1. Introduction**

Additional information is available at the end of the chapter

### **Cystic Fibrosis Pulmonary Exacerbation – Natural History, Causative Factors and Management**

Iara Maria Sequeiros and Nabil Jarad

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54336

**1. Introduction**

Cystic Fibrosis (CF) is the most common life-threatening inherited disease in the United Kingdom, affecting over 9,000 people, 56% of which are 16 years or older (UK CF Registry Annual Data Report 2009). Life expectancy currently remains well below the general popu‐ lation, but with improvement of care most CF patients born after 1980 are expected to reach adulthood. Much of the morbidity and mortality associated with CF are related to the respi‐ ratory system (Goss & Burns, 2007).

Recurrent acute infective pulmonary exacerbations are one of the most important features of CF. Pulmonary exacerbations are common events throughout the lifetime of a patient with CF. Frequent exacerbations are associated with impaired quality of life and accelerated de‐ cline in lung function (Marshall, 2004; Britto et al., 2002; Liou et al., 2001; Ramsey, 1996). Statistical models have demonstrated that frequent pulmonary exacerbations are also associ‐ ated with premature mortality (Liou et al., 2001).

Preventing and treating pulmonary exacerbations promptly and appropriately are therefore key therapeutic goals of the CF community that clinicians endeavour to employ in the effort to positively impact on long term outcomes, survival and quality of life of CF patients.

#### **2. The pathophysiology of CF lung disease**

Despite CF being one of the most studied and understood genetic diseases, the exact patho‐ physiology of recurrent CF pulmonary exacerbations is still not completely understood. The most accepted hypothesis to explain how the primary defect in CF, the mutations in the

cystic fibrosis transmembrane conductance regulator gene (CFTR), leads to the colonisation of the airways by bacteria, chronic infection and inflammation and ultimately airway injury is the "low volume hypothesis" (Boucher, 2004).

The volume of the periciliary airway surface fluid is normally precisely regulated and is critical for efficient mucus flow and elimination. In the normal airway epithelium, the amount of fluid is determined by a combination of ion channels that promote the secretion of salt and water, such as the CFTR and alternative anion channels, and others that promote absorption of so‐ dium and fluid, such as epithelial sodium channels (ENaC) in the apical cell membrane. The

Cystic Fibrosis Pulmonary Exacerbation – Natural History, Causative Factors and Management

http://dx.doi.org/10.5772/54336

175

In the CF epithelium, in consequence of the CFTR mutations, the CFTR gene product is faul‐ ty, whilst in normal conditions it is a transmembrane protein that functions as a chloride channel. The CFTR down regulatory function of ENaC activity is absent or compromised, resulting in abnormal ion transport properties due to the combined defects of accelerated so‐ dium transport and failure to secrete chloride, resulting in overall increased absorption of sodium and fluid from the airway surface and consequently reduced, low volume of the air‐

The loss of functional height of the periciliary layer in which the cilia beat, defined by the length of extended cilia, interferes with cilia motion and causes impaired ciliary function, re‐ sulting in slower mucociliary clearance with accumulation of mucus with trapped debris, microorganisms and airway secretory products, such as cytokines (e.g., interleukin-8), neu‐ trophil proteases and growth factors that promote airway inflammation and mucus cell hy‐ perplasia. In consequence of increased number of mucus secreting goblet cells, there is persistent mucin secretion, which generates high volume of thick, inspissated mucus, which adheres to and obstructs the airways and is resistant to cough clearance and is an ideal site and reservoir for bacterial growth. The poor liquid content and hypoxic environment in these thick mucus plaques promote biofilm bacterial formation and bacterial overgrowth. Mucus stasis and the presence of accumulated secretory products and bacteria triggers the recruitment of neutrophils to the airways and the release of pro-inflammatory cytokines, which lead to a vicious cycle of inflammation, airway obstruction, chronic infection, repeat‐ ed pulmonary exacerbations and resultant airway tissue damage and loss of lung function

**3. Microbiology in the CF lung disease and relationship with pulmonary**

Pulmonary exacerbations in CF are caused by recognised typical pathogens that are acquired following a characteristic age-dependent pattern (figure 2). Whilst still very young, CF patients suffer their first infections most frequently caused by *Staphylococcus aureus* and *Haemophilus in‐ fluenzae*. *S. aureus* is often the first organism isolated from children with CF, but the role of this bacteria in the pathogenesis of CF remains under debate as studies reveal conflicting results in regards to clinical benefits from anti-staphylococcal therapy (Smyth & Walters, 2003; Lyczak et al., 2002). Non-typeable *H. influenzae* is also commonly cultured from CF children as early as in their first year of life, but although it can cause exacerbations in patients with non-CF bron‐

chiectasis (Barker, 2002), its role in CF is still unclear (Goss & Burns, 2007).

CFTR also acts as a conductance regulator, influencing and down regulating the latter.

way surface liquid (Guggino & Guggino, 2000).

(Frizzell & Pilewski, 2004).

**exacerbations**

The airways are covered by airway surface liquid, composed by two separate fluid layers, a su‐ perficial mucus layer and an underlying lubricating pericellular environment near the cell sur‐ face. A thin layer of surfactant separates the mucus and the periciliary fluid layer (figure 1).

The mucus layer is produced by goblet cells and submucosal glands and is composed of wa‐ ter, carbohydrates, proteins and lipids, extending from the intermediate airways to the up‐ per airways. It normally is composed of a network of mucous glycoproteins or mucin. Its adhesive characteristic means that the mucus layer binds and traps deposited particles and its viscoelastic properties facilitate cilia movement into transporting mucus and also its clearance by cough. Mucus containing cellular debris and possible trapped microorganisms is transported from the lower respiratory tract to the pharynx by mucociliary clearance and air flow (Rubin, 2002). Mucus clearance is considered a most important innate airway de‐ fence mechanism (Randell & Boucher, 2006; Knowles & Boucher, 2002), and disruption of the normal mucus secretion and mucociliary clearance impairs lung defence and increases the risk of infection.

The periciliary fluid provides a watery environment that enables the cilia to beat and propel the mucus away towards the upper airways. Appropriate clearance and hygiene maintenance of the respiratory tract requires coordinated interaction of the mucus and periciliary layers and for this to be effective, adequate hydration of the near-cell airway surface is critical.

**Figure 1.** The fluid surface area in normal patients (a) and in patients with CF (b). The epithelial cells are covered by two layers of fluid – a serous layer (white band) and a mucous layer (grey area). In CF patients and due to the disease process, both layers shrink and the shape of the cilia and its movement are adversely affected.

The volume of the periciliary airway surface fluid is normally precisely regulated and is critical for efficient mucus flow and elimination. In the normal airway epithelium, the amount of fluid is determined by a combination of ion channels that promote the secretion of salt and water, such as the CFTR and alternative anion channels, and others that promote absorption of so‐ dium and fluid, such as epithelial sodium channels (ENaC) in the apical cell membrane. The CFTR also acts as a conductance regulator, influencing and down regulating the latter.

cystic fibrosis transmembrane conductance regulator gene (CFTR), leads to the colonisation of the airways by bacteria, chronic infection and inflammation and ultimately airway injury

The airways are covered by airway surface liquid, composed by two separate fluid layers, a su‐ perficial mucus layer and an underlying lubricating pericellular environment near the cell sur‐ face. A thin layer of surfactant separates the mucus and the periciliary fluid layer (figure 1).

The mucus layer is produced by goblet cells and submucosal glands and is composed of wa‐ ter, carbohydrates, proteins and lipids, extending from the intermediate airways to the up‐ per airways. It normally is composed of a network of mucous glycoproteins or mucin. Its adhesive characteristic means that the mucus layer binds and traps deposited particles and its viscoelastic properties facilitate cilia movement into transporting mucus and also its clearance by cough. Mucus containing cellular debris and possible trapped microorganisms is transported from the lower respiratory tract to the pharynx by mucociliary clearance and air flow (Rubin, 2002). Mucus clearance is considered a most important innate airway de‐ fence mechanism (Randell & Boucher, 2006; Knowles & Boucher, 2002), and disruption of the normal mucus secretion and mucociliary clearance impairs lung defence and increases

The periciliary fluid provides a watery environment that enables the cilia to beat and propel the mucus away towards the upper airways. Appropriate clearance and hygiene maintenance of the respiratory tract requires coordinated interaction of the mucus and periciliary layers and

**Figure 1.** The fluid surface area in normal patients (a) and in patients with CF (b). The epithelial cells are covered by two layers of fluid – a serous layer (white band) and a mucous layer (grey area). In CF patients and due to the disease

process, both layers shrink and the shape of the cilia and its movement are adversely affected.

for this to be effective, adequate hydration of the near-cell airway surface is critical.

is the "low volume hypothesis" (Boucher, 2004).

174 Respiratory Disease and Infection - A New Insight

the risk of infection.

In the CF epithelium, in consequence of the CFTR mutations, the CFTR gene product is faul‐ ty, whilst in normal conditions it is a transmembrane protein that functions as a chloride channel. The CFTR down regulatory function of ENaC activity is absent or compromised, resulting in abnormal ion transport properties due to the combined defects of accelerated so‐ dium transport and failure to secrete chloride, resulting in overall increased absorption of sodium and fluid from the airway surface and consequently reduced, low volume of the air‐ way surface liquid (Guggino & Guggino, 2000).

The loss of functional height of the periciliary layer in which the cilia beat, defined by the length of extended cilia, interferes with cilia motion and causes impaired ciliary function, re‐ sulting in slower mucociliary clearance with accumulation of mucus with trapped debris, microorganisms and airway secretory products, such as cytokines (e.g., interleukin-8), neu‐ trophil proteases and growth factors that promote airway inflammation and mucus cell hy‐ perplasia. In consequence of increased number of mucus secreting goblet cells, there is persistent mucin secretion, which generates high volume of thick, inspissated mucus, which adheres to and obstructs the airways and is resistant to cough clearance and is an ideal site and reservoir for bacterial growth. The poor liquid content and hypoxic environment in these thick mucus plaques promote biofilm bacterial formation and bacterial overgrowth.

Mucus stasis and the presence of accumulated secretory products and bacteria triggers the recruitment of neutrophils to the airways and the release of pro-inflammatory cytokines, which lead to a vicious cycle of inflammation, airway obstruction, chronic infection, repeat‐ ed pulmonary exacerbations and resultant airway tissue damage and loss of lung function (Frizzell & Pilewski, 2004).

#### **3. Microbiology in the CF lung disease and relationship with pulmonary exacerbations**

Pulmonary exacerbations in CF are caused by recognised typical pathogens that are acquired following a characteristic age-dependent pattern (figure 2). Whilst still very young, CF patients suffer their first infections most frequently caused by *Staphylococcus aureus* and *Haemophilus in‐ fluenzae*. *S. aureus* is often the first organism isolated from children with CF, but the role of this bacteria in the pathogenesis of CF remains under debate as studies reveal conflicting results in regards to clinical benefits from anti-staphylococcal therapy (Smyth & Walters, 2003; Lyczak et al., 2002). Non-typeable *H. influenzae* is also commonly cultured from CF children as early as in their first year of life, but although it can cause exacerbations in patients with non-CF bron‐ chiectasis (Barker, 2002), its role in CF is still unclear (Goss & Burns, 2007).

Other relevant pathogens in CF usually cultured later in the course of the disease include *Burkholderia cepacia* complex, *Stenotrophomonas maltophilia* and *Achromobacter xylosoxidans*. The *B. cepacia* complex has at least 10 distinct genomovars and is related with rapid clinical deterioration. The most virulent and transmissible form is the genomovar III, B. cenocepa‐ cia, due to its well recognised association with severe necrotising pneumonia and death first described over 20 years ago (Isles et al., 1984). *S. maltophilia* and *A. xylosoxidans* are more prevalent than the *B. cepacia* complex, but seem to be less virulent, although their role in the pathogenesis of the CF lung is not yet completely understood (Goss et al., 2002).

mucoid *P. aeruginosa* tend to exhibit greater in vivo sensitivity to antibiotics than non-

Cystic Fibrosis Pulmonary Exacerbation – Natural History, Causative Factors and Management

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177

**3.** The biofilm formation is highly immunogenic and tends to accelerate structural lung

The incidence and clinical impact of mucoid *P. aeruginosa* (figure 3a) in the sputum of a co‐ hort of patients with CF according to age. The emergence of these bacteria is associated with decline in FEV1 (figure 3b) and worsening of radiograph appearance (figure 3c). (Adapted

> 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 **Age (years)**

> > **FEV1**

6 7 8 9 10 11 12 13 14 15 16 17 **Age (years)**

**Wisconsin Chest Radiograph Score**

**6 7 8 9 10 11 12 13 14 15 16 17 18 Age (years)**

**Figure 3.** (a): Proportion (%) of *P. aeruginosa* with mucoid strains per age group. (b): Change in FEV1 for the same CF

damage leading to significant histological and radiological changes.

mucoid strains in the same patient.

 (a)

**Score**

**FEV1 (% Predicted)**

 (b)

(c)

group as in 3a. (c): Change in the chest radiograph appearance in the same CF group as in 3a

Worse

from Li et al., 2005).

**Figure 2.** Prevalence of respiratory pathogens according to patient's age. Data from Cystic Fibrosis Foundation Patient Registry, 2010 Annual Data Report.

*Pseudomonas aeruginosa* is considered to be the most important pathogen in CF. By the time patients reach adulthood, up to 80% of them become infected with *P. aeruginosa*. The trans‐ formation of *P. aeruginosa* from the non-mucoid to the mucoid strains, which often occurs during adolescence years (figure 3a), is pivotal and is known to be associated with clinical (figure 3b) and radiological deterioration (figure 3c) (Li et al., 2005). At initial colonisation, *P. aeruginosa* presents a phenotype similar to environmental strains, but this changes dramati‐ cally with time and prolonged infection as *P. aeruginosa* has the potent ability of quorum sensing which leads to biofilm formation (Drenkard & Ausubel, 2002). Biofilm is a matrix of proteins, carbohydrate and collagen and muco-polysaccharide within which the bacteria re‐ side. This layer is often called alginate formation. The formation of alginate biofilm has 3 im‐ portant consequences:


mucoid *P. aeruginosa* tend to exhibit greater in vivo sensitivity to antibiotics than nonmucoid strains in the same patient.

**3.** The biofilm formation is highly immunogenic and tends to accelerate structural lung damage leading to significant histological and radiological changes.

Other relevant pathogens in CF usually cultured later in the course of the disease include *Burkholderia cepacia* complex, *Stenotrophomonas maltophilia* and *Achromobacter xylosoxidans*. The *B. cepacia* complex has at least 10 distinct genomovars and is related with rapid clinical deterioration. The most virulent and transmissible form is the genomovar III, B. cenocepa‐ cia, due to its well recognised association with severe necrotising pneumonia and death first described over 20 years ago (Isles et al., 1984). *S. maltophilia* and *A. xylosoxidans* are more prevalent than the *B. cepacia* complex, but seem to be less virulent, although their role in the

**Figure 2.** Prevalence of respiratory pathogens according to patient's age. Data from Cystic Fibrosis Foundation Patient

*Pseudomonas aeruginosa* is considered to be the most important pathogen in CF. By the time patients reach adulthood, up to 80% of them become infected with *P. aeruginosa*. The trans‐ formation of *P. aeruginosa* from the non-mucoid to the mucoid strains, which often occurs during adolescence years (figure 3a), is pivotal and is known to be associated with clinical (figure 3b) and radiological deterioration (figure 3c) (Li et al., 2005). At initial colonisation, *P. aeruginosa* presents a phenotype similar to environmental strains, but this changes dramati‐ cally with time and prolonged infection as *P. aeruginosa* has the potent ability of quorum sensing which leads to biofilm formation (Drenkard & Ausubel, 2002). Biofilm is a matrix of proteins, carbohydrate and collagen and muco-polysaccharide within which the bacteria re‐ side. This layer is often called alginate formation. The formation of alginate biofilm has 3 im‐

**1.** The bacteria are protected from antibiotics, which increases the minimal-inhibitory con‐

**2.** The film reduces the activity of aminoglycosides and beta-lactam antibiotics by chang‐ ing the pH of the respiratory mucosa and by production of beta-lactamase – ironically,

Registry, 2010 Annual Data Report.

176 Respiratory Disease and Infection - A New Insight

portant consequences:

centration.

pathogenesis of the CF lung is not yet completely understood (Goss et al., 2002).

The incidence and clinical impact of mucoid *P. aeruginosa* (figure 3a) in the sputum of a co‐ hort of patients with CF according to age. The emergence of these bacteria is associated with decline in FEV1 (figure 3b) and worsening of radiograph appearance (figure 3c). (Adapted from Li et al., 2005).

**Figure 3.** (a): Proportion (%) of *P. aeruginosa* with mucoid strains per age group. (b): Change in FEV1 for the same CF group as in 3a. (c): Change in the chest radiograph appearance in the same CF group as in 3a

#### **4. Acute pulmonary exacerbations in CF**

Pulmonary exacerbations in CF are an important feature of the disease. Frequency of pulmo‐ nary exacerbations is associated with accelerated decline in lung function tests, impaired quality of life and premature mortality. Nevertheless, defining what constitutes a pulmona‐ ry exacerbation remains controversial.




exacerbation.










following 7 symptoms during the study.






parenteral antibiotics for any 4 of the following 12 signs or symptoms:



**Table 3.** Ramsey et al. (1999) Inhaled Tobramycin Study: Pulmonary exacerbation indicated by at least 2 of the






**Table 1.** Signs and symptoms of pulmonary exacerbations as agreed by the consensus document of the US Cystic Fibrosis Foundation, 1994. Typically a combination of 3 or more signs and symptoms represent a pulmonary

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179

**Table 2.** Fuchs et al. (1994) Pulmozyme Study: "Exacerbation of respiratory symptoms": a patient treated with


To date there is not yet a consensus or a standardised diagnostic criteria as to what clinically characterises pulmonary exacerbations in CF (Abbot et al., 2009; Dakin et al., 2001), but all descriptions point to that patients present with a combination of symptoms. Although, not stated clearly, the implication is that symptoms during exacerbations are greater than symp‐ toms during 'disease stability'.

Pulmonary exacerbations are usually described by patients in subjective terms as an increase in one or more respiratory symptoms, such as cough, sputum volume, viscosity and colour change, breathlessness; altered systemic symptoms, such as increased fatigue, decreased ap‐ petite and energy levels. This is usually, but not always associated with an increase in objec‐ tive clinical signs and changes in biomarkers. These included: weight loss, tachypnoea, tachycardia, decreased lung function parameters, chest radiographic changes and raised se‐ rum inflammatory markers (Ramsey et al., 1999; Fuchs et al., 1994).

The lack of a standardised definition is also present when it comes to classifying pulmonary exacerbations according to their severity. Pulmonary exacerbations are broadly divided into: a) mild exacerbations requiring treatment with oral antibiotics and b) severe exacerbations needing treatment with intravenous (IV) antibiotics. It also remains unclear if mild exacerba‐ tions are the originators of severe infections, if they are earlier milder versions of severe pul‐ monary exacerbations or in fact differentiated clinical episodes with no correlation between mild and severe infections (Goss & Burns, 2007).

Despite the lack of agreement, several diagnostic systems are described in the literature and used in CF clinical trials, such as the Fuchs et al. (1994) Pulmozyme® study and the Ramsey et al. (1999) inhaled Tobramycin study diagnostic criteria, and the consensus document by the US CF Foundation, 1994 (tables 1, 2 and 3).

These definitions, often called "symptom-related definitions", although widely used, have 3 shortcomings:


#### - Increased cough

**4. Acute pulmonary exacerbations in CF**

ry exacerbation remains controversial.

178 Respiratory Disease and Infection - A New Insight

toms during 'disease stability'.

Pulmonary exacerbations in CF are an important feature of the disease. Frequency of pulmo‐ nary exacerbations is associated with accelerated decline in lung function tests, impaired quality of life and premature mortality. Nevertheless, defining what constitutes a pulmona‐

To date there is not yet a consensus or a standardised diagnostic criteria as to what clinically characterises pulmonary exacerbations in CF (Abbot et al., 2009; Dakin et al., 2001), but all descriptions point to that patients present with a combination of symptoms. Although, not stated clearly, the implication is that symptoms during exacerbations are greater than symp‐

Pulmonary exacerbations are usually described by patients in subjective terms as an increase in one or more respiratory symptoms, such as cough, sputum volume, viscosity and colour change, breathlessness; altered systemic symptoms, such as increased fatigue, decreased ap‐ petite and energy levels. This is usually, but not always associated with an increase in objec‐ tive clinical signs and changes in biomarkers. These included: weight loss, tachypnoea, tachycardia, decreased lung function parameters, chest radiographic changes and raised se‐

The lack of a standardised definition is also present when it comes to classifying pulmonary exacerbations according to their severity. Pulmonary exacerbations are broadly divided into: a) mild exacerbations requiring treatment with oral antibiotics and b) severe exacerbations needing treatment with intravenous (IV) antibiotics. It also remains unclear if mild exacerba‐ tions are the originators of severe infections, if they are earlier milder versions of severe pul‐ monary exacerbations or in fact differentiated clinical episodes with no correlation between

Despite the lack of agreement, several diagnostic systems are described in the literature and used in CF clinical trials, such as the Fuchs et al. (1994) Pulmozyme® study and the Ramsey et al. (1999) inhaled Tobramycin study diagnostic criteria, and the consensus document by

These definitions, often called "symptom-related definitions", although widely used, have 3

**1.** They do not account for differences of CF pulmonary exacerbations in children and adults. For example, the presence of fever and acute changes in chest radiographs are

**2.** They do not quantify symptom severity and do not give allowance for increased severi‐ ty of a single symptom, which may make patients seek medical consultation and receive treatment. For example, marked increase in breathlessness, significant increase in cough production of viscous sputum or increased lethargy alone could be a single symptom

**3.** The above definitions do not stipulate that increase in symptoms should be sustained and should be beyond the natural fluctuation of symptoms encountered in most CF patients.

that may constitute an exacerbation requiring treatment with antibiotics.

rum inflammatory markers (Ramsey et al., 1999; Fuchs et al., 1994).

mild and severe infections (Goss & Burns, 2007).

the US CF Foundation, 1994 (tables 1, 2 and 3).

often seen in children, but not in adults.

shortcomings:



**Table 1.** Signs and symptoms of pulmonary exacerbations as agreed by the consensus document of the US Cystic Fibrosis Foundation, 1994. Typically a combination of 3 or more signs and symptoms represent a pulmonary exacerbation.

#### - Change in sputum


**Table 2.** Fuchs et al. (1994) Pulmozyme Study: "Exacerbation of respiratory symptoms": a patient treated with parenteral antibiotics for any 4 of the following 12 signs or symptoms:


**Table 3.** Ramsey et al. (1999) Inhaled Tobramycin Study: Pulmonary exacerbation indicated by at least 2 of the following 7 symptoms during the study.

<sup>-</sup> Fever (oral temperature >38°C)

In a retrospective analysis of 77 pulmonary exacerbations in 88 adult patients, the authors found that IV antibiotics were given to 18 patients (24.4%) whose symptoms did not meet the US CF criteria (table 1) (Jeffcote et al., 2004). This would suggest that nearly one quarter of patients presented with sufficiently severe symptoms to warrant treatment with IV antibi‐ otics, although the combination of factors did not meet the pre-designed criteria.

nosed was higher than that in CF patients and the number of hospitalisation was much re‐ duced in COPD patients (but not in CF patients) compared to a similar period without daily

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181

**Figure 4.** An example of the daily recording of a 23 male CF patient. The Y axis is the score of each symptom (cough, sputum, breathlessness and fatigue). For FEV1, the Y axis is the value in litres. The time line on the X axis is divided in weeks. Note that only 9 days were recorded by the patients out of possible 49 days (7 weeks). This patient was with‐

monitoring the year prior to the study.

drawn from the study.

For this reason, we explored another definition of pulmonary exacerbations, which resem‐ bled the internationally accepted definition of exacerbation in chronic obstructive pulmona‐ ry disease (COPD). In such patients, pulmonary exacerbations are defined as "an event in the course of the disease in which there is a sustained worsening of the patient's respiratory symptoms from the stable state that is beyond normal day-to-day variations, that is acute in onset and necessitates escalation of treatment". This is often termed as "an action-defini‐ tion" in which an exacerbation is regarded to be present when the following 3 criteria are met:


This definition has its problems too. Patients have different thresholds for seeking medical care and therefore some may present more frequently than others, despite similar severity of symptoms. Another problem is that in countries where patients have to pay for their treat‐ ment, financial restraint may prevent patients from seeking medical treatment for exacerba‐ tion of symptoms.

To try to resolve this issue, Sarfaraz et al. (2010) conducted a study using electronic remote daily telemonitoring of symptoms in a cohort of adult CF patients. The study highlighted several issues in the management of CF exacerbations. Out of the 51 patients enrolled in the study, 32 (63%) patients were subsequently excluded as their recordings were not frequent enough to form a baseline data or insufficient to look into the natural history of the disease (Figure 4). Furthermore, for the remaining 19 patients who completed the study, an average of only 60% of the total number of study days was recorded.

Despite these significant deficiencies, there were enough CF pulmonary exacerbations for analyses. The authors noticed that 75% of all CF exacerbations had a "prodromal" phase in which one or more than one symptom increased in the 2 weeks prior to exacerbations (Figure 5).

In order to examine whether the problem with daily recording was related to the introduc‐ tion of a modern system, a parallel study in COPD patients was carried out (Sund et al., 2009). A systematic comparison between the 2 groups was later published (Jarad & Sund, 2011). The comparison revealed that the dropout rate was much smaller for COPD patients, who recorded daily data in 77% of the total number of study days (figure 6). In accordance with the higher recording rate by COPD patients, the number of early exacerbations diag‐ nosed was higher than that in CF patients and the number of hospitalisation was much re‐ duced in COPD patients (but not in CF patients) compared to a similar period without daily monitoring the year prior to the study.

In a retrospective analysis of 77 pulmonary exacerbations in 88 adult patients, the authors found that IV antibiotics were given to 18 patients (24.4%) whose symptoms did not meet the US CF criteria (table 1) (Jeffcote et al., 2004). This would suggest that nearly one quarter of patients presented with sufficiently severe symptoms to warrant treatment with IV antibi‐

For this reason, we explored another definition of pulmonary exacerbations, which resem‐ bled the internationally accepted definition of exacerbation in chronic obstructive pulmona‐ ry disease (COPD). In such patients, pulmonary exacerbations are defined as "an event in the course of the disease in which there is a sustained worsening of the patient's respiratory symptoms from the stable state that is beyond normal day-to-day variations, that is acute in onset and necessitates escalation of treatment". This is often termed as "an action-defini‐ tion" in which an exacerbation is regarded to be present when the following 3 criteria are

**2.** The symptoms are subjectively more than the patient's own baseline symptoms –

**3.** The symptoms are objectively compelling to the physicians as to be deemed to require

This definition has its problems too. Patients have different thresholds for seeking medical care and therefore some may present more frequently than others, despite similar severity of symptoms. Another problem is that in countries where patients have to pay for their treat‐ ment, financial restraint may prevent patients from seeking medical treatment for exacerba‐

To try to resolve this issue, Sarfaraz et al. (2010) conducted a study using electronic remote daily telemonitoring of symptoms in a cohort of adult CF patients. The study highlighted several issues in the management of CF exacerbations. Out of the 51 patients enrolled in the study, 32 (63%) patients were subsequently excluded as their recordings were not frequent enough to form a baseline data or insufficient to look into the natural history of the disease (Figure 4). Furthermore, for the remaining 19 patients who completed the study, an average

Despite these significant deficiencies, there were enough CF pulmonary exacerbations for analyses. The authors noticed that 75% of all CF exacerbations had a "prodromal" phase in which one or more than one symptom increased in the 2 weeks prior to exacerbations (Figure

In order to examine whether the problem with daily recording was related to the introduc‐ tion of a modern system, a parallel study in COPD patients was carried out (Sund et al., 2009). A systematic comparison between the 2 groups was later published (Jarad & Sund, 2011). The comparison revealed that the dropout rate was much smaller for COPD patients, who recorded daily data in 77% of the total number of study days (figure 6). In accordance with the higher recording rate by COPD patients, the number of early exacerbations diag‐

otics, although the combination of factors did not meet the pre-designed criteria.

**1.** The increase in respiratory symptoms is sustained (lasting 2-3 days).

enough so that the patient seeks medical treatment.

of only 60% of the total number of study days was recorded.

escalation of treatment with antibiotics.

180 Respiratory Disease and Infection - A New Insight

met:

tion of symptoms.

5).

**Figure 4.** An example of the daily recording of a 23 male CF patient. The Y axis is the score of each symptom (cough, sputum, breathlessness and fatigue). For FEV1, the Y axis is the value in litres. The time line on the X axis is divided in weeks. Note that only 9 days were recorded by the patients out of possible 49 days (7 weeks). This patient was with‐ drawn from the study.

**Figure 5.** A 24 year old female CF patient who had a pulmonary exacerbation (marked with the red vertical line) de‐ fined by the decline in FEV1. Note that there was a "prodromal phase" manifested by increase in cough and fatigue within the 2 weeks prior to the exacerbation.

**Figure 6.** A recording from a patient with COPD. The red line represents an exacerbation defined as 15% decline in FEV1 below the baseline. Note the patient performed a daily recording without interruption allowing a proper identi‐

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183

Differences between the two groups may not only reflect differences in disease processes and age groups. They may also disclose the differences in the system of care for CF patients who tend to have a privileged access to hospital care through direct contact with a CF multi‐ disciplinary team. This is not available to COPD patients, whose contact with healthcare

fication of acute exacerbation.

takes place through a general practitioner.

**Figure 6.** A recording from a patient with COPD. The red line represents an exacerbation defined as 15% decline in FEV1 below the baseline. Note the patient performed a daily recording without interruption allowing a proper identi‐ fication of acute exacerbation.

Differences between the two groups may not only reflect differences in disease processes and age groups. They may also disclose the differences in the system of care for CF patients who tend to have a privileged access to hospital care through direct contact with a CF multi‐ disciplinary team. This is not available to COPD patients, whose contact with healthcare takes place through a general practitioner.

**Figure 5.** A 24 year old female CF patient who had a pulmonary exacerbation (marked with the red vertical line) de‐ fined by the decline in FEV1. Note that there was a "prodromal phase" manifested by increase in cough and fatigue

within the 2 weeks prior to the exacerbation.

182 Respiratory Disease and Infection - A New Insight

#### **5. Causes of pulmonary exacerbations**

Whilst still not completely understood, causes of pulmonary exacerbations are most likely multi-factorial, and may differ amongst patients.

In addition, this study concords with previous studies that have demonstrated factors associat‐ ed with pulmonary exacerbations, such as young age, female gender, lower FEV1, CFRD and

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185

Conversely inhaled antibiotics such as aminoglycosides, oral macrolides, mucolytics and in‐ haled hypertonic saline have been shown to reduce the rate of pulmonary exacerbations (Gibson et al., 2003; Saiman et al., 2003; Ramsey et al. 1999; Fuchs et al., 1994), presumably by reducing the bacterial load and inflammation and clearing the airways, reducing the vol‐

> **Histogram of the numbers of pulmonary exacerbations**

**0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16**

Number of exacerbations

**Figure 7.** A histogram of the number of exacerbations experienced in one year by 680 patients in the South West of

**Figure 8.** Patients with and without chronic infection with *P. aeruginosa* in 4 annual exacerbations category. Note that the proportion of those who are infected is greater with increased frequency of annual exacerbations. PExs = number

**Number of patients**

previous history of multiple exacerbations (Block et al., 2006; Marshall et al., 2005).

ume of retained secretions.

England.

of pulmonary exacerbations per year.

Nevertheless, CF pulmonary exacerbations are probably a consequence of a loss of equilibrium between host defence mechanisms and the airway microorganisms. It is believed that viruses, including respiratory syncytial virus, have a role in pulmonary exacerbations (Hiatt et al., 1999), and whilst these have also been associated with the acquisition of new organisms, for the majority of adult CF patients a new pulmonary exacerbations seems to be caused much more commonly by a clonal expansion of already present colonising bacteria (Aaron et al., 2004).

Only rarely are pulmonary exacerbations a consequence of newly acquired bacteria. The in‐ crease in bacterial load in conjunction with an inflammatory response of the airways result in a local influx of polymorphonucleocytes and the release of inflammatory mediators such as interleukins – (IL) 8, 6, 1β, tumour necrosis factor alpha (TNF-α), leukotriene B4 and free neutrophil elastase.

Treatment with antimicrobial agents is associated with decreased levels of inflammatory mediators and bacterial density and improvement of symptoms and lung function parame‐ ters (Colombo et al., 2005; Ordonez et al., 2003; Sagel et al., 2001; Bonfield et al., 1995; Kon‐ stan et al., 1994; Konstan et al., 1993). This suggests that the increased density of microbes plays an important role in pulmonary exacerbations. What remains unclear is the lack of ability of powerful antibiotics given at great doses in eradicating pulmonary microbes in CF patients. Rather it would seem that the role of antibiotics is akin to cutting the grass in a front garden lawn, only for the grass to grow again.

#### **6. Epidemiology and risk factors**

CF pulmonary exacerbations are common and the impact they have on patient care, espe‐ cially in terms of labour intensiveness, is the main reason for the establishment of CF centres and teams. In addition, treatment of pulmonary exacerbations accounts for a significant part of the cost of CF care.

In a cross sectional study on adult CF patients in the South West of England, Jarad & Giles (2008) found that a significant number of patients did not suffer from any pulmonary exac‐ erbation. Many patients, however, suffered from at least one exacerbation every year (figure 7). When examining risk factors of exacerbations, reduced FEV1 (figure 8), infection with *P. aeruginosa* (figure 9) and cystic fibrosis related diabetes (CFRD) were correlated with in‐ creased rate of exacerbations (figure 10) (adapted from Jarad & Giles, 2008). Remarkably, ge‐ netic profile, diagnostic sweat chloride, body mass index, age and gender did not correlate with frequency of pulmonary exacerbations.

In addition, this study concords with previous studies that have demonstrated factors associat‐ ed with pulmonary exacerbations, such as young age, female gender, lower FEV1, CFRD and previous history of multiple exacerbations (Block et al., 2006; Marshall et al., 2005).

**5. Causes of pulmonary exacerbations**

184 Respiratory Disease and Infection - A New Insight

multi-factorial, and may differ amongst patients.

front garden lawn, only for the grass to grow again.

**6. Epidemiology and risk factors**

with frequency of pulmonary exacerbations.

neutrophil elastase.

of the cost of CF care.

Whilst still not completely understood, causes of pulmonary exacerbations are most likely

Nevertheless, CF pulmonary exacerbations are probably a consequence of a loss of equilibrium between host defence mechanisms and the airway microorganisms. It is believed that viruses, including respiratory syncytial virus, have a role in pulmonary exacerbations (Hiatt et al., 1999), and whilst these have also been associated with the acquisition of new organisms, for the majority of adult CF patients a new pulmonary exacerbations seems to be caused much more commonly by a clonal expansion of already present colonising bacteria (Aaron et al., 2004).

Only rarely are pulmonary exacerbations a consequence of newly acquired bacteria. The in‐ crease in bacterial load in conjunction with an inflammatory response of the airways result in a local influx of polymorphonucleocytes and the release of inflammatory mediators such as interleukins – (IL) 8, 6, 1β, tumour necrosis factor alpha (TNF-α), leukotriene B4 and free

Treatment with antimicrobial agents is associated with decreased levels of inflammatory mediators and bacterial density and improvement of symptoms and lung function parame‐ ters (Colombo et al., 2005; Ordonez et al., 2003; Sagel et al., 2001; Bonfield et al., 1995; Kon‐ stan et al., 1994; Konstan et al., 1993). This suggests that the increased density of microbes plays an important role in pulmonary exacerbations. What remains unclear is the lack of ability of powerful antibiotics given at great doses in eradicating pulmonary microbes in CF patients. Rather it would seem that the role of antibiotics is akin to cutting the grass in a

CF pulmonary exacerbations are common and the impact they have on patient care, espe‐ cially in terms of labour intensiveness, is the main reason for the establishment of CF centres and teams. In addition, treatment of pulmonary exacerbations accounts for a significant part

In a cross sectional study on adult CF patients in the South West of England, Jarad & Giles (2008) found that a significant number of patients did not suffer from any pulmonary exac‐ erbation. Many patients, however, suffered from at least one exacerbation every year (figure 7). When examining risk factors of exacerbations, reduced FEV1 (figure 8), infection with *P. aeruginosa* (figure 9) and cystic fibrosis related diabetes (CFRD) were correlated with in‐ creased rate of exacerbations (figure 10) (adapted from Jarad & Giles, 2008). Remarkably, ge‐ netic profile, diagnostic sweat chloride, body mass index, age and gender did not correlate Conversely inhaled antibiotics such as aminoglycosides, oral macrolides, mucolytics and in‐ haled hypertonic saline have been shown to reduce the rate of pulmonary exacerbations (Gibson et al., 2003; Saiman et al., 2003; Ramsey et al. 1999; Fuchs et al., 1994), presumably by reducing the bacterial load and inflammation and clearing the airways, reducing the vol‐ ume of retained secretions.

**Figure 7.** A histogram of the number of exacerbations experienced in one year by 680 patients in the South West of England.

**Figure 8.** Patients with and without chronic infection with *P. aeruginosa* in 4 annual exacerbations category. Note that the proportion of those who are infected is greater with increased frequency of annual exacerbations. PExs = number of pulmonary exacerbations per year.

Fig9

**7. What affects the time until the subsequent CF pulmonary**

was 112 days, although this varied considerably (figure 11).

treatment were found to be the two independent risk factors.

Time from the end of one pulmonary exacerbation until the subsequent exacerbation (time until the next exacerbation) is a significant health outcome and one of the significant end points in many CF clinical trials. Shorter periods until the following exacerbation is a marker of increased impact of disease on patients. In a prospective study on 170 exacerbations in 58 adult CF patients, (Sequeiros & Jarad, 2012), the median time until subsequent exacerbations

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187

**Figure 11.** A histogram of time until the next exacerbation on the X axis. Median time (red vertical line) was 121 days, although this varied considerably. Those patients who did not have an exacerbation during the subsequent year after the first pulmonary exacerbation were regarded to have a time until the next exacerbation of 360 days (adapted from

Factors affecting shorter periods until the following pulmonary exacerbation were exam‐ ined, including age, lung disease severity, CF related complications (CFRD, allergic bron‐ cho-pulmonary aspergillosis (ABPA)), chronic infection with *P. aeruginosa* and site of administration of antibiotics – either home or in hospital. In addition, symptom scores, lung function tests and inflammatory biomarkers at the end of the IV treatment were examined. When analysing individual variables, patients with lower FEV1, greater symptom scores and higher C-reactive protein (CRP) at the end of the exacerbation treatment were associat‐ ed with shorter time until the next exacerbation. Also patients with ABPA and CFRD had a shorter time until the next exacerbations than those without. After adjustments to confound‐ ing factors, however, older CF patients and those with lower FEV1 at the end of course of

**exacerbations?**

Sequeiros & Jarad, 2012).

**Figure 9.** Annual exacerbation frequency increases in patients with lower FEV1. N = number of patients per group. PExs = number of pulmonary exacerbations per year.

**Figure 10.** Proportion of patients with CFRD and without CFRD according to annual exacerbation frequency. PExs = number of pulmonary exacerbations per year.

### **7. What affects the time until the subsequent CF pulmonary exacerbations?**

Time from the end of one pulmonary exacerbation until the subsequent exacerbation (time until the next exacerbation) is a significant health outcome and one of the significant end points in many CF clinical trials. Shorter periods until the following exacerbation is a marker of increased impact of disease on patients. In a prospective study on 170 exacerbations in 58 adult CF patients, (Sequeiros & Jarad, 2012), the median time until subsequent exacerbations was 112 days, although this varied considerably (figure 11).

Fig9

**40**

number of pulmonary exacerbations per year.

PExs = number of pulmonary exacerbations per year.

**50**

**60**

**70**

**80**

**90**

186 Respiratory Disease and Infection - A New Insight

**FEV1 (% Predicted)**

**n=169**

**Pcorr = <0.002**

**n=150**

 **No PExs 1-2 PExs 3-4 PExs Over 4 PExs**

**Figure 9.** Annual exacerbation frequency increases in patients with lower FEV1. N = number of patients per group.

**Figure 10.** Proportion of patients with CFRD and without CFRD according to annual exacerbation frequency. PExs =

**n=73**

**n=43**

**Figure 11.** A histogram of time until the next exacerbation on the X axis. Median time (red vertical line) was 121 days, although this varied considerably. Those patients who did not have an exacerbation during the subsequent year after the first pulmonary exacerbation were regarded to have a time until the next exacerbation of 360 days (adapted from Sequeiros & Jarad, 2012).

Factors affecting shorter periods until the following pulmonary exacerbation were exam‐ ined, including age, lung disease severity, CF related complications (CFRD, allergic bron‐ cho-pulmonary aspergillosis (ABPA)), chronic infection with *P. aeruginosa* and site of administration of antibiotics – either home or in hospital. In addition, symptom scores, lung function tests and inflammatory biomarkers at the end of the IV treatment were examined.

When analysing individual variables, patients with lower FEV1, greater symptom scores and higher C-reactive protein (CRP) at the end of the exacerbation treatment were associat‐ ed with shorter time until the next exacerbation. Also patients with ABPA and CFRD had a shorter time until the next exacerbations than those without. After adjustments to confound‐ ing factors, however, older CF patients and those with lower FEV1 at the end of course of treatment were found to be the two independent risk factors.

#### **8. Management of pulmonary exacerbations**

The mainstay of treatment of pulmonary exacerbations is administration of antibiotics. For mild exacerbations oral antibiotics are usually administered – mainly quinolones. This ap‐ proach is based on little evidence, since a large proportion of patient have quinolone resist‐ ant *P. aeruginosa*.

clinical recovery in 72% and 75% of patients respectively. A recent guideline committee for treatment of CF pulmonary exacerbations that studied the literature of management of exac‐ erbations found no evidence for the optimal duration of treatment of CF pulmonary exacer‐ bations (Flume et al., 2009). The committee recommended that future studies should examine short and long term subjective and objective outcomes of management of exacerba‐

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Risk factors and efficacy of extending the course of antibiotics have been prospectively ex‐ amined (Sequeiros & Jarad, 2012). As in previous studies, nearly 23% of 168 pulmonary ex‐ acerbations in 58 adult CF patients did not recover after 2 weeks of IV antibiotic treatment, needing extension of duration of treatment. For those prescribed extended courses, most pa‐ tients required additional 7 days of treatment, but some needed doubling of the duration of treatment (figure 12). Unlike previous studies, a validated symptom score was used and bio‐

**Figure 12.** A histogram of the number of patients (Y axis) and duration of treatment for exacerbations for patients needing extension of IV treatment (X axis). Most patients needed additional 7 days, but some needed at least dou‐

Risk factors for extending the IV course were found to be more severe lung disease and per‐ sistent respiratory symptoms and systemic inflammation as assessed by CRP at the end of treatment. The extension of treatment beyond 14 days resulted in improvement of symp‐ toms, but not of FEV1. Extension of treatment did not result in increased time to the next

tions according to duration of treatment.

markers measured at the end of treatment were examined.

bling the duration of treatment (Adapted from Sequeiros & Jarad, 2012).

exacerbation.

More severe exacerbations are prescribed IV antibiotics. As most patients with frequent ex‐ acerbations commonly grow *P. aeruginosa* in their sputum, CF physicians prescribe two anti‐ biotics aiming to effectively decrease the bacterial load and reduce the probability of developing antibiotic resistance. The choice of antibiotic class range would often include an aminoglycoside (tobramycin, gentamycin and amikacin) and a beta-lactam (ceftazidime, az‐ treonam, pipracillin or ticarcillin) or a quinolone (ciprofloxacin). In more recent years, carbo‐ penims (imipenim or meropenim) are being more frequently used. Occasionally IV colistine is prescribed in cases of bacterial resistance. The duration of treatment is often 2 weeks.

The doses of antibiotics given for CF patients are often larger than those given to other respi‐ ratory and non-respiratory infections. This is, theoretically, thought to be necessary due the high bacterial load in the damaged and inflamed lungs and the difficulty of antibiotics pene‐ trating the thick layer of inspissated sputum to reach the bacteria – in particular when mu‐ coid *P. aeruginosa* is present.

Lack of evidence of how to optimally manage CF pulmonary exacerbations is, in part, due to the fact that the determinants of the successful outcome of treatment of an exacerbation have not yet been clearly identified. In most CF centres improvement in general clinical status and in lung function tests are accepted to determine the 'end' of the exacerbation. For those patients who do not show sufficient improvement in the opinion of the treating physicians, the course of antibiotics is either extended or the combination of antibiotics is changed. De‐ spite its shortcomings, this approach has been endorsed in a consensus document published by the UK CF Trust (UK CF Trust Antibiotic Guidelines 2008).

For pulmonary exacerbations treated with IV antibiotics, the combination of choice is often determined on arbitrary grounds. Previous *in vitro* sensitivity and previous clinical response normally determine the choice of antibiotics. However, several retrospective and prospec‐ tive studies found that the concordance of *in vitro* sensitivity did not affect any of the out‐ comes of the CF exacerbations, including lung function tests and the time until the subsequent pulmonary exacerbation (Jarad et al., 2007; Foweraker et al., 2005).

Frequent and longer courses of IV treatment, particularly with aminoglycosides, have been shown to be associated with renal impairment (Smyth et al., 2008; Al Aloul et al., 2005) and ototoxicity (Mulheran et al., 2001; Scott et al., 2001), as well as increased rate of antibiotic al‐ lergy (Moss et al., 1984). In addition, extension of duration of IV antibiotic treatment is asso‐ ciated with added volume of work to patients and CF staff and in incremental cost pressure of CF care.

Two retrospective studies (Sanders et al., 2010; Rosenberg & Scharamm, 1993) found that ad‐ ministration of antibiotics for 14 days in adolescent and young adult CF patients resulted in clinical recovery in 72% and 75% of patients respectively. A recent guideline committee for treatment of CF pulmonary exacerbations that studied the literature of management of exac‐ erbations found no evidence for the optimal duration of treatment of CF pulmonary exacer‐ bations (Flume et al., 2009). The committee recommended that future studies should examine short and long term subjective and objective outcomes of management of exacerba‐ tions according to duration of treatment.

**8. Management of pulmonary exacerbations**

188 Respiratory Disease and Infection - A New Insight

ant *P. aeruginosa*.

coid *P. aeruginosa* is present.

of CF care.

The mainstay of treatment of pulmonary exacerbations is administration of antibiotics. For mild exacerbations oral antibiotics are usually administered – mainly quinolones. This ap‐ proach is based on little evidence, since a large proportion of patient have quinolone resist‐

More severe exacerbations are prescribed IV antibiotics. As most patients with frequent ex‐ acerbations commonly grow *P. aeruginosa* in their sputum, CF physicians prescribe two anti‐ biotics aiming to effectively decrease the bacterial load and reduce the probability of developing antibiotic resistance. The choice of antibiotic class range would often include an aminoglycoside (tobramycin, gentamycin and amikacin) and a beta-lactam (ceftazidime, az‐ treonam, pipracillin or ticarcillin) or a quinolone (ciprofloxacin). In more recent years, carbo‐ penims (imipenim or meropenim) are being more frequently used. Occasionally IV colistine is prescribed in cases of bacterial resistance. The duration of treatment is often 2 weeks.

The doses of antibiotics given for CF patients are often larger than those given to other respi‐ ratory and non-respiratory infections. This is, theoretically, thought to be necessary due the high bacterial load in the damaged and inflamed lungs and the difficulty of antibiotics pene‐ trating the thick layer of inspissated sputum to reach the bacteria – in particular when mu‐

Lack of evidence of how to optimally manage CF pulmonary exacerbations is, in part, due to the fact that the determinants of the successful outcome of treatment of an exacerbation have not yet been clearly identified. In most CF centres improvement in general clinical status and in lung function tests are accepted to determine the 'end' of the exacerbation. For those patients who do not show sufficient improvement in the opinion of the treating physicians, the course of antibiotics is either extended or the combination of antibiotics is changed. De‐ spite its shortcomings, this approach has been endorsed in a consensus document published

For pulmonary exacerbations treated with IV antibiotics, the combination of choice is often determined on arbitrary grounds. Previous *in vitro* sensitivity and previous clinical response normally determine the choice of antibiotics. However, several retrospective and prospec‐ tive studies found that the concordance of *in vitro* sensitivity did not affect any of the out‐ comes of the CF exacerbations, including lung function tests and the time until the

Frequent and longer courses of IV treatment, particularly with aminoglycosides, have been shown to be associated with renal impairment (Smyth et al., 2008; Al Aloul et al., 2005) and ototoxicity (Mulheran et al., 2001; Scott et al., 2001), as well as increased rate of antibiotic al‐ lergy (Moss et al., 1984). In addition, extension of duration of IV antibiotic treatment is asso‐ ciated with added volume of work to patients and CF staff and in incremental cost pressure

Two retrospective studies (Sanders et al., 2010; Rosenberg & Scharamm, 1993) found that ad‐ ministration of antibiotics for 14 days in adolescent and young adult CF patients resulted in

subsequent pulmonary exacerbation (Jarad et al., 2007; Foweraker et al., 2005).

by the UK CF Trust (UK CF Trust Antibiotic Guidelines 2008).

Risk factors and efficacy of extending the course of antibiotics have been prospectively ex‐ amined (Sequeiros & Jarad, 2012). As in previous studies, nearly 23% of 168 pulmonary ex‐ acerbations in 58 adult CF patients did not recover after 2 weeks of IV antibiotic treatment, needing extension of duration of treatment. For those prescribed extended courses, most pa‐ tients required additional 7 days of treatment, but some needed doubling of the duration of treatment (figure 12). Unlike previous studies, a validated symptom score was used and bio‐ markers measured at the end of treatment were examined.

**Figure 12.** A histogram of the number of patients (Y axis) and duration of treatment for exacerbations for patients needing extension of IV treatment (X axis). Most patients needed additional 7 days, but some needed at least dou‐ bling the duration of treatment (Adapted from Sequeiros & Jarad, 2012).

Risk factors for extending the IV course were found to be more severe lung disease and per‐ sistent respiratory symptoms and systemic inflammation as assessed by CRP at the end of treatment. The extension of treatment beyond 14 days resulted in improvement of symp‐ toms, but not of FEV1. Extension of treatment did not result in increased time to the next exacerbation.

#### **9. What measurements determine the outcome of CF pulmonary exacerbations?**

The aim of management of CF pulmonary exacerbations is to restore patients to pre-exacer‐ bation clinical status. In most CF centres, determining the end of an exacerbation is done ar‐ bitrarily during clinical consultation.

**10. Oral corticosteroids as an adjuvant to antibiotics**

would be thought to be a logical approach.

stay compared with placebo-treated patients.

CF exacerbations, and the issue remains contentious.

pared to adult CF patients (figure 14).

matic group (Elborn et al., 2000).

tions (Hester et al., 2007).

As discussed above, in addition to excessive bacterial growth, exuberant local lung inflam‐ mation is considered to play an important role in CF pulmonary exacerbations. In this con‐ text, using corticosteroids in addition to antibiotics during the treatment of exacerbations

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191

Managing exacerbations with corticosteroids and antibiotics is the norm in patients with COPD. In a prospective study on inpatients with severe COPD who were hospitalised due to acute exacerbations, Davies et al. (1999) found that adding 30mg prednisolone for 14 days to usual treatment resulted in improvement of FEV1 and shortening of length of hospital

For CF patients, an open label study found that adding oral corticosteroids resulted in an improvement of FEV1 in a small cohort of adult CF patients (Dovey et al., 2007). Further‐ more, a national UK survey performed in the authors' unit, found that all UK CF physicians had used adjuvant corticosteroids to different extents in managing CF pulmonary exacerba‐

This is important because of the increased propensity of CF patients to develop diabetes and osteoporosis by virtue of the CF disease process. Corticosteroids are bound to adversely af‐ fect the likelihood of these two complications, (increase the incidence and severity), as a sig‐ nificant negative side effect of the drug. To date there are no large trials to answer the question of whether adding corticosteroids to antibiotics improves outcome of treatment of

**11. Elective courses of antibiotics versus symptomatic treatments**

Elective regular administration of IV antibiotics several times per year has been a practice adopted by many CF teams to improve symptoms and reduce decline in lung function tests. In a national UK survey of CF centres (Higgs & Jarad, 2005), the authors found that greater proportion of paediatric patients received regular (elective) courses of IV antibiotics com‐

This is despite the fact that a previous study showed no differences in spirometry im‐ provement in patients who received elective 3 monthly anti-pseudomonal antibiotic treatment compared to those who received conventional symptomatic treatment, trig‐ gered by increased symptoms. The elective group received an average of 4 courses of antibiotics per year, compared with an average of 3 courses per year in the sympto‐

Sequeiros et al. (2009) examined several clinical parameters throughout the duration of treat‐ ment of exacerbations with IV antibiotics to assess which one best correlated with the pa‐ tient's clinical picture. These included spirometry, airway calibre and airway resistance assessed by high frequency test. A novel symptom scoring system was also used, which has been more recently validated (Jarad & Sequeiros, 2012).

The symptom score was the parameter that changed most frequently at the end of treatment of exacerbations, more so than FEV1 and FVC (figure 13). As for airway calibre and resist‐ ance, these did not significantly change at the end of exacerbations. Changes in symptom score correlated with changes in FEV1 and FVC. The authors concluded that the novel symptom scoring system is sensitive to change with treatment and can be a useful tool for the assessment of treatment of pulmonary exacerbations.

**Figure 13.** Change after 14 days of IV antibiotics in symptom score (lower score is better health status), FEV1 and FVC in a cohort of adult CF patients treated for acute pulmonary exacerbations. The symptom score improved in a higher proportion of patients in comparison with FEV1 and FVC.

#### **10. Oral corticosteroids as an adjuvant to antibiotics**

**9. What measurements determine the outcome of CF pulmonary**

The aim of management of CF pulmonary exacerbations is to restore patients to pre-exacer‐ bation clinical status. In most CF centres, determining the end of an exacerbation is done ar‐

Sequeiros et al. (2009) examined several clinical parameters throughout the duration of treat‐ ment of exacerbations with IV antibiotics to assess which one best correlated with the pa‐ tient's clinical picture. These included spirometry, airway calibre and airway resistance assessed by high frequency test. A novel symptom scoring system was also used, which has

The symptom score was the parameter that changed most frequently at the end of treatment of exacerbations, more so than FEV1 and FVC (figure 13). As for airway calibre and resist‐ ance, these did not significantly change at the end of exacerbations. Changes in symptom score correlated with changes in FEV1 and FVC. The authors concluded that the novel symptom scoring system is sensitive to change with treatment and can be a useful tool for

**Figure 13.** Change after 14 days of IV antibiotics in symptom score (lower score is better health status), FEV1 and FVC in a cohort of adult CF patients treated for acute pulmonary exacerbations. The symptom score improved in a higher

**exacerbations?**

bitrarily during clinical consultation.

190 Respiratory Disease and Infection - A New Insight

been more recently validated (Jarad & Sequeiros, 2012).

the assessment of treatment of pulmonary exacerbations.

proportion of patients in comparison with FEV1 and FVC.

As discussed above, in addition to excessive bacterial growth, exuberant local lung inflam‐ mation is considered to play an important role in CF pulmonary exacerbations. In this con‐ text, using corticosteroids in addition to antibiotics during the treatment of exacerbations would be thought to be a logical approach.

Managing exacerbations with corticosteroids and antibiotics is the norm in patients with COPD. In a prospective study on inpatients with severe COPD who were hospitalised due to acute exacerbations, Davies et al. (1999) found that adding 30mg prednisolone for 14 days to usual treatment resulted in improvement of FEV1 and shortening of length of hospital stay compared with placebo-treated patients.

For CF patients, an open label study found that adding oral corticosteroids resulted in an improvement of FEV1 in a small cohort of adult CF patients (Dovey et al., 2007). Further‐ more, a national UK survey performed in the authors' unit, found that all UK CF physicians had used adjuvant corticosteroids to different extents in managing CF pulmonary exacerba‐ tions (Hester et al., 2007).

This is important because of the increased propensity of CF patients to develop diabetes and osteoporosis by virtue of the CF disease process. Corticosteroids are bound to adversely af‐ fect the likelihood of these two complications, (increase the incidence and severity), as a sig‐ nificant negative side effect of the drug. To date there are no large trials to answer the question of whether adding corticosteroids to antibiotics improves outcome of treatment of CF exacerbations, and the issue remains contentious.

#### **11. Elective courses of antibiotics versus symptomatic treatments**

Elective regular administration of IV antibiotics several times per year has been a practice adopted by many CF teams to improve symptoms and reduce decline in lung function tests. In a national UK survey of CF centres (Higgs & Jarad, 2005), the authors found that greater proportion of paediatric patients received regular (elective) courses of IV antibiotics com‐ pared to adult CF patients (figure 14).

This is despite the fact that a previous study showed no differences in spirometry im‐ provement in patients who received elective 3 monthly anti-pseudomonal antibiotic treatment compared to those who received conventional symptomatic treatment, trig‐ gered by increased symptoms. The elective group received an average of 4 courses of antibiotics per year, compared with an average of 3 courses per year in the sympto‐ matic group (Elborn et al., 2000).

Self-administration of antibiotics was first introduced 30 years ago. This practice has been facilitated by improvement in technology and increased familiarity of patients and CF teams

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193

For patients with available peripheral venous access and infrequent exacerbations, antibiot‐ ics are administered via a peripherally inserted central catheter (PIC line) (figure 15). If such lines are inserted in the non-dominant arm, patients can self-administer their antibiotics.

**Figure 15.** Insertion of a PIC line in the left arm of a right handed CF patient. The patient is able to self-administer

Over the years, venous access frequently becomes more difficult to attain due to repeated use and occlusion of peripheral veins. In such patients, placement of a long-term totally im‐ plantable venous access system is an option. One of these methods, a port-a-cath, consists of a reservoir surgically inserted under the skin in the upper chest or arm, with the use of seda‐ tion or general anaesthesia. The reservoir is covered by a silicone mesh and appears as a bump under the skin. It leads to a long venous catheter, which is inserted into a central vein, usually a subclavian vein, and terminates in the superior vena cava. It is completely internal, so bathing and swimming are not a problem, although contact sports are contra-indicated

Ports and peripherally inserted lines have their own complications and require specific care, such anti-septic manipulation and monthly local anticoagulation to prevent blockage from blot clots. Despite careful maintenance, infection and blockage are not uncommon. Irreversi‐

(a) (b) (c)

due to risk of trauma and dislodgement of the device (figure 16).

ble occlusion of ports almost inevitably necessitates complete replacement.

with antibiotics.

antibiotics using his right hand.

**Figure 14.** Larger proportion of paediatric CF patients received elective IV antibiotics in comparison to adult CF pa‐ tients (Higgs & Jarad, 2005).

#### **12. Self-administration of IV antibiotics**

Delivery of healthcare for CF patients has changed significantly over the past 20 years. Pre‐ viously all patients were managed in hospital, but there has been a drive for the manage‐ ment of chronic conditions at home. Although not specifically suggested, management of acute exacerbations of clinical conditions such as COPD, bronchiectasis and CF is also fre‐ quently being done at home.

CF patients with pulmonary exacerbations requiring IV antibiotics place a great strain on the capacity of hospitals in terms of the available number of beds, on their manpower and other financial resources with repeated admissions. Accommodation and boarding for pa‐ tients and, sometimes, members of their families account for the largest fraction of hospital costs for inpatients. Equipment and drugs make up the largest proportion of home therapy costs (Wolter et al., 1997).

Self-administration of antibiotics was first introduced 30 years ago. This practice has been facilitated by improvement in technology and increased familiarity of patients and CF teams with antibiotics.

**P= 0.036**

**Adult CF Units Paediatric CF Units**

**Figure 14.** Larger proportion of paediatric CF patients received elective IV antibiotics in comparison to adult CF pa‐

Delivery of healthcare for CF patients has changed significantly over the past 20 years. Pre‐ viously all patients were managed in hospital, but there has been a drive for the manage‐ ment of chronic conditions at home. Although not specifically suggested, management of acute exacerbations of clinical conditions such as COPD, bronchiectasis and CF is also fre‐

CF patients with pulmonary exacerbations requiring IV antibiotics place a great strain on the capacity of hospitals in terms of the available number of beds, on their manpower and other financial resources with repeated admissions. Accommodation and boarding for pa‐ tients and, sometimes, members of their families account for the largest fraction of hospital costs for inpatients. Equipment and drugs make up the largest proportion of home therapy

**0**

**12. Self-administration of IV antibiotics**

**10**

**20**

**30**

**40**

**50**

**60**

**Percentage of patients taking regular antibiotics**

192 Respiratory Disease and Infection - A New Insight

tients (Higgs & Jarad, 2005).

quently being done at home.

costs (Wolter et al., 1997).

**70**

**80**

**90**

**100**

**110**

For patients with available peripheral venous access and infrequent exacerbations, antibiot‐ ics are administered via a peripherally inserted central catheter (PIC line) (figure 15). If such lines are inserted in the non-dominant arm, patients can self-administer their antibiotics.

**Figure 15.** Insertion of a PIC line in the left arm of a right handed CF patient. The patient is able to self-administer antibiotics using his right hand.

Over the years, venous access frequently becomes more difficult to attain due to repeated use and occlusion of peripheral veins. In such patients, placement of a long-term totally im‐ plantable venous access system is an option. One of these methods, a port-a-cath, consists of a reservoir surgically inserted under the skin in the upper chest or arm, with the use of seda‐ tion or general anaesthesia. The reservoir is covered by a silicone mesh and appears as a bump under the skin. It leads to a long venous catheter, which is inserted into a central vein, usually a subclavian vein, and terminates in the superior vena cava. It is completely internal, so bathing and swimming are not a problem, although contact sports are contra-indicated due to risk of trauma and dislodgement of the device (figure 16).

Ports and peripherally inserted lines have their own complications and require specific care, such anti-septic manipulation and monthly local anticoagulation to prevent blockage from blot clots. Despite careful maintenance, infection and blockage are not uncommon. Irreversi‐ ble occlusion of ports almost inevitably necessitates complete replacement.

pital expenses, especially if the treatment centre/hospital is at a considerable distance from the patient's home. After numerous admissions throughout their lives, patients and their families become acquainted with many aspects of IV drug administration and often want to start selfadministration of these medications, avoiding hospital admissions (Gilbert et al., 1988). Rea‐

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195

The superior outcome of hospital management over home treatment has been attributed to closer supervision and direct input by the multidisciplinary team, including physiothera‐ pists, dieticians and nursing staff, throughout the period of hospital stay (Thornton et al., 2004; Pond et al., 1994), ensuring increased adherence to treatment. Albeit unproven, bed rest during exacerbations has also been widely regarded as another reason for the favoura‐

Conversely, there are numerous reasons why home treatment could be clinically less effec‐ tive in treating exacerbations in CF patients (table 5). Considerable commitment is required from patients who are on home based treatment, as, in addition to their treatment schedules, they may wish to maintain their domestic routines and social lives, as well as fulfil educa‐ tional and work commitments. Continuing with normal life and not taking time off work or school would mean maintaining higher general activity levels. These patients are probably not getting the amount of rest they need as part of their treatment (Thornton et al., 2005). Self-performed physiotherapy may not be as effective during exacerbations compared to the treatment provided by a professional physiotherapist and calorie intake may suffer without

**Table 5.** Reasons why healthcare professionals are concerned about the practice of home IV treatment.

sons for patients' preference for home treatment are outlined in table 4.



ble outcome of hospital treatment.

daily encouragement (Pond et al., 1994).




**Table 4.** Reasons why patients prefer home IV antibiotic treatment.





**Figure 16.** The port-a-cath. (a): schematic representation of the position of a port-a-cath. (b): accessing the device chamber by a specifically designed needle. (c) and (d): a needle is inserted into the port-a-cath chamber and a patient self-administering IV antibiotic via his port-a-cath.

#### **13. Managing cystic fibrosis pulmonary exacerbations at home versus in hospital**

Home therapy is becoming a preferred treatment option for patients with CF suffering from pulmonary exacerbations in the UK and other parts of the world. With the widespread prac‐ tice of home based IV antibiotic therapy, concern has been expressed by CF healthcare workers about whether the outcome of care for those treated at home by self-administering IV antibiotics might be inferior to that of patients treated in hospital.

Hospital management is not favoured by most CF patients, who prefer home therapy (Thorn‐ ton et al., 2005; Pond et al., 1994; Strandvik et al., 1992; Donati et al., 1987). Hospital treatment is probably disruptive to patients and their families, taking patients away from school or work commitments and social activities for considerable amounts of time. There are also financial strains on patients due to earning losses as a result of time off from work and travelling to hos‐ pital expenses, especially if the treatment centre/hospital is at a considerable distance from the patient's home. After numerous admissions throughout their lives, patients and their families become acquainted with many aspects of IV drug administration and often want to start selfadministration of these medications, avoiding hospital admissions (Gilbert et al., 1988). Rea‐ sons for patients' preference for home treatment are outlined in table 4.



(a) (b)

(c) (d)

IV antibiotics might be inferior to that of patients treated in hospital.

self-administering IV antibiotic via his port-a-cath.

194 Respiratory Disease and Infection - A New Insight

**hospital**

**Figure 16.** The port-a-cath. (a): schematic representation of the position of a port-a-cath. (b): accessing the device chamber by a specifically designed needle. (c) and (d): a needle is inserted into the port-a-cath chamber and a patient

**13. Managing cystic fibrosis pulmonary exacerbations at home versus in**

Home therapy is becoming a preferred treatment option for patients with CF suffering from pulmonary exacerbations in the UK and other parts of the world. With the widespread prac‐ tice of home based IV antibiotic therapy, concern has been expressed by CF healthcare workers about whether the outcome of care for those treated at home by self-administering

Hospital management is not favoured by most CF patients, who prefer home therapy (Thorn‐ ton et al., 2005; Pond et al., 1994; Strandvik et al., 1992; Donati et al., 1987). Hospital treatment is probably disruptive to patients and their families, taking patients away from school or work commitments and social activities for considerable amounts of time. There are also financial strains on patients due to earning losses as a result of time off from work and travelling to hos‐


**Table 4.** Reasons why patients prefer home IV antibiotic treatment.

The superior outcome of hospital management over home treatment has been attributed to closer supervision and direct input by the multidisciplinary team, including physiothera‐ pists, dieticians and nursing staff, throughout the period of hospital stay (Thornton et al., 2004; Pond et al., 1994), ensuring increased adherence to treatment. Albeit unproven, bed rest during exacerbations has also been widely regarded as another reason for the favoura‐ ble outcome of hospital treatment.

Conversely, there are numerous reasons why home treatment could be clinically less effec‐ tive in treating exacerbations in CF patients (table 5). Considerable commitment is required from patients who are on home based treatment, as, in addition to their treatment schedules, they may wish to maintain their domestic routines and social lives, as well as fulfil educa‐ tional and work commitments. Continuing with normal life and not taking time off work or school would mean maintaining higher general activity levels. These patients are probably not getting the amount of rest they need as part of their treatment (Thornton et al., 2005). Self-performed physiotherapy may not be as effective during exacerbations compared to the treatment provided by a professional physiotherapist and calorie intake may suffer without daily encouragement (Pond et al., 1994).

<sup>-</sup> Reduced medical input

<sup>-</sup> Reduced input from physiotherapists and dieticians

<sup>-</sup> Reliance on patients to diagnose own complications

<sup>-</sup> Possible lack of compliance with the IV treatment

<sup>-</sup> Lack of rest

**Table 5.** Reasons why healthcare professionals are concerned about the practice of home IV treatment.

Some antibiotic regimens for home treatment are adapted to make administration more con‐ venient and more compatible with work and school hours (Pond et al., 1994). This includes twice daily beta-lactam antibiotics versus the recommended thrice-daily regime.

and convenience (Pond et al., 1994). Selection of patients that are considered competent and safe for self-administered home treatment should be made carefully. A hygienic environ‐ ment and adequate knowledge of preparation and administration of antibiotics should be ensured, as well as regular physiotherapy, rest and suitable nutrition, which are crucial for the successful treatment of CF exacerbations. Better still is the availability of support from family and friends, who are familiar with home treatment methods, as well as from the CF

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197

Patients need to be aware of the perceived reasons for an inferior outcome of home treat‐

**P=0.02**

**P=0.01**

**Standard Home Intensive Home Hospital**

**Figure 17.** Changes in FEV1 with treatment of exacerbations treated at home with and without intensive assistance

and in hospital. Best outcome is seen in the hospital treatment group (Sequeiros & Jarad, 2010).

ment and the CF community should definitively address these.

**PNS**

multidisciplinary team.

**-0.5 -0.4 -0.3 -0.2 -0.1 -0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3**

**Change in FEV1 (L)**

Another important issue is adherence, which is recognised as being potentially poor in CF (Dodd & Webb, 2000) and may be worse in some patients on home IV treatment. Although assessed by the multidisciplinary team for competency in terms of self-administration of drugs, the level of adherence of patients to treatment is not truly known. This is a widely anecdotally known phenomenon, often revealed when considerable amounts of unused an‐ tibiotics and other drugs are returned by patients and their families to the caring CF centre.

The conflict between patients' preference for home treatment and health providers' concern to achieve a favourable outcome of care during stages of clinical instability in CF is ongoing. This is currently handled in variable ways by different CF centres. Most centres feel that they have to offer some kind of home treatment, although a small number do not. Others prefer a happy medium of starting treatment in hospital and then discharging patients a few days later to complete the antibiotic course at home. Some CF centres prefer not to treat pa‐ tients at home for two successive exacerbations.

More recently, Collaco et al. (2010) published a large retrospective study with a total of 834 treated exacerbations in both paediatric and adult patients in the United States. They con‐ clude that similar decline in long term FEV1 was observed regardless whether antibiotics were administered in hospital or at home, with equivalence also found in regards to interval duration in between successive exacerbation episodes. Interestingly though, subjects treated in hospital had a statistically significant greater improvement of lung function immediately after treatment (immediate recovery) in comparison to the home treated group, despite simi‐ lar pre-treatment spirometry. Also, patients treated in hospital had shorter total number of days of IV antibiotic treatment, implying that patients overcame their exacerbations quicker when treated in hospital (12.7 days versus 18.9 days), which of course impacts on quality of life, drug toxicity, antibiotic resistance and healthcare costs.

Given the controversy around this subject, Sequeiros & Jarad (2010) prospectively exam‐ ined the effect of home treatment with intensive assistance from CF nurses, physiothera‐ pists and dieticians on patient outcomes. The authors compared outcomes of this intensive intervention with outcomes of exacerbations treated at home without intensive assistance and in hospital.

The study showed that, unlike previous studies, those who were treated in hospital had ini‐ tially poorer quality of life and were underweight compared to those who were treated at, but these recuperated to match post treatment levels similar to home based patients. The re‐ search also showed that, despite intensive home intervention, outcomes did not differ from standard home treatment and were inferior to those treated in hospital. The study has been published in abstract form (Sequeiros & Jarad, 2010) and is presently being prepared as a full manuscript for publication (figure 17).

Ideally, outcome of care for home treatment should be at least equal to outcome for hospital treatment and clinical improvement not sacrificed on the basis of economic considerations and convenience (Pond et al., 1994). Selection of patients that are considered competent and safe for self-administered home treatment should be made carefully. A hygienic environ‐ ment and adequate knowledge of preparation and administration of antibiotics should be ensured, as well as regular physiotherapy, rest and suitable nutrition, which are crucial for the successful treatment of CF exacerbations. Better still is the availability of support from family and friends, who are familiar with home treatment methods, as well as from the CF multidisciplinary team.

Some antibiotic regimens for home treatment are adapted to make administration more con‐ venient and more compatible with work and school hours (Pond et al., 1994). This includes

Another important issue is adherence, which is recognised as being potentially poor in CF (Dodd & Webb, 2000) and may be worse in some patients on home IV treatment. Although assessed by the multidisciplinary team for competency in terms of self-administration of drugs, the level of adherence of patients to treatment is not truly known. This is a widely anecdotally known phenomenon, often revealed when considerable amounts of unused an‐ tibiotics and other drugs are returned by patients and their families to the caring CF centre. The conflict between patients' preference for home treatment and health providers' concern to achieve a favourable outcome of care during stages of clinical instability in CF is ongoing. This is currently handled in variable ways by different CF centres. Most centres feel that they have to offer some kind of home treatment, although a small number do not. Others prefer a happy medium of starting treatment in hospital and then discharging patients a few days later to complete the antibiotic course at home. Some CF centres prefer not to treat pa‐

More recently, Collaco et al. (2010) published a large retrospective study with a total of 834 treated exacerbations in both paediatric and adult patients in the United States. They con‐ clude that similar decline in long term FEV1 was observed regardless whether antibiotics were administered in hospital or at home, with equivalence also found in regards to interval duration in between successive exacerbation episodes. Interestingly though, subjects treated in hospital had a statistically significant greater improvement of lung function immediately after treatment (immediate recovery) in comparison to the home treated group, despite simi‐ lar pre-treatment spirometry. Also, patients treated in hospital had shorter total number of days of IV antibiotic treatment, implying that patients overcame their exacerbations quicker when treated in hospital (12.7 days versus 18.9 days), which of course impacts on quality of

Given the controversy around this subject, Sequeiros & Jarad (2010) prospectively exam‐ ined the effect of home treatment with intensive assistance from CF nurses, physiothera‐ pists and dieticians on patient outcomes. The authors compared outcomes of this intensive intervention with outcomes of exacerbations treated at home without intensive

The study showed that, unlike previous studies, those who were treated in hospital had ini‐ tially poorer quality of life and were underweight compared to those who were treated at, but these recuperated to match post treatment levels similar to home based patients. The re‐ search also showed that, despite intensive home intervention, outcomes did not differ from standard home treatment and were inferior to those treated in hospital. The study has been published in abstract form (Sequeiros & Jarad, 2010) and is presently being prepared as a

Ideally, outcome of care for home treatment should be at least equal to outcome for hospital treatment and clinical improvement not sacrificed on the basis of economic considerations

twice daily beta-lactam antibiotics versus the recommended thrice-daily regime.

tients at home for two successive exacerbations.

196 Respiratory Disease and Infection - A New Insight

life, drug toxicity, antibiotic resistance and healthcare costs.

assistance and in hospital.

full manuscript for publication (figure 17).

Patients need to be aware of the perceived reasons for an inferior outcome of home treat‐ ment and the CF community should definitively address these.

**Figure 17.** Changes in FEV1 with treatment of exacerbations treated at home with and without intensive assistance and in hospital. Best outcome is seen in the hospital treatment group (Sequeiros & Jarad, 2010).

#### **14. Conclusion**

CF exacerbations are complex and still not completely understood. However, they re‐ main an important part of the CF lung disease due to their great negative impact on quality of life, resultant decline in lung function and mortality.

[6] Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, Berger M. (1995) Inflammatory cytokines in cystic fibrosis lungs. *American Journal of Respira‐*

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[7] Boucher RC. (2004) New concepts of the pathogenesis of cystic fibrosis lung disease.

[8] Britto MT, Kotagal UR, Hornung RW, Atherton HD, Tsevat J, Wilmott RW. (2002) Impact of recent pulmonary exacerbations on quality of life in patients with cystic

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[10] Colombo C, Constantini D, Rocchi A, Cariani L, Garlaschi ML, Tirelli S, Calori G, Co‐ preni E, Conese M. (2005) Cytokine levels in sputum of cystic fibrosis patients before

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[15] Dakin C, Henry RL, Field P, Morton J. (2001) Defining an exacerbation of pulmonary

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[19] Drenkard E, Ausubel FM. (2002) Pseudomonas biofilm formation and antibiotic re‐

Available at: www.cftrust.org.uk/aboutcf/publications/cfregistryreports/

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section 1, Bethesda, Maryland.

111:28-33.

This chapter addresses the natural history, causes and aspects of management, and progno‐ sis of CF exacerbations pertinent mainly to adult CF patients. Until introduction of more ef‐ fective and disease modifying treatments for CF, better understanding and management of CF exacerbations remain an important goal for the CF community.

Prevention, accurate identification and treatment of pulmonary exacerbations are key to the improved survival and quality of life of CF patients. Efforts should be made to standardize treatments and ensure high standards of care throughout different CF centres worldwide.

#### **Author details**

Iara Maria Sequeiros and Nabil Jarad

University Hospitals Bristol NHS Foundation Trust , United Kingdom

#### **References**


[6] Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, Berger M. (1995) Inflammatory cytokines in cystic fibrosis lungs. *American Journal of Respira‐ tory and Critical Care Medicine*, 152:2111-2118.

**14. Conclusion**

198 Respiratory Disease and Infection - A New Insight

**Author details**

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CF exacerbations are complex and still not completely understood. However, they re‐ main an important part of the CF lung disease due to their great negative impact on

This chapter addresses the natural history, causes and aspects of management, and progno‐ sis of CF exacerbations pertinent mainly to adult CF patients. Until introduction of more ef‐ fective and disease modifying treatments for CF, better understanding and management of

Prevention, accurate identification and treatment of pulmonary exacerbations are key to the improved survival and quality of life of CF patients. Efforts should be made to standardize treatments and ensure high standards of care throughout different CF centres worldwide.

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**Section 3**

**Helminthic Infections of Lung**

**Helminthic Infections of Lung**

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204 Respiratory Disease and Infection - A New Insight

**Chapter 8**

**Helminthic Infections and Asthma: Still a Challenge for**

Asthma is one of the most common chronic diseases in the world. It is estimated that around 300 million people in the world currently have asthma [1]. Asthma has become more common in both children and adults around the world in recent decades. The increase in the prevalence of this disease has been associated with an increase in atopic sensitization, and is paralleled by similar increases in other allergic disorders such as eczema and rhinitis [1]. The rate of asthma increases as communities adopt western lifestyles and become urbanized. With the projected increase in the proportion of the world's population that is urban from 45% to 59% in 2025, there is likely to be a marked increase in the number of asthmatics worldwide over the next two decades [2]. Nevertheless the prevalence of asthma in rural developing countries has been under estimated for many years [3]. In many areas of the world persons with asthma do not have access to basic asthma medications or medical care and are not included in any statistical survey [4]. Increasing the economic wealth and improving the distribution of re‐ sources between and within countries represent important priorities to enable better health care to be provided. The burden of asthma in many countries is of sufficient magnitude to warrant its recognition as a priority disorder in government health strategies. Particular re‐ sources need to be provided to improve the care of disadvantaged groups with high morbidity. Resources also need to be provided to address preventable factors [1, 2]. It is estimated that asthma accounts for about 1 in every 250 deaths worldwide. Many of the deaths are prevent‐ able, being due to suboptimal long-term medical care and delay in obtaining help during the final attack. The economic cost of asthma is considerable both in terms of direct medical costs (such as hospital admissions and cost of pharmaceuticals) and indirect medical costs (such as time lost from work and premature death) [1]. Therefore there is a greater understanding of

> © 2013 Hagel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Developing Countries**

Isabel Hagel, Maira Cabrera and

Additional information is available at the end of the chapter

Maria Cristina Di Prisco

http://dx.doi.org/10.5772/54690

**1. Introduction**

### **Helminthic Infections and Asthma: Still a Challenge for Developing Countries**

Isabel Hagel, Maira Cabrera and Maria Cristina Di Prisco

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54690

#### **1. Introduction**

Asthma is one of the most common chronic diseases in the world. It is estimated that around 300 million people in the world currently have asthma [1]. Asthma has become more common in both children and adults around the world in recent decades. The increase in the prevalence of this disease has been associated with an increase in atopic sensitization, and is paralleled by similar increases in other allergic disorders such as eczema and rhinitis [1]. The rate of asthma increases as communities adopt western lifestyles and become urbanized. With the projected increase in the proportion of the world's population that is urban from 45% to 59% in 2025, there is likely to be a marked increase in the number of asthmatics worldwide over the next two decades [2]. Nevertheless the prevalence of asthma in rural developing countries has been under estimated for many years [3]. In many areas of the world persons with asthma do not have access to basic asthma medications or medical care and are not included in any statistical survey [4]. Increasing the economic wealth and improving the distribution of re‐ sources between and within countries represent important priorities to enable better health care to be provided. The burden of asthma in many countries is of sufficient magnitude to warrant its recognition as a priority disorder in government health strategies. Particular re‐ sources need to be provided to improve the care of disadvantaged groups with high morbidity. Resources also need to be provided to address preventable factors [1, 2]. It is estimated that asthma accounts for about 1 in every 250 deaths worldwide. Many of the deaths are prevent‐ able, being due to suboptimal long-term medical care and delay in obtaining help during the final attack. The economic cost of asthma is considerable both in terms of direct medical costs (such as hospital admissions and cost of pharmaceuticals) and indirect medical costs (such as time lost from work and premature death) [1]. Therefore there is a greater understanding of

© 2013 Hagel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the factors that cause asthma which may lead to novel public health and pharmacological measures to reduce the prevalence of asthma seems to be a worldwide priority.

of the adaptive immune system (CD4 + T-lymphocytes, and antigen-specific IgE secreting Blymphocytes). The innate and adaptive cells are of Th2 class, secreting the cytokines IL-13 and IL-4 prominently or responding to these cytokines through their transduction molecule STAT6 [12, 13]. Thus, the input of these cells into the bronchus and the release or secretion of many mediators (e.g. heparin, reactive lipids or eicosanoids, and enzymes including tryptase and chitinase) lead to increased permeability of blood vessels and consequent edema, increased mucus production, and exaggerated smooth muscle contraction [13] causing airflow obstruc‐ tion and the symptoms described above. Also, continued inflammation results in remodeling of the airway in which it is thought that TGF-β cytokine may drive the metaplasia of the epi‐ thelium, increased vascularity, thickening of the basement membrane, and muscular hyper‐

Helminthic Infections and Asthma: Still a Challenge for Developing Countries

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209

The induction of adaptive immunity requires antigen-presenting cells (APCs). It is well known that dendritic cells (DCs) are the main type of APC involved in the induction of Th2 responses to allergens in asthma [16]. In the lung, DCs can be found throughout the conducting airways, interstitium, vasculature and pleura and in bronchial lymph nodes. Lung DCs express many receptors, including Toll-like receptors, Nod-like receptors and C-type lectin receptors up reg‐ ulate the expression of several co-stimulatory molecules (such as CD80 and CD86) and che‐ mokines (such as CCL17 and CCL22) that attract T cells, eosinophils and basophils into the lungs[16-19]. In humans, monocyte-derived conventional DCs promote Th2 responses by se‐ creting pro-inflammatory cytokines and up-regulating the expression of co-stimulatory mol‐ ecules after antigen stimulation[20] suggesting that lung DCs are necessary for Th2 cell

As mentioned above, various inflammatory cells, such as basophils, eosinophils and mast cells are recruited to airways after allergen challenge. Although the main focus in asthma has been on their roles as inflammatory cells, increasing data suggest that these cells also function as APCs to initiate or enhance Th2 responses. Basophils, which are circulating granulocytes that express the high-affinity IgE receptor FcεRI, amplify immediate hypersensitivity responses by releasing histamine-containing granules and by producing large quantities of IL-4 [13]. More‐ over, several studies have highlighted a crucial previously unknown role for basophils as APCs that drive Th2 responses through their expression of major histocompatibility complex (MHC) class II and co stimulatory molecules [21]. Also it has been proposed that MHC class II- de‐ pendent interactions between basophils, which are prominent at sites of allergic inflammation, and CD4+ T cells may have an important role in the induction of Th2-mediated inflammation [22, 23]. Another circulating granulocyte that is prominent at sites of allergic inflammation is the eosinophil. After being stimulated, eosinophils have an important pro-inflammatory role by producing leukotrienes, as well as Th1 cytokines (interferon-γ and IL-2) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13 and TNF-α which contribute to airway inflammation [11]. In addition, eosinophils, like basophils, can also function as APCs [24]. Other relevant components of air‐ way inflammation are the mast cells which express FcεRI and c-Kit and reside in tissues near mucosal surfaces and blood vessels. Mast cells can initiate immediate hypersensitivity reac‐ tions by de-granulating in response to both adaptive (IgE-mediated) and innate immune sig‐ nals. For example, mast cells can be activated through cross-linking of antigen-specific IgE

trophy, leading to lasting airflow obstruction and breathlessness [14, 15].

stimulation during airway inflammation.

Among the many factors influencing the prevalence of asthma in developing countries from the tropics are geo-helminthic infections [3], including those caused by *Ascaris lumbricoides*, *Trichuris trichiura* and hookworm (*Ancylostoma duodenale* and *Necator americanus*). These infec‐ tions have a worldwide distribution being present in almost all geographic and climatic re‐ gions. The prevalence of these infections tends to be highest in warm, moist climates; also they are closely correlated with poor environmental hygiene and lack of access to health services [5, 6]. The estimated global prevalence of *A. lumbricoides*, *T. trichiura* and hookworm are 1.5 billion, 1.3 billion and 900 million, respectively, and greater than 2 billion humans are infected with at least one of these parasites [5, 6]. Hookworm is transmitted through skin contact with free-living larvae in the soil, whereas *A. lumbricoides* and *T. trichiura* are transmitted through ingestion of embryonated eggs from the environment. *A. lumbricoides* and hookworm undergo a phase of larval migration through the lungs, whereas *T. trichiura* has a purely enteric life cycle. Infections with *A. lumbricoides* and *T. trichiura* peak in prevalence and intensity between 5 and 15 years of age in endemic areas and there is a decline in both epidemiological parameters in adulthood [5, 6]. Morbidity, including important effects on growth, nutrition and mental development in childhood tends to be greatest in heavily infected children and vulnerable adults (e.g. women of child-bearing age) with long exposure histories [6]. Epidemiological observations have provided evidence that geo-helminthic infections can influence the devel‐ opment of allergic diseases such as asthma. Different studies carried out mostly in rural areas from different countries have shown that infection by intestinal helminths stimulates the risk of asthma [7, 8, 9], whereas other studies in similar populations, have shown an inverse asso‐ ciation between asthma and the infection of these parasites [10]. Whether helminthic infections increase or decrease the risk of asthma may depend on environmental factors determining time of exposure and the prevalence and intensity of the infection [3]. On the other hand the individual capacity of the immune system to recognize certain allergenic components of the many molecules involved in host parasite interactions as well as the possible role of parasite mediators to inhibit inflammatory processes may be important aspects in the modulation of the pathogenesis of asthma by these parasites.

#### **2. Immunological aspects of asthma**

It is well accepted that asthma is a chronic inflammatory disorder of the bronchial mu‐ cosa [11]. The symptoms include chest tightness, wheeze, and cough, and often variable obstruction of airflow through the bronchi. The clinical manifestation of asthma is the result of three events within the airways: reversible obstruction, airway hyper-reactivity and inflammation. Among these factors, airway inflammation is believed to play a ma‐ jor role in the development of the disease [11].

The bronchial inflammation process of asthma is stimulated by cells of the innate immune system (dendritic cells, mast cells, eosinophils, neutrophils, macrophages and NK-cells) and of the adaptive immune system (CD4 + T-lymphocytes, and antigen-specific IgE secreting Blymphocytes). The innate and adaptive cells are of Th2 class, secreting the cytokines IL-13 and IL-4 prominently or responding to these cytokines through their transduction molecule STAT6 [12, 13]. Thus, the input of these cells into the bronchus and the release or secretion of many mediators (e.g. heparin, reactive lipids or eicosanoids, and enzymes including tryptase and chitinase) lead to increased permeability of blood vessels and consequent edema, increased mucus production, and exaggerated smooth muscle contraction [13] causing airflow obstruc‐ tion and the symptoms described above. Also, continued inflammation results in remodeling of the airway in which it is thought that TGF-β cytokine may drive the metaplasia of the epi‐ thelium, increased vascularity, thickening of the basement membrane, and muscular hyper‐ trophy, leading to lasting airflow obstruction and breathlessness [14, 15].

the factors that cause asthma which may lead to novel public health and pharmacological

Among the many factors influencing the prevalence of asthma in developing countries from the tropics are geo-helminthic infections [3], including those caused by *Ascaris lumbricoides*, *Trichuris trichiura* and hookworm (*Ancylostoma duodenale* and *Necator americanus*). These infec‐ tions have a worldwide distribution being present in almost all geographic and climatic re‐ gions. The prevalence of these infections tends to be highest in warm, moist climates; also they are closely correlated with poor environmental hygiene and lack of access to health services [5, 6]. The estimated global prevalence of *A. lumbricoides*, *T. trichiura* and hookworm are 1.5 billion, 1.3 billion and 900 million, respectively, and greater than 2 billion humans are infected with at least one of these parasites [5, 6]. Hookworm is transmitted through skin contact with free-living larvae in the soil, whereas *A. lumbricoides* and *T. trichiura* are transmitted through ingestion of embryonated eggs from the environment. *A. lumbricoides* and hookworm undergo a phase of larval migration through the lungs, whereas *T. trichiura* has a purely enteric life cycle. Infections with *A. lumbricoides* and *T. trichiura* peak in prevalence and intensity between 5 and 15 years of age in endemic areas and there is a decline in both epidemiological parameters in adulthood [5, 6]. Morbidity, including important effects on growth, nutrition and mental development in childhood tends to be greatest in heavily infected children and vulnerable adults (e.g. women of child-bearing age) with long exposure histories [6]. Epidemiological observations have provided evidence that geo-helminthic infections can influence the devel‐ opment of allergic diseases such as asthma. Different studies carried out mostly in rural areas from different countries have shown that infection by intestinal helminths stimulates the risk of asthma [7, 8, 9], whereas other studies in similar populations, have shown an inverse asso‐ ciation between asthma and the infection of these parasites [10]. Whether helminthic infections increase or decrease the risk of asthma may depend on environmental factors determining time of exposure and the prevalence and intensity of the infection [3]. On the other hand the individual capacity of the immune system to recognize certain allergenic components of the many molecules involved in host parasite interactions as well as the possible role of parasite mediators to inhibit inflammatory processes may be important aspects in the modulation of

It is well accepted that asthma is a chronic inflammatory disorder of the bronchial mu‐ cosa [11]. The symptoms include chest tightness, wheeze, and cough, and often variable obstruction of airflow through the bronchi. The clinical manifestation of asthma is the result of three events within the airways: reversible obstruction, airway hyper-reactivity and inflammation. Among these factors, airway inflammation is believed to play a ma‐

The bronchial inflammation process of asthma is stimulated by cells of the innate immune system (dendritic cells, mast cells, eosinophils, neutrophils, macrophages and NK-cells) and

measures to reduce the prevalence of asthma seems to be a worldwide priority.

208 Respiratory Disease and Infection - A New Insight

the pathogenesis of asthma by these parasites.

**2. Immunological aspects of asthma**

jor role in the development of the disease [11].

The induction of adaptive immunity requires antigen-presenting cells (APCs). It is well known that dendritic cells (DCs) are the main type of APC involved in the induction of Th2 responses to allergens in asthma [16]. In the lung, DCs can be found throughout the conducting airways, interstitium, vasculature and pleura and in bronchial lymph nodes. Lung DCs express many receptors, including Toll-like receptors, Nod-like receptors and C-type lectin receptors up reg‐ ulate the expression of several co-stimulatory molecules (such as CD80 and CD86) and che‐ mokines (such as CCL17 and CCL22) that attract T cells, eosinophils and basophils into the lungs[16-19]. In humans, monocyte-derived conventional DCs promote Th2 responses by se‐ creting pro-inflammatory cytokines and up-regulating the expression of co-stimulatory mol‐ ecules after antigen stimulation[20] suggesting that lung DCs are necessary for Th2 cell stimulation during airway inflammation.

As mentioned above, various inflammatory cells, such as basophils, eosinophils and mast cells are recruited to airways after allergen challenge. Although the main focus in asthma has been on their roles as inflammatory cells, increasing data suggest that these cells also function as APCs to initiate or enhance Th2 responses. Basophils, which are circulating granulocytes that express the high-affinity IgE receptor FcεRI, amplify immediate hypersensitivity responses by releasing histamine-containing granules and by producing large quantities of IL-4 [13]. More‐ over, several studies have highlighted a crucial previously unknown role for basophils as APCs that drive Th2 responses through their expression of major histocompatibility complex (MHC) class II and co stimulatory molecules [21]. Also it has been proposed that MHC class II- de‐ pendent interactions between basophils, which are prominent at sites of allergic inflammation, and CD4+ T cells may have an important role in the induction of Th2-mediated inflammation [22, 23]. Another circulating granulocyte that is prominent at sites of allergic inflammation is the eosinophil. After being stimulated, eosinophils have an important pro-inflammatory role by producing leukotrienes, as well as Th1 cytokines (interferon-γ and IL-2) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13 and TNF-α which contribute to airway inflammation [11]. In addition, eosinophils, like basophils, can also function as APCs [24]. Other relevant components of air‐ way inflammation are the mast cells which express FcεRI and c-Kit and reside in tissues near mucosal surfaces and blood vessels. Mast cells can initiate immediate hypersensitivity reac‐ tions by de-granulating in response to both adaptive (IgE-mediated) and innate immune sig‐ nals. For example, mast cells can be activated through cross-linking of antigen-specific IgE bound to FcεRI [25] or in response to Toll-like receptor agonists, or to cytokines such as IL-33 [26]. In addition to producing histamine and leukotrienes, mast cells produce cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-16, tumor necrosis factor and transforming growth factor-β) and chemokines (such as IL-8, lymphotactin, CCL1 (TCA-3), CCL5 (RANTES), CCL2 (MCP-1) and CCL3 (MIP1-α)[27].

The capacity of helminths, to stimulate inflammatory responses has been well documented and they are probably related to the complex lifecycle and the antigenic composition of this nematode. For example, there is evidence that after penetration of the intestinal mucosa *A. suum* larvae migrate to the liver, inducing the formation of granulomas, extensive inflamma‐ tion and tissue injury [41]. Surviving larvae reach the lungs and generate an inflammatory infiltrate in the airways dominated by severe per-alveolar eosinophilia [41, 42]. Pulmonary eosinophilia due to the passage of helminth larvae through the lungs is referred to as Loeffler's syndrome [43]*.* Eosinophilic pulmonary infiltrates and respiratory symptoms due to *A. lum‐ bricoides or A. suum* are generally part of a self-limited process due to the transient nature of larval passage through the lungs in the *Ascaris* life cycle [44]. The majority of patients remain asymptomatic, however 8 to 15% of infected individuals display morbidity, with respiratory symptoms occurring 9 to 12 days after the ingestion of eggs and lasting 5 to 10 days [45, 46]. Symptoms may include cough, dyspnea, wheeze, and hemoptysis and may progress to frank respiratory distress [43]. Peripheral eosinophilia is often present at the onset of symptoms, but the peak level of eosinophilia will have a delay of several days from presentation [47, 48]. Also, *Strongyloides stercoralis* may cause pulmonary symptoms and infiltrates as a manifestation of chronic infection or as a result of hyper-infection in immune-compromised hosts [49]. The unique life cycle of *S. stercoralis* allows a chronic infection of extended duration to occur due to the ability of new filariform larvae to continuously infect the human host through the per‐ ianal skin or bowel mucosa [50]. Patients with chronic infection may have repeated episodes of fever and pneumonitis that may be mistaken for recurrent bacterial pneumonias [51]. Eo‐ sinophilia, though often absent during the acute episodes, may occur during the intervening period between episodes [51]. Pulmonary involvement of strongyloidiasis has been reported

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The mechanisms by which these parasites induce airway inflammation are still not well elu‐ cidated. High levels of polyclonal and specific IgE against adult stages of the parasite are a characteristic of *A. lumbricoides* infection [52]. It has been shown that *Ascaris* can induce allergic sensitization in animal models [42] and in human beings, including immediate cutaneus hyperreactivity [52] and airway response after aerosol challenge with parasite extracts [53] suggest‐ ing the presence of an allergenic component on the stimulation of the respiratory symptoms by these parasites. Helminths harbor an arsenal of many pro- allergenic molecules which may be involved in airway inflammatory processes. One of the most studied is the *A. lumbricoides* body fluid antigen -1 (ABA-1) [54-57] which constitutes the most important of the group of antigens of the denominated family of nematode polyprotein allergens (NPA) for its capacity to stimulate strong IgE responses [58]. Recognition of ABA-1 may have a genetic basis [59, 60]. For example, evidences from studies carried out in Colombian endemic areas have shown that polymorphisms of LIG4 and TNFSF13B of the 13q33 region are associated with high levels of specific IgE to ABA-1 in *A. lumbricoides* infected children [60, 61]. However, the possible role of this allergen in airway inflammation remains unclear. Another important allergenic com‐ ponent of helminths is tropomyosin, which is a microfilament associated protein present in all eukaryotic cells, essential in the process of muscle work, proper action of the movement ap‐ paratus and the basic functionality of filaments within the cytoskeleton [62]. There is a high degree of homology among tropomyosins even of phylogenetically distant species of inver‐

as an asthma mimic.

An important mediator of airway inflammation is Nitric Oxide (NO) which it is a diffusible gas that can activate biochemical process either on the same cell that produced it or on neigh‐ boring cells [28]. NO is synthesized from L-arginine by enzyme nitric oxide synthase (NOS), of which there are three isoforms : NOS I or neuronal NOS (nNOS) was originally isolated from rat and porcine cerebellum; NOS II or inducible NOS (iNOS) from activated macrophag‐ es; NOS III or endothelial NOS (eNOS) from endothelial cells [28]. High NO levels could be harmful for asthmatic patients because at elevated concentrations, NO lead to the formation of reactive nitrogen species (RNS) and subsequent oxidation and nitration of proteins, which negatively affect protein functions that are biologically relevant to chronic inflammation in the asthmatic bronchial tissues [28,29]. In asthmatic patients, NO is mainly produced by iNOS expressed in bronchial epithelial cells and some inflammatory cells [30]. NO is also produced by neutrophils and macrophages in response to IFN-γ and a second signal provided by a PAMP ligand or TNF-α. iNOS expression is induced by these signals, this enzyme promotes the oxidation of the guadino nitrogen of L-arginine, resulting in the production of NO and citruline [31]. In addition, other mechanisms have been associated with NO production. It has been demonstrated that the ligation of CD23 (low affinity receptor for IgE FcεRII) on human macrophages is a strong inducer of NO [32]. Indeed, the cross-linking of CD23 by IgE, (IgE immune complexes or by specific monoclonal antibodies) induces pro-inflammatory response, including NO production [33]. Therefore, allergic sensitization inside the lower airways may account for NO production. In this context, it has been reported that aeroallergen sensitization correlated with exhaled NO (eNO) in mild to moderated asthmatic subjects [34]. Also, the latephase influx of eosinophils may contribute to NO production at the respiratory mucosa.

#### **3. Stimulation of airway inflammation by helminths parasites**

It is important to point out that there are close similarities between the allergic inflammatory responses stimulated in the host by environmental allergens (described above) with the im‐ mune responses elicited by parasite antigens. Gut inflammation stimulated by intestinal nem‐ atode also include innate (mastocytes, basophils and eosinophils) and adaptive cells of Th2 class which secret preferentially IL-13 and IL-4 cytokines. Pro- inflammatory cells like masto‐ cytes and eosinophils stimulated by these cytokines may release many mediators (heparin, reactive lipids or eicosanoids, and enzymes including tryptase and chitinase) leading to in‐ creased permeability of blood vessels, increased mucus production and smooth muscle con‐ traction [35]. Also, inflammation induced by most helminths in the host is associated with NO production through somatic and excretory-secretory antigens of adult worm and larvae [36- 40]. These mechanisms may contribute to make a hostile microenvironment in the gut for the parasites promoting worm expulsion.

The capacity of helminths, to stimulate inflammatory responses has been well documented and they are probably related to the complex lifecycle and the antigenic composition of this nematode. For example, there is evidence that after penetration of the intestinal mucosa *A. suum* larvae migrate to the liver, inducing the formation of granulomas, extensive inflamma‐ tion and tissue injury [41]. Surviving larvae reach the lungs and generate an inflammatory infiltrate in the airways dominated by severe per-alveolar eosinophilia [41, 42]. Pulmonary eosinophilia due to the passage of helminth larvae through the lungs is referred to as Loeffler's syndrome [43]*.* Eosinophilic pulmonary infiltrates and respiratory symptoms due to *A. lum‐ bricoides or A. suum* are generally part of a self-limited process due to the transient nature of larval passage through the lungs in the *Ascaris* life cycle [44]. The majority of patients remain asymptomatic, however 8 to 15% of infected individuals display morbidity, with respiratory symptoms occurring 9 to 12 days after the ingestion of eggs and lasting 5 to 10 days [45, 46]. Symptoms may include cough, dyspnea, wheeze, and hemoptysis and may progress to frank respiratory distress [43]. Peripheral eosinophilia is often present at the onset of symptoms, but the peak level of eosinophilia will have a delay of several days from presentation [47, 48]. Also, *Strongyloides stercoralis* may cause pulmonary symptoms and infiltrates as a manifestation of chronic infection or as a result of hyper-infection in immune-compromised hosts [49]. The unique life cycle of *S. stercoralis* allows a chronic infection of extended duration to occur due to the ability of new filariform larvae to continuously infect the human host through the per‐ ianal skin or bowel mucosa [50]. Patients with chronic infection may have repeated episodes of fever and pneumonitis that may be mistaken for recurrent bacterial pneumonias [51]. Eo‐ sinophilia, though often absent during the acute episodes, may occur during the intervening period between episodes [51]. Pulmonary involvement of strongyloidiasis has been reported as an asthma mimic.

bound to FcεRI [25] or in response to Toll-like receptor agonists, or to cytokines such as IL-33 [26]. In addition to producing histamine and leukotrienes, mast cells produce cytokines (IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-16, tumor necrosis factor and transforming growth factor-β) and chemokines (such as IL-8, lymphotactin, CCL1 (TCA-3), CCL5 (RANTES), CCL2

An important mediator of airway inflammation is Nitric Oxide (NO) which it is a diffusible gas that can activate biochemical process either on the same cell that produced it or on neigh‐ boring cells [28]. NO is synthesized from L-arginine by enzyme nitric oxide synthase (NOS), of which there are three isoforms : NOS I or neuronal NOS (nNOS) was originally isolated from rat and porcine cerebellum; NOS II or inducible NOS (iNOS) from activated macrophag‐ es; NOS III or endothelial NOS (eNOS) from endothelial cells [28]. High NO levels could be harmful for asthmatic patients because at elevated concentrations, NO lead to the formation of reactive nitrogen species (RNS) and subsequent oxidation and nitration of proteins, which negatively affect protein functions that are biologically relevant to chronic inflammation in the asthmatic bronchial tissues [28,29]. In asthmatic patients, NO is mainly produced by iNOS expressed in bronchial epithelial cells and some inflammatory cells [30]. NO is also produced by neutrophils and macrophages in response to IFN-γ and a second signal provided by a PAMP ligand or TNF-α. iNOS expression is induced by these signals, this enzyme promotes the oxidation of the guadino nitrogen of L-arginine, resulting in the production of NO and citruline [31]. In addition, other mechanisms have been associated with NO production. It has been demonstrated that the ligation of CD23 (low affinity receptor for IgE FcεRII) on human macrophages is a strong inducer of NO [32]. Indeed, the cross-linking of CD23 by IgE, (IgE immune complexes or by specific monoclonal antibodies) induces pro-inflammatory response, including NO production [33]. Therefore, allergic sensitization inside the lower airways may account for NO production. In this context, it has been reported that aeroallergen sensitization correlated with exhaled NO (eNO) in mild to moderated asthmatic subjects [34]. Also, the latephase influx of eosinophils may contribute to NO production at the respiratory mucosa.

**3. Stimulation of airway inflammation by helminths parasites**

It is important to point out that there are close similarities between the allergic inflammatory responses stimulated in the host by environmental allergens (described above) with the im‐ mune responses elicited by parasite antigens. Gut inflammation stimulated by intestinal nem‐ atode also include innate (mastocytes, basophils and eosinophils) and adaptive cells of Th2 class which secret preferentially IL-13 and IL-4 cytokines. Pro- inflammatory cells like masto‐ cytes and eosinophils stimulated by these cytokines may release many mediators (heparin, reactive lipids or eicosanoids, and enzymes including tryptase and chitinase) leading to in‐ creased permeability of blood vessels, increased mucus production and smooth muscle con‐ traction [35]. Also, inflammation induced by most helminths in the host is associated with NO production through somatic and excretory-secretory antigens of adult worm and larvae [36- 40]. These mechanisms may contribute to make a hostile microenvironment in the gut for the

(MCP-1) and CCL3 (MIP1-α)[27].

210 Respiratory Disease and Infection - A New Insight

parasites promoting worm expulsion.

The mechanisms by which these parasites induce airway inflammation are still not well elu‐ cidated. High levels of polyclonal and specific IgE against adult stages of the parasite are a characteristic of *A. lumbricoides* infection [52]. It has been shown that *Ascaris* can induce allergic sensitization in animal models [42] and in human beings, including immediate cutaneus hyperreactivity [52] and airway response after aerosol challenge with parasite extracts [53] suggest‐ ing the presence of an allergenic component on the stimulation of the respiratory symptoms by these parasites. Helminths harbor an arsenal of many pro- allergenic molecules which may be involved in airway inflammatory processes. One of the most studied is the *A. lumbricoides* body fluid antigen -1 (ABA-1) [54-57] which constitutes the most important of the group of antigens of the denominated family of nematode polyprotein allergens (NPA) for its capacity to stimulate strong IgE responses [58]. Recognition of ABA-1 may have a genetic basis [59, 60]. For example, evidences from studies carried out in Colombian endemic areas have shown that polymorphisms of LIG4 and TNFSF13B of the 13q33 region are associated with high levels of specific IgE to ABA-1 in *A. lumbricoides* infected children [60, 61]. However, the possible role of this allergen in airway inflammation remains unclear. Another important allergenic com‐ ponent of helminths is tropomyosin, which is a microfilament associated protein present in all eukaryotic cells, essential in the process of muscle work, proper action of the movement ap‐ paratus and the basic functionality of filaments within the cytoskeleton [62]. There is a high degree of homology among tropomyosins even of phylogenetically distant species of inver‐ tebrates, but not with vertebrate tropomyosins [62]. Invertebrate tropomyosins induce IgE antibodies and are potent allergens for humans whereas vertebrate ones were reported to be non-allergenic [62, 63]. Tropomyosin from *A. lumbricoides* presents a high degree of sequence identity to those from other invertebrates, including cockroach, mites, and shrimp [64]. It is expressed in high levels in the third stage larvae (L3), which is the stage of pulmonary passage of the parasite and stimulates strongly the production of IgE [64], high positivity to skin prick tests and histamine release from basophils[64, 65]. *A.lumbricoides* tropomyosin cross-reacts with other invertebrate tropomyosins [64] thus enhancing the allergic response to other envi‐ ronmental allergens containing tropomyosin (cockroach, mites, and shrimp) [64, 65] which are known to induce airway inflammation and asthma [65]. Other important nematode allergens are chitins, an important component for egg shell integrity and for the structure of the rigid pharynx, including the buccal cavity and grinder, a specialized cuticle that is shed and resynthesized during molting [66]. There is evidence that chitins are involved in Th2 type in‐ flammation. For example, experimental models of asthma using mice have shown that the intranasal administration of chitin induced an accumulation of eosinophils and basophils [67]. Exposure to chitin also stimulated the alternative activation of macrophages as indicated by the presence of arginase-expressing cells in the lungs as early as 6 h post intranasal adminis‐ tration of chitin [67]. The recruitment of innate immune cells has shown to be dependent both upon expression of the high affinity receptor for leucotriene B4, BLT1 and upon the presence of macrophages [68] suggesting a possible role of chitin in innate immune cell recruitment, leading to preferential Th2 type responses. In humans, The non-chitinolytic chitinase YKL-40 has been linked to allergic inflammation. YKL-40 levels are elevated in the serum and bron‐ choalveolar lavage (BAL) fluid of asthmatics [69]. Furthermore, increased YKL-40 levels cor‐ related with asthma severity and are elevated in response to allergen challenge. Nematode proteases which constitute a group of highly evolutionary conserved molecules may exhibit allergenic properties such as other proteases like house dust mite-derived *Der p 1*, domestic cats- derived *Fel d1*, and fungal allergens [70, 71]. These allergens most often gain access to host tissues at mucosal sites and in some individuals elicit potent Th2 cytokine responses, reflecting the hallmarks of the immune response to helminthic infection including eosinophil‐ ia, goblet cell hyperplasia and elevated serum IgE levels as explained above. Basophils which are innate immune cells able to rapidly secrete IL-4 *in vitro* following stimulation with anti-IgE [72] as well as in response to allergens and helminth products [73], would be involved in the innate sensing of these products which may lead to the initiation of adaptive Th2 cytokine responses to parasite and/or environmental allergens. For example, immunization of mice with the cysteine protease allergen, papain, resulted in the transient recruitment of basophils to lymph nodes that peaked 1 day prior to the peak of IL-4 producing CD4+ T cells [74]. These papain-elicited basophils within the lymph node were shown to express thymic stromal lym‐ phopoietin (TSLP), an IL-7-like cytokine produced predominantly by epithelial cells and im‐ plicated in CD4+ Th2 cell differentiation [74]. Given their multiple roles in regulating inflammation, cellular trafficking and epithelial barrier function members of the protease-ac‐ tivated receptor (PAR) family [75] would be potential molecular targets for helminth proteases.

shown that antigenic extracts of *A. suum* potentiate allergic responses to ovalbumin in pigs (76, 77). It has been also reported that co-administration of hen egg lysozyme with the excre‐ tory ⁄ secretory products of *N. brasiliensis* in mice results in the generation of egg-lysozymespecific lymphocyte proliferation, IL-4 release and IgG1 antibody responses [78]. Furthermore, it has been shown that unidentified components in the body fluid of *A. suum* promotes a Th2 response and are adjuvant for specific IgE synthesis to some parasitic allergens like ABA-1 [79]. Because, in addition to this allergen, *A. lumbricoides* extract has at least 11 human-IgE-binding components, the adjuvant effect may be more generalized in humans beings, and because of co-exposure with other environmental allergens, this could happen for cross-reactive and noncross-reactive mite allergens [61].These observations are consistent with early studies carried out in atopic Venezuelan children in which an elevated percentage of skin positivity as well as high levels of specific serum IgE toward aero-allergens, were found among children infected

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with *A. lumbricoides* compared to their non parasitized counterparts [80].

**helminthic infections and asthma**

**4. Epidemiological studies showing a positive association between**

Human immune response to infections with helminths parasites may differ according to the profile of the infection. As mentioned above, acute or seasonal infections may include primary infections and repeated or intermittent infections without long period of continuous infections and may result from infrequent short exposures or intermittent exposure after treatment in endemic areas [3, 5]. It has been proposed that mild, seasonal helminthic infections stimulate preferentially inflammatory Th2 type immune responses among parasitized populations [3, 81] characterized by the production of high levels of serum specific IgE and allergic reactivity toward parasite soluble antigens [82] which may lead to the development of bronchial hyper reactivity and asthma [83, 84] particularly among atopic individuals [85]. This situation may be reflected on the increased prevalence of asthma and allergic diseases observed in many low and middle-income countries [86, 87] undergoing parasite eradication programs [5, 6]. For example, epidemiological studies carried out in different rural communities in China, have shown a strong association between Ascaris lumbricoides infection and the development of asthma [88]. These early results are consistent with a meta-analysis of many of studies inves‐ tigating the association between the presence of geohelminth eggs in stool samples and asthma providing some evidence for parasite-specific effects [7]. In this work in which thirty-three studies were taken in account, *Ascaris lumbricoides* was associated with significantly increased odds of asthma (OR, 1.34; 95% CI, 1.05–1.71; 20 studies. Further studies carried out in urban Brazil have shown positive associations between Ascaris lumbricoides infection with recent wheeze [9] which in turn have been associated to allergic sensitization toward A. lumbricoides antigens [9]. Other studies carried out in Costa Rica have shown a relationship between sen‐ sitization (defined as a positive IgE) to A. lumbricoides and measures of asthma morbidity and severity in a population with low prevalence of parasitic infection but high prevalence of par‐ asitic exposure [89]. In this work, a cross-sectional study of 439 children (ages 6 to 14 years) with asthma was carried out and linear regression and logistic regression were used for the

Regardless of the intrinsic allergenic potential of these parasites, there are evidences that active infection can potentiate the allergic response to non-related antigens. For example, it has been shown that antigenic extracts of *A. suum* potentiate allergic responses to ovalbumin in pigs (76, 77). It has been also reported that co-administration of hen egg lysozyme with the excre‐ tory ⁄ secretory products of *N. brasiliensis* in mice results in the generation of egg-lysozymespecific lymphocyte proliferation, IL-4 release and IgG1 antibody responses [78]. Furthermore, it has been shown that unidentified components in the body fluid of *A. suum* promotes a Th2 response and are adjuvant for specific IgE synthesis to some parasitic allergens like ABA-1 [79]. Because, in addition to this allergen, *A. lumbricoides* extract has at least 11 human-IgE-binding components, the adjuvant effect may be more generalized in humans beings, and because of co-exposure with other environmental allergens, this could happen for cross-reactive and noncross-reactive mite allergens [61].These observations are consistent with early studies carried out in atopic Venezuelan children in which an elevated percentage of skin positivity as well as high levels of specific serum IgE toward aero-allergens, were found among children infected with *A. lumbricoides* compared to their non parasitized counterparts [80].

tebrates, but not with vertebrate tropomyosins [62]. Invertebrate tropomyosins induce IgE antibodies and are potent allergens for humans whereas vertebrate ones were reported to be non-allergenic [62, 63]. Tropomyosin from *A. lumbricoides* presents a high degree of sequence identity to those from other invertebrates, including cockroach, mites, and shrimp [64]. It is expressed in high levels in the third stage larvae (L3), which is the stage of pulmonary passage of the parasite and stimulates strongly the production of IgE [64], high positivity to skin prick tests and histamine release from basophils[64, 65]. *A.lumbricoides* tropomyosin cross-reacts with other invertebrate tropomyosins [64] thus enhancing the allergic response to other envi‐ ronmental allergens containing tropomyosin (cockroach, mites, and shrimp) [64, 65] which are known to induce airway inflammation and asthma [65]. Other important nematode allergens are chitins, an important component for egg shell integrity and for the structure of the rigid pharynx, including the buccal cavity and grinder, a specialized cuticle that is shed and resynthesized during molting [66]. There is evidence that chitins are involved in Th2 type in‐ flammation. For example, experimental models of asthma using mice have shown that the intranasal administration of chitin induced an accumulation of eosinophils and basophils [67]. Exposure to chitin also stimulated the alternative activation of macrophages as indicated by the presence of arginase-expressing cells in the lungs as early as 6 h post intranasal adminis‐ tration of chitin [67]. The recruitment of innate immune cells has shown to be dependent both upon expression of the high affinity receptor for leucotriene B4, BLT1 and upon the presence of macrophages [68] suggesting a possible role of chitin in innate immune cell recruitment, leading to preferential Th2 type responses. In humans, The non-chitinolytic chitinase YKL-40 has been linked to allergic inflammation. YKL-40 levels are elevated in the serum and bron‐ choalveolar lavage (BAL) fluid of asthmatics [69]. Furthermore, increased YKL-40 levels cor‐ related with asthma severity and are elevated in response to allergen challenge. Nematode proteases which constitute a group of highly evolutionary conserved molecules may exhibit allergenic properties such as other proteases like house dust mite-derived *Der p 1*, domestic cats- derived *Fel d1*, and fungal allergens [70, 71]. These allergens most often gain access to host tissues at mucosal sites and in some individuals elicit potent Th2 cytokine responses, reflecting the hallmarks of the immune response to helminthic infection including eosinophil‐ ia, goblet cell hyperplasia and elevated serum IgE levels as explained above. Basophils which are innate immune cells able to rapidly secrete IL-4 *in vitro* following stimulation with anti-IgE [72] as well as in response to allergens and helminth products [73], would be involved in the innate sensing of these products which may lead to the initiation of adaptive Th2 cytokine responses to parasite and/or environmental allergens. For example, immunization of mice with the cysteine protease allergen, papain, resulted in the transient recruitment of basophils to lymph nodes that peaked 1 day prior to the peak of IL-4 producing CD4+ T cells [74]. These papain-elicited basophils within the lymph node were shown to express thymic stromal lym‐ phopoietin (TSLP), an IL-7-like cytokine produced predominantly by epithelial cells and im‐ plicated in CD4+ Th2 cell differentiation [74]. Given their multiple roles in regulating inflammation, cellular trafficking and epithelial barrier function members of the protease-ac‐ tivated receptor (PAR) family [75] would be potential molecular targets for helminth proteases.

212 Respiratory Disease and Infection - A New Insight

Regardless of the intrinsic allergenic potential of these parasites, there are evidences that active infection can potentiate the allergic response to non-related antigens. For example, it has been

#### **4. Epidemiological studies showing a positive association between helminthic infections and asthma**

Human immune response to infections with helminths parasites may differ according to the profile of the infection. As mentioned above, acute or seasonal infections may include primary infections and repeated or intermittent infections without long period of continuous infections and may result from infrequent short exposures or intermittent exposure after treatment in endemic areas [3, 5]. It has been proposed that mild, seasonal helminthic infections stimulate preferentially inflammatory Th2 type immune responses among parasitized populations [3, 81] characterized by the production of high levels of serum specific IgE and allergic reactivity toward parasite soluble antigens [82] which may lead to the development of bronchial hyper reactivity and asthma [83, 84] particularly among atopic individuals [85]. This situation may be reflected on the increased prevalence of asthma and allergic diseases observed in many low and middle-income countries [86, 87] undergoing parasite eradication programs [5, 6]. For example, epidemiological studies carried out in different rural communities in China, have shown a strong association between Ascaris lumbricoides infection and the development of asthma [88]. These early results are consistent with a meta-analysis of many of studies inves‐ tigating the association between the presence of geohelminth eggs in stool samples and asthma providing some evidence for parasite-specific effects [7]. In this work in which thirty-three studies were taken in account, *Ascaris lumbricoides* was associated with significantly increased odds of asthma (OR, 1.34; 95% CI, 1.05–1.71; 20 studies. Further studies carried out in urban Brazil have shown positive associations between Ascaris lumbricoides infection with recent wheeze [9] which in turn have been associated to allergic sensitization toward A. lumbricoides antigens [9]. Other studies carried out in Costa Rica have shown a relationship between sen‐ sitization (defined as a positive IgE) to A. lumbricoides and measures of asthma morbidity and severity in a population with low prevalence of parasitic infection but high prevalence of par‐ asitic exposure [89]. In this work, a cross-sectional study of 439 children (ages 6 to 14 years) with asthma was carried out and linear regression and logistic regression were used for the multivariate statistical analysis. Sensitization to A. lumbricoides was associated with having at least 1 positive skin test to allergens (odds ratio, 5.15; 95% CI, 2.36-11.21; P <.0001), increased total serum IgE and eosinophils in peripheral blood, reductions in FEV (1) and FEV (1)/forced vital capacity, increased airway responsiveness and bronchodilator responsiveness, and hos‐ pitalizations for asthma in the previous year (odds ratio, 3.08; 95% CI, 1.23-7.68; P = 0.02). Similarly, it has been also reported that sensitization to A. lumbricoides is associated with increased severity of asthma among Romanian children [90]. This association was mediated by a high degree of atopy among the asthmatic children sensitized to A. lumbricoides and belonging to a population with a low prevalence of helminthiasis. In according to these find‐ ings, studies carried out in Venezuela have shown strong associations between A. Lumbri‐ coides infection and bronchial hyper-reactivity [84]. In this work, 470 school children from different rural and urban communities were evaluated. It was found that in rural children, bronchial hyper reactivity was associated with increased specific levels of anti- IgE (p<0.0001) and skin test positivity toward A lumbricoides antigens (p<0.0001). The percentage of FVE1 predictive values correlated inversely (p<0.0001) with anti-A lumbricoides IgE levels. Elevated numbers of circulating CD3+CD4+ and CD20+CD23+ cells were found in rural children with bronchial hyper reactivity compared to their asymptomatic counterparts. They correlated positively with anti-A lumbricoides IgE levels (p<0.005 and p<0.0001 respectively). In contrast, in urban children, bronchial hyper reactivity was associated with elevated anti-D pteronissinus IgE levels (p=0, 0089), skin hyper reactivity towards this aero allergen (p=0,003) and to an increase in the number of CD3+CD8+ (p<0.0001). These results were consistent with previous work showing that monthly treatments of parasitized asthmatic children with anti-helminthic drugs in Venezuela may reduce BHR, symptoms of wheeze and the need for asthma medica‐ tions [2]. Taking together these findings suggest the importance of the atopic condition on the airway inflammatory response stimulated by these parasites [91]. On the other hand, studies performed in slum children from endemic areas have shown that parasitized non-asthmatic children can significant respond to bronchodilator inhalation, and that this response can be reversed by anti-helminthic treatment suggesting that these parasites cause bronchoconstric‐ tion [92]. Moreover, in a more recent study it was shown that infestation with *Ascaris lumbri‐ coides* in Brazilian children increased the risk of non-atopic asthma [8] such that children with high load infestation (≥100 eggs/g) have been found to be five times more likely to have BHR than children with low load or no infestation [8]. Thus, regardless the atopic status of the individuals, the link between helminth infestation and non-atopic asthma could be mediated by the stimulation of transient inflammatory responses by parasite antigens in the pulmonary phase of the helminth life cycle such as those described according to the Loeffler's syndrome.

ment that allows the parasite to survive. Nematode parasites may enhance survival by direct‐ ing the immune response to that of a less appropriate type. For example interference with the Th1/-Th2 response balance, the production of high levels of regulatory cytokines such as IL-10 and TGF- β which may lead to a general suppression of T and B cell responses and also mimicry of host proteins which direct the immune response to tolerance have been reported [93]. It has been proposed that because these suppressing mechanisms are not parasite- specific, they may affect the development of allergic reactions in chronically exposed populations [94, 95]. It has been proposed that the effect of geo-helminths on the suppression of atopy is more important early in life causing a deviated Th2 immune phenotype that is not changed later in life, after elimination of the infection [96]. Moreover, there is evidence that maternal helminthic infec‐ tions could affect infant immunity [97, 98] raising the possibility that the immunologic effects of infection start in the fetus. Further, the inverse association between chronic helminthic in‐ fections and allergic disorders among school children from different rural populations from Venezuela, Gambia, Ethiopia, Taiwan, Ecuador and Ghana has been well documented [99].

Helminthic Infections and Asthma: Still a Challenge for Developing Countries

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The exact mechanisms by which these parasites dampen allergic responses are probably mul‐ tiple. Chronic helminthic infections may protect against allergic disease because of their pro‐ found suppression of the host immune system, leading to a general T-cell hypo responsiveness that is facilitated by the activity of regulatory T (Treg) and B cells and the modulation effects of innate immune cells such as macrophages, dendritic cells (DCs) and local stromal cells, resulting in an anti-inflammatory environment characterized by increased levels of interleukin (IL)-10 and transforming growth factor (TGF)-β [100]. In humans, several studies have shown that Treg cell activity (both by natural CD4+CD25+FoxP3+ and adaptive CD4+IL10+ Tr1 cells) protects against allergic disease [100,101, 102]. Indeed, successful allergen specific immuno‐ therapy in humans which leads to a reduction in allergic symptoms has been associated with the emergence of IL-10–producing Treg cells which may be involved in the increase in IgG4 and IgA responses and a simultaneous decrease in IgE [103]. Several studies carried out in distinct experimental models have revealed a number of active molecules in extracts of hel‐ minths that can modulate the immune system of the host. Early work conducted by Itami et al [104] have demonstrated that high molecular weight components purified by gel filtration chromatography from an *A. suum* adult worm extract were able to suppress the murine anti‐ body production to a bystander antigen. This effect was attributed to a 200 KD a protein com‐ ponent called PAS-1. The protein was affinity purified using a monoclonal antibody (MAIP-1) produced against high molecular weight suppressive components. Pas-1 was shown to be capable of down regulating antibody production Th2 secretion; eosinophils recruitment and airway hyper-responsiveness induced by *A. suum* allergens [105]. This effect was mediated by the stimulating capacity of Pas-1 on the production of regulatory cytokines such as IL-10. Probably the best-characterized groups of helminth immunomodulators are the cystatins (cys‐ teine protease inhibitors) [106] which can inhibit antigen processing and presentation [107], interfering with antigen-specific T cell responses and the proliferation of T and B cells. They can also modulate cytokine responses. Particularly they are involved in the up regulation of IL-10 that leads to the down regulation of co-stimulatory surface molecules of macrophages [108]. These properties contribute to induction of an anti-inflammatory environment, con‐ comitant with a strong inhibition of cellular proliferation. Also, carbohydrates linked to pro‐

#### **5. The other side of the coin: Chronic helminthic infection may suppress allergic manifestation and the development of asthma**

Because parasites are in constant attack by a range of effective immune mechanisms, they have developed effective evasion mechanisms which may vary from simple avoidance to a more active modulation of the immune response in order to establish a non inflammatory environ‐ ment that allows the parasite to survive. Nematode parasites may enhance survival by direct‐ ing the immune response to that of a less appropriate type. For example interference with the Th1/-Th2 response balance, the production of high levels of regulatory cytokines such as IL-10 and TGF- β which may lead to a general suppression of T and B cell responses and also mimicry of host proteins which direct the immune response to tolerance have been reported [93]. It has been proposed that because these suppressing mechanisms are not parasite- specific, they may affect the development of allergic reactions in chronically exposed populations [94, 95]. It has been proposed that the effect of geo-helminths on the suppression of atopy is more important early in life causing a deviated Th2 immune phenotype that is not changed later in life, after elimination of the infection [96]. Moreover, there is evidence that maternal helminthic infec‐ tions could affect infant immunity [97, 98] raising the possibility that the immunologic effects of infection start in the fetus. Further, the inverse association between chronic helminthic in‐ fections and allergic disorders among school children from different rural populations from Venezuela, Gambia, Ethiopia, Taiwan, Ecuador and Ghana has been well documented [99].

multivariate statistical analysis. Sensitization to A. lumbricoides was associated with having at least 1 positive skin test to allergens (odds ratio, 5.15; 95% CI, 2.36-11.21; P <.0001), increased total serum IgE and eosinophils in peripheral blood, reductions in FEV (1) and FEV (1)/forced vital capacity, increased airway responsiveness and bronchodilator responsiveness, and hos‐ pitalizations for asthma in the previous year (odds ratio, 3.08; 95% CI, 1.23-7.68; P = 0.02). Similarly, it has been also reported that sensitization to A. lumbricoides is associated with increased severity of asthma among Romanian children [90]. This association was mediated by a high degree of atopy among the asthmatic children sensitized to A. lumbricoides and belonging to a population with a low prevalence of helminthiasis. In according to these find‐ ings, studies carried out in Venezuela have shown strong associations between A. Lumbri‐ coides infection and bronchial hyper-reactivity [84]. In this work, 470 school children from different rural and urban communities were evaluated. It was found that in rural children, bronchial hyper reactivity was associated with increased specific levels of anti- IgE (p<0.0001) and skin test positivity toward A lumbricoides antigens (p<0.0001). The percentage of FVE1 predictive values correlated inversely (p<0.0001) with anti-A lumbricoides IgE levels. Elevated numbers of circulating CD3+CD4+ and CD20+CD23+ cells were found in rural children with bronchial hyper reactivity compared to their asymptomatic counterparts. They correlated positively with anti-A lumbricoides IgE levels (p<0.005 and p<0.0001 respectively). In contrast, in urban children, bronchial hyper reactivity was associated with elevated anti-D pteronissinus IgE levels (p=0, 0089), skin hyper reactivity towards this aero allergen (p=0,003) and to an increase in the number of CD3+CD8+ (p<0.0001). These results were consistent with previous work showing that monthly treatments of parasitized asthmatic children with anti-helminthic drugs in Venezuela may reduce BHR, symptoms of wheeze and the need for asthma medica‐ tions [2]. Taking together these findings suggest the importance of the atopic condition on the airway inflammatory response stimulated by these parasites [91]. On the other hand, studies performed in slum children from endemic areas have shown that parasitized non-asthmatic children can significant respond to bronchodilator inhalation, and that this response can be reversed by anti-helminthic treatment suggesting that these parasites cause bronchoconstric‐ tion [92]. Moreover, in a more recent study it was shown that infestation with *Ascaris lumbri‐ coides* in Brazilian children increased the risk of non-atopic asthma [8] such that children with high load infestation (≥100 eggs/g) have been found to be five times more likely to have BHR than children with low load or no infestation [8]. Thus, regardless the atopic status of the individuals, the link between helminth infestation and non-atopic asthma could be mediated by the stimulation of transient inflammatory responses by parasite antigens in the pulmonary phase of the helminth life cycle such as those described according to the Loeffler's syndrome.

214 Respiratory Disease and Infection - A New Insight

**5. The other side of the coin: Chronic helminthic infection may suppress**

Because parasites are in constant attack by a range of effective immune mechanisms, they have developed effective evasion mechanisms which may vary from simple avoidance to a more active modulation of the immune response in order to establish a non inflammatory environ‐

**allergic manifestation and the development of asthma**

The exact mechanisms by which these parasites dampen allergic responses are probably mul‐ tiple. Chronic helminthic infections may protect against allergic disease because of their pro‐ found suppression of the host immune system, leading to a general T-cell hypo responsiveness that is facilitated by the activity of regulatory T (Treg) and B cells and the modulation effects of innate immune cells such as macrophages, dendritic cells (DCs) and local stromal cells, resulting in an anti-inflammatory environment characterized by increased levels of interleukin (IL)-10 and transforming growth factor (TGF)-β [100]. In humans, several studies have shown that Treg cell activity (both by natural CD4+CD25+FoxP3+ and adaptive CD4+IL10+ Tr1 cells) protects against allergic disease [100,101, 102]. Indeed, successful allergen specific immuno‐ therapy in humans which leads to a reduction in allergic symptoms has been associated with the emergence of IL-10–producing Treg cells which may be involved in the increase in IgG4 and IgA responses and a simultaneous decrease in IgE [103]. Several studies carried out in distinct experimental models have revealed a number of active molecules in extracts of hel‐ minths that can modulate the immune system of the host. Early work conducted by Itami et al [104] have demonstrated that high molecular weight components purified by gel filtration chromatography from an *A. suum* adult worm extract were able to suppress the murine anti‐ body production to a bystander antigen. This effect was attributed to a 200 KD a protein com‐ ponent called PAS-1. The protein was affinity purified using a monoclonal antibody (MAIP-1) produced against high molecular weight suppressive components. Pas-1 was shown to be capable of down regulating antibody production Th2 secretion; eosinophils recruitment and airway hyper-responsiveness induced by *A. suum* allergens [105]. This effect was mediated by the stimulating capacity of Pas-1 on the production of regulatory cytokines such as IL-10. Probably the best-characterized groups of helminth immunomodulators are the cystatins (cys‐ teine protease inhibitors) [106] which can inhibit antigen processing and presentation [107], interfering with antigen-specific T cell responses and the proliferation of T and B cells. They can also modulate cytokine responses. Particularly they are involved in the up regulation of IL-10 that leads to the down regulation of co-stimulatory surface molecules of macrophages [108]. These properties contribute to induction of an anti-inflammatory environment, con‐ comitant with a strong inhibition of cellular proliferation. Also, carbohydrates linked to pro‐ teins and lipids of nematodes, particularly those of phosphorylcholine (PC)-modified carbohydrates [109] have attracted significant attention in the past years due to their immu‐ nogenic and immunomodulatory properties. Structural features of glycolipids including oli‐ gosaccharide backbone, substitution with PC, and ceramide composition are shared between all the parasitic nematode species with widespread anatomical location in the worm [109] suggesting the importance of these components in host parasite interactions. The secreted filarial nematode glycoprotein ES-62 constitutes a suitable example. Through PC modifica‐ tions, ES-62 can inhibit the proliferation of CD4+ T cells and conventional B2 cells *in vivo*, and reduces CD4+ cell IL-4 and IFN-γ production [110]. Conversely, ES-62 promotes proliferation and IL-10 production by peritoneal B1 cells [111]. It has been proposed that inhibition of proinflammatory Th1 responses occurs as ES-62 interacts with toll-like receptor (TLR) 4 through its PC residues [112], also in mast cells the interaction of TLR 4 with ES-62 results in the inhibition of degranulation and release of inflammatory mediators [113]. On the other hand, like proteins, glycolipids can be target of antibody responses. In the case of helminths antibody reactivity to lipids has been described in schistosomiasis [114] and more recently in *A. lumbricoides* infection [115]. Epidemiological studies using *Ascaris* derived glicolipids have shown that children carrying heavy infections show highest IgG reactivity glycolipids com‐ pared to lightly or non-infected children [115]. In the same study IgG antibody reactivity to both glyco proteins and glycolipids were directed to the PC moiety as determined by either removal of this group or a competition assay suggesting that *A. lumbricoides* specific glycolipids have antigenic properties. The mechanism by which glycolipids can stimulate IgE and IgG responses is not clear. It is possible that antibodies could develop directly to glycolipids through activation of CD1d which is a non classical MHC lipid presenting molecule. Never‐ theless, cross reactivity between glycolipids and PC present on proteins may also occur [115]. The immunomodulatory effects exhibited by PC- substituted molecules can be seen as a con‐ tribution to equilibrium in host -parasite interactions in which expanding of TH2 type re‐ sponses enables the parasite to survive preventing harmful pro-inflammatory mechanisms in the host. Since PC substituted molecules from nematode differ clearly from those from the host, they would be a suitable target for the development of new anti inflammatory drugs.

has adopted several resolutions calling for the control or elimination of these diseases, and for the implementation of a number of large-scale control and elimination programs. However, de‐ spite such WHA/WHO resolutions, the control of morbidity and the elimination of these infec‐ tions are still a big challenge for global health programs. Some of the identified obstacles include the current scarcity of tools for updated disease mapping, the development of new anthelmin‐ tic drugs and vaccines, the improvement of sensitive diagnostic tools and the monitoring of the progress of control interventions and quantification of changes in incidence of infection and dis‐ ease [116]. However and according to the WHA/WHO resolution, mass chemotherapy have been widely implemented in rural areas of many developing countries in which sanitary limita‐ tions are far to be overcome [6]. Under these conditions, this approach would reduce worm bur‐ dens without elimination of the infection in endemic areas, which gradually will change the profile of the infection from a chronic pattern, with moderate to heavy worm burdens [5] to a more mild and seasonal pattern [5], thereby disrupting the regulatory, anti-inflammatory ef‐ fect of chronic infections on the immune response, allowing allergic sensitization in atopic par‐ asitized individuals [117, 118]. Thus, the development of diagnosis protocols facilitating rapid identification of atopic individuals among rural populations is an immediate challenge to ach‐ ieve control of parasites without affecting the health status of a significant proportion of these populations. On the other hand, the presence of respiratory symptoms as a consequence of in‐ flammation due to the parasite migratory phase in non atopic individuals [8] must also be con‐ sidered. For these purposes, large birth cohort studies designed according to specific epidemiological objectives and based on results from cross-sectional studies, small longitudi‐ nal and pilot intervention studies would help to elucidate the role of the many parameters in‐ volved in host- parasite interactions contributing to the pathogenesis of asthma. Also, the identification of conserved features of helminth products that interact with innate immune cells to co-ordinate adaptive anti-parasite responses as well as of potent parasite derived allergens is a key challenge to improve the technology used in the diagnosis and monitoring of allergic dis‐ eases in the tropics. Noteworthy is the cross-reactivity of helminth antigens with environmen‐ tal allergens which may explain the high prevalence of IgE sensitization to invertebrate allergens leading to the development of asthma and other allergic diseases [61]. Finally, the identification and characterization of individual helminth-derived immunomodulatory mole‐ cules that selectively induce regulatory immune responses will provide potential candidates

Helminthic Infections and Asthma: Still a Challenge for Developing Countries

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217

for immunotherapy [119] and must be the subject for future research programs.

Isabel Hagel, Maira Cabrera and Maria Cristina Di Prisco

\*Address all correspondence to: isabelhagel@yahoo.com

CDCH, PG: 09-7946-2010/1.

**Author details**

This work has been supported by the Central University of Venezuela through the project:

Institute of Biomedicine, Faculty of medicine, Central University of Venezuela, Venezuela

#### **6. Future challenges and research perspectives**

As mentioned above, the prevalence of asthma in rural developing countries has been under es‐ timated for many years and in many areas of the world in which persons with asthma do not have access to basic asthma medications or medical care and are not included in any statistical survey. Thus adequate control of asthma in developing countries would require improve‐ ments in health care and the development of technologies to obtain the information needed to identify high-risk groups (disease mapping) [3]. This goal would be difficult to achieve if coun‐ tries do not allocate resources to enable better health care. On the other hand, since many years several global efforts have been made to address the health effects of human parasitism by hel‐ minths which results from poverty and exert a well known detrimental impact on the health sta‐ tus of children continuously exposed to these parasites. The World Health Assembly (WHA) has adopted several resolutions calling for the control or elimination of these diseases, and for the implementation of a number of large-scale control and elimination programs. However, de‐ spite such WHA/WHO resolutions, the control of morbidity and the elimination of these infec‐ tions are still a big challenge for global health programs. Some of the identified obstacles include the current scarcity of tools for updated disease mapping, the development of new anthelmin‐ tic drugs and vaccines, the improvement of sensitive diagnostic tools and the monitoring of the progress of control interventions and quantification of changes in incidence of infection and dis‐ ease [116]. However and according to the WHA/WHO resolution, mass chemotherapy have been widely implemented in rural areas of many developing countries in which sanitary limita‐ tions are far to be overcome [6]. Under these conditions, this approach would reduce worm bur‐ dens without elimination of the infection in endemic areas, which gradually will change the profile of the infection from a chronic pattern, with moderate to heavy worm burdens [5] to a more mild and seasonal pattern [5], thereby disrupting the regulatory, anti-inflammatory ef‐ fect of chronic infections on the immune response, allowing allergic sensitization in atopic par‐ asitized individuals [117, 118]. Thus, the development of diagnosis protocols facilitating rapid identification of atopic individuals among rural populations is an immediate challenge to ach‐ ieve control of parasites without affecting the health status of a significant proportion of these populations. On the other hand, the presence of respiratory symptoms as a consequence of in‐ flammation due to the parasite migratory phase in non atopic individuals [8] must also be con‐ sidered. For these purposes, large birth cohort studies designed according to specific epidemiological objectives and based on results from cross-sectional studies, small longitudi‐ nal and pilot intervention studies would help to elucidate the role of the many parameters in‐ volved in host- parasite interactions contributing to the pathogenesis of asthma. Also, the identification of conserved features of helminth products that interact with innate immune cells to co-ordinate adaptive anti-parasite responses as well as of potent parasite derived allergens is a key challenge to improve the technology used in the diagnosis and monitoring of allergic dis‐ eases in the tropics. Noteworthy is the cross-reactivity of helminth antigens with environmen‐ tal allergens which may explain the high prevalence of IgE sensitization to invertebrate allergens leading to the development of asthma and other allergic diseases [61]. Finally, the identification and characterization of individual helminth-derived immunomodulatory mole‐ cules that selectively induce regulatory immune responses will provide potential candidates for immunotherapy [119] and must be the subject for future research programs.

This work has been supported by the Central University of Venezuela through the project: CDCH, PG: 09-7946-2010/1.

#### **Author details**

teins and lipids of nematodes, particularly those of phosphorylcholine (PC)-modified carbohydrates [109] have attracted significant attention in the past years due to their immu‐ nogenic and immunomodulatory properties. Structural features of glycolipids including oli‐ gosaccharide backbone, substitution with PC, and ceramide composition are shared between all the parasitic nematode species with widespread anatomical location in the worm [109] suggesting the importance of these components in host parasite interactions. The secreted filarial nematode glycoprotein ES-62 constitutes a suitable example. Through PC modifica‐ tions, ES-62 can inhibit the proliferation of CD4+ T cells and conventional B2 cells *in vivo*, and reduces CD4+ cell IL-4 and IFN-γ production [110]. Conversely, ES-62 promotes proliferation and IL-10 production by peritoneal B1 cells [111]. It has been proposed that inhibition of proinflammatory Th1 responses occurs as ES-62 interacts with toll-like receptor (TLR) 4 through its PC residues [112], also in mast cells the interaction of TLR 4 with ES-62 results in the inhibition of degranulation and release of inflammatory mediators [113]. On the other hand, like proteins, glycolipids can be target of antibody responses. In the case of helminths antibody reactivity to lipids has been described in schistosomiasis [114] and more recently in *A. lumbricoides* infection [115]. Epidemiological studies using *Ascaris* derived glicolipids have shown that children carrying heavy infections show highest IgG reactivity glycolipids com‐ pared to lightly or non-infected children [115]. In the same study IgG antibody reactivity to both glyco proteins and glycolipids were directed to the PC moiety as determined by either removal of this group or a competition assay suggesting that *A. lumbricoides* specific glycolipids have antigenic properties. The mechanism by which glycolipids can stimulate IgE and IgG responses is not clear. It is possible that antibodies could develop directly to glycolipids through activation of CD1d which is a non classical MHC lipid presenting molecule. Never‐ theless, cross reactivity between glycolipids and PC present on proteins may also occur [115]. The immunomodulatory effects exhibited by PC- substituted molecules can be seen as a con‐ tribution to equilibrium in host -parasite interactions in which expanding of TH2 type re‐ sponses enables the parasite to survive preventing harmful pro-inflammatory mechanisms in the host. Since PC substituted molecules from nematode differ clearly from those from the host, they would be a suitable target for the development of new anti inflammatory drugs.

216 Respiratory Disease and Infection - A New Insight

**6. Future challenges and research perspectives**

As mentioned above, the prevalence of asthma in rural developing countries has been under es‐ timated for many years and in many areas of the world in which persons with asthma do not have access to basic asthma medications or medical care and are not included in any statistical survey. Thus adequate control of asthma in developing countries would require improve‐ ments in health care and the development of technologies to obtain the information needed to identify high-risk groups (disease mapping) [3]. This goal would be difficult to achieve if coun‐ tries do not allocate resources to enable better health care. On the other hand, since many years several global efforts have been made to address the health effects of human parasitism by hel‐ minths which results from poverty and exert a well known detrimental impact on the health sta‐ tus of children continuously exposed to these parasites. The World Health Assembly (WHA)

Isabel Hagel, Maira Cabrera and Maria Cristina Di Prisco

\*Address all correspondence to: isabelhagel@yahoo.com

Institute of Biomedicine, Faculty of medicine, Central University of Venezuela, Venezuela

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**Section 4**

**Smoking Cessation and Lung**

### **Smoking Cessation and Lung**

**Chapter 9**

**Psychological Approaches to**

Jennifer Lira-Mandujano,

http://dx.doi.org/10.5772/53265

**1. Introduction**

been questioned [2-4].

**Increase Tobacco Abstinence in Patients with**

M. Carmen Míguez-Varela and Sara E. Cruz-Morales

Additional information is available at the end of the chapter

**Chronic Obstructive Pulmonary Disease: A Review**

In the last century, the definition of COPD was considered as a disease constituted by chron‐ ic bronchitis and pulmonary emphysema; however, nowadays these terms are in disuse. In the last decades emerged the Global Initiative for Chronic Obstructive Lung Disease (GOLD), a group formed by researches with the objective of count with a diagnostic guide to study this disease. According to the GOLD, the COPD is a common preventable and treatable disease, characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases, exacerbations and co-morbidities contribute to the overall severity in individual patients [1]. However, some of the parameters and the definition have

The prevalence of COPD in developed countries ranges from 3 to 6% in subjects over 50 years of age. According to the National Center for Health Statistics (NCHS) in the United States, prevalence of COPD was stable from 1998 through 2009 and was significantly higher among women than in men, and the prevalence was higher in old groups. The prevalence was high among non-Hispanic white (5.7%) and Puerto Rican (6.9%) adults, Mexican-Amer‐ ican adults had the lowest COPD prevalence (2.6%) [5]. The prevalence of COPD appears to be increasing not only in many developed countries, but also in Latin America. COPD occu‐ pies the fourth place in terms of mortality around the world, in Mexico is currently located between the sixth and the fourth. In 2005 there was more than 11 000 deaths in men, against just over 9 000 in women, recent studies show that the prevalence is equal between men and

and reproduction in any medium, provided the original work is properly cited.

© 2013 Lira-Mandujano et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary Disease: A Review**

Jennifer Lira-Mandujano, M. Carmen Míguez-Varela and Sara E. Cruz-Morales

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53265

#### **1. Introduction**

In the last century, the definition of COPD was considered as a disease constituted by chron‐ ic bronchitis and pulmonary emphysema; however, nowadays these terms are in disuse. In the last decades emerged the Global Initiative for Chronic Obstructive Lung Disease (GOLD), a group formed by researches with the objective of count with a diagnostic guide to study this disease. According to the GOLD, the COPD is a common preventable and treatable disease, characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases, exacerbations and co-morbidities contribute to the overall severity in individual patients [1]. However, some of the parameters and the definition have been questioned [2-4].

The prevalence of COPD in developed countries ranges from 3 to 6% in subjects over 50 years of age. According to the National Center for Health Statistics (NCHS) in the United States, prevalence of COPD was stable from 1998 through 2009 and was significantly higher among women than in men, and the prevalence was higher in old groups. The prevalence was high among non-Hispanic white (5.7%) and Puerto Rican (6.9%) adults, Mexican-Amer‐ ican adults had the lowest COPD prevalence (2.6%) [5]. The prevalence of COPD appears to be increasing not only in many developed countries, but also in Latin America. COPD occu‐ pies the fourth place in terms of mortality around the world, in Mexico is currently located between the sixth and the fourth. In 2005 there was more than 11 000 deaths in men, against just over 9 000 in women, recent studies show that the prevalence is equal between men and

© 2013 Lira-Mandujano et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

women [6]. According the European Federation of Allergies and Airways Diseases (EFA), in Europe the prevalence of COPD varies among the different countries, having in the ex‐ tremes more than 10% Germany and 2% in Netherlands: with an annual mortality of 0.28 per 1000 in Germany and 0.30 per 1000 in Austria [7]. An important problem is that COPD prevalence varies across the world, mainly to the different definitions of the disease, leading to an over-diagnosis or under-diagnosis.

>70%). A ratio less than normal defines the patient as having COPD. The diagnosis of COPD is made when the FEV1/FVC ratio is <70%. The GOLD criteria also require that values be after bronchodilator medication has been given to make the diagnosis, and the NICE criteria require FEV1%. According to the ERS criteria, it is FEV1% predicted that defines when a pa‐ tient has COPD, that is, when FEV1% predicted is < 88% for men, or < 89% for women [1].

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

http://dx.doi.org/10.5772/53265

233

At the beginning, the airflow obstruction first causes breathlessness that reduces the forced expiratory volume in one second (FEV1) to about 1 liter, which is less than half the normal value. Then, the condition progresses persistently over five or more years, with further loss of FEV, causing more and more distressing disability and, finally, death from respiratory failure [11]. The (FEV1) declines normally with aging by approximately 30 mL/yr, but in vulnerable smokers, the decline is greater (about 60 mL/yr), resulting in the development of COPD. Smoking cessation usually restores the normal or near-normal rate of FEV1 decline. Therefore, smoking cessation is a critical component for the preven‐ tion of COPD progression. FEV1 is an index in the definition of COPD and classification of its severity. FEV1 is a good predictor of exercise tolerance and correlates with survival and quality of life. More rapid FEV1 decline is also predictive of morbidity, mortality,

With regard to lung diseases, COPD is which is more associated with cigarette smoking. Smoking cessation reduces the risk of COPD, improves the prognosis [11,13], prevents pro‐ gression of the disease [14], and reduces exacerbations of COPD [15-17]. However, COPD patients find it difficult to quit smoking; in fact, the majority of new patients to the hospital for exacerbation of COPD continue smoking. COPD patients have a long smoking history, higher tobacco consumption, higher level of CO in the exhaled air, severe dependence to nicotine, and most have experienced numerous unsuccessful previous quit attempts [18,19]. This makes smoking cessation even more difficult for this group. Smoke quitting not only reduces the risk of COPD but also improve some symptoms like cough, coughing, shortness of breath and immune response, which leads to fewer respiratory infections to occur [20].

There are different drug treatments to quit smoking, however, when pharmacological treat‐ ments are combined with psychological techniques, the effectiveness increased significantly in patients with COPD [20-22]. Coronini-Cronberg et al. [21] pointed out that the main prob‐ lems associated with the relapse of smoking in patients with COPD are the lack of motiva‐ tion to quit smoking, poor communication with healthcare professionals, and the misleading

In patients with COPD, the comorbidity with anxiety and depression becomes more rele‐ vant. For that reason, it is important to review the available literature related to psychologi‐ cal techniques to quit smoking, specifically focused on psychological techniques to increase motivation, development of skills to deal with situations to prevent relapse, and the man‐ agement of anxiety, depression, and stress. Therefore, the objective of this study was to con‐

and hospitalization rates [12].

**3. Smoking cessation**

information of the effects of smoking on health.

Smoking contributes too many health problems including cancer, cardiovascular disease, and lung diseases, among others. In respiratory illnesses smoking increase the probability of bronchitis, emphysema, chronic obstructive lung disease and pneumonia [8]. Diagnoses of these illnesses are more common in smokers than in non-smokers; in fact is the most impor‐ tant risk factor of COPD and their health complications [1,9]. Therefore, in a person with the diagnosis COPD, if it is smoking, the intervention plan should include the elimination of the tobacco, since it is the most effective measurement in the prevention of this illness, provok‐ ing a delay in the loss of the pulmonary function and improving the survival [10].

#### **2. Pathophysiology**

The exposure to noxious particles produces lung inflammation, this chronic inflammation produces several airways disease as obstructive bronchiolitis and parenchymal destruction (known as emphysema) and impair defense mechanisms, all these changes result in a pro‐ gressive airflow limitation [1].

The main risk factors for COPD are tobacco smoking, occupational dusts and chemicals, in‐ door and outdoor air pollution. In countries as Mexico, Nepal, New Guinea and Colombia, exposure to wood smoke also cause COPD. Inhalation in the work environment of dusts, gases, fumes and chemicals are other risk factors. For example in United States 19% of pa‐ tients with COPD had an occupational exposure.

The symptoms more frequently observed include dyspnea, chronic cough and excessive sputum production or expectoration. However, COPD is not just simply a "smoker's cough", but an under-diagnosed, life threatening lung disease that may progressively lead to death. Physical examination includes cyanosis of lips and fingers, breathing with pursed lips (more common in patients with emphysema), use of accessory muscles of respiration: scalene and sternocleidomastoid (in cases of severe COPD), engorgement jugular, decrease in respirato‐ ry or abolished (in severe stages of COPD) noise, vibrations reduced vowels (advanced stage), there may be wheezing.

The spirometry is a simple diagnostic test that confirms the presence of COPD, it measures the amount and speed of air inhaled and exhaled. Spirometry measures the forced expirato‐ ry volume in one second (FEV1), which is the greatest volume of air that can be breathed out in the first second of a large breath; also measures the forced vital capacity (FVC), which is the greatest volume of air that can be breathed out in a completely large breath (OMS). Nor‐ mally, at least 70% of the FVC comes out in the first second (i.e. the FEV1/FVC ratio is >70%). A ratio less than normal defines the patient as having COPD. The diagnosis of COPD is made when the FEV1/FVC ratio is <70%. The GOLD criteria also require that values be after bronchodilator medication has been given to make the diagnosis, and the NICE criteria require FEV1%. According to the ERS criteria, it is FEV1% predicted that defines when a pa‐ tient has COPD, that is, when FEV1% predicted is < 88% for men, or < 89% for women [1].

At the beginning, the airflow obstruction first causes breathlessness that reduces the forced expiratory volume in one second (FEV1) to about 1 liter, which is less than half the normal value. Then, the condition progresses persistently over five or more years, with further loss of FEV, causing more and more distressing disability and, finally, death from respiratory failure [11]. The (FEV1) declines normally with aging by approximately 30 mL/yr, but in vulnerable smokers, the decline is greater (about 60 mL/yr), resulting in the development of COPD. Smoking cessation usually restores the normal or near-normal rate of FEV1 decline. Therefore, smoking cessation is a critical component for the preven‐ tion of COPD progression. FEV1 is an index in the definition of COPD and classification of its severity. FEV1 is a good predictor of exercise tolerance and correlates with survival and quality of life. More rapid FEV1 decline is also predictive of morbidity, mortality, and hospitalization rates [12].

#### **3. Smoking cessation**

women [6]. According the European Federation of Allergies and Airways Diseases (EFA), in Europe the prevalence of COPD varies among the different countries, having in the ex‐ tremes more than 10% Germany and 2% in Netherlands: with an annual mortality of 0.28 per 1000 in Germany and 0.30 per 1000 in Austria [7]. An important problem is that COPD prevalence varies across the world, mainly to the different definitions of the disease, leading

Smoking contributes too many health problems including cancer, cardiovascular disease, and lung diseases, among others. In respiratory illnesses smoking increase the probability of bronchitis, emphysema, chronic obstructive lung disease and pneumonia [8]. Diagnoses of these illnesses are more common in smokers than in non-smokers; in fact is the most impor‐ tant risk factor of COPD and their health complications [1,9]. Therefore, in a person with the diagnosis COPD, if it is smoking, the intervention plan should include the elimination of the tobacco, since it is the most effective measurement in the prevention of this illness, provok‐

The exposure to noxious particles produces lung inflammation, this chronic inflammation produces several airways disease as obstructive bronchiolitis and parenchymal destruction (known as emphysema) and impair defense mechanisms, all these changes result in a pro‐

The main risk factors for COPD are tobacco smoking, occupational dusts and chemicals, in‐ door and outdoor air pollution. In countries as Mexico, Nepal, New Guinea and Colombia, exposure to wood smoke also cause COPD. Inhalation in the work environment of dusts, gases, fumes and chemicals are other risk factors. For example in United States 19% of pa‐

The symptoms more frequently observed include dyspnea, chronic cough and excessive sputum production or expectoration. However, COPD is not just simply a "smoker's cough", but an under-diagnosed, life threatening lung disease that may progressively lead to death. Physical examination includes cyanosis of lips and fingers, breathing with pursed lips (more common in patients with emphysema), use of accessory muscles of respiration: scalene and sternocleidomastoid (in cases of severe COPD), engorgement jugular, decrease in respirato‐ ry or abolished (in severe stages of COPD) noise, vibrations reduced vowels (advanced

The spirometry is a simple diagnostic test that confirms the presence of COPD, it measures the amount and speed of air inhaled and exhaled. Spirometry measures the forced expirato‐ ry volume in one second (FEV1), which is the greatest volume of air that can be breathed out in the first second of a large breath; also measures the forced vital capacity (FVC), which is the greatest volume of air that can be breathed out in a completely large breath (OMS). Nor‐ mally, at least 70% of the FVC comes out in the first second (i.e. the FEV1/FVC ratio is

ing a delay in the loss of the pulmonary function and improving the survival [10].

to an over-diagnosis or under-diagnosis.

232 Respiratory Disease and Infection - A New Insight

**2. Pathophysiology**

gressive airflow limitation [1].

stage), there may be wheezing.

tients with COPD had an occupational exposure.

With regard to lung diseases, COPD is which is more associated with cigarette smoking. Smoking cessation reduces the risk of COPD, improves the prognosis [11,13], prevents pro‐ gression of the disease [14], and reduces exacerbations of COPD [15-17]. However, COPD patients find it difficult to quit smoking; in fact, the majority of new patients to the hospital for exacerbation of COPD continue smoking. COPD patients have a long smoking history, higher tobacco consumption, higher level of CO in the exhaled air, severe dependence to nicotine, and most have experienced numerous unsuccessful previous quit attempts [18,19]. This makes smoking cessation even more difficult for this group. Smoke quitting not only reduces the risk of COPD but also improve some symptoms like cough, coughing, shortness of breath and immune response, which leads to fewer respiratory infections to occur [20].

There are different drug treatments to quit smoking, however, when pharmacological treat‐ ments are combined with psychological techniques, the effectiveness increased significantly in patients with COPD [20-22]. Coronini-Cronberg et al. [21] pointed out that the main prob‐ lems associated with the relapse of smoking in patients with COPD are the lack of motiva‐ tion to quit smoking, poor communication with healthcare professionals, and the misleading information of the effects of smoking on health.

In patients with COPD, the comorbidity with anxiety and depression becomes more rele‐ vant. For that reason, it is important to review the available literature related to psychologi‐ cal techniques to quit smoking, specifically focused on psychological techniques to increase motivation, development of skills to deal with situations to prevent relapse, and the man‐ agement of anxiety, depression, and stress. Therefore, the objective of this study was to con‐ duct a review of the effectiveness of the treatments to quit smoking in patients with COPD, identifying the main difficulties to maintain abstinence and propose strategies to eliminate such obstacles.

cotherapy. It is concluded that a combination of psychosocial and pharmacological interven‐ tions are superior to none apply any treatment. However, few studies have evaluated in the context of clinical trials the effectiveness of psychological interventions in patients with

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

http://dx.doi.org/10.5772/53265

235

Hilberink, Jacobs, Bottema of Vries, and Grol [25] conducted in the Netherlands a random‐ ized controlled trial in patients with COPD (392 smokers) to evaluate the type of interven‐ tion on smoking cessation rates, the interventions were a minimal intensity intervention for smoking cessation or the usual care. The intervention was educational and included patient support by health professionals. In the first visit, it was spoken of symptoms, health status and treatment, smoking behavior and motivational state for smoking cessation; also a bro‐ chure and a video designed for people with COPD were given. Based on the stages of change [26], the patients were assigned in three categories: 1) preparers (wanted to quit in the next month), 2) contemplators (wanted to quit in the next 6 months), and 3) precontem‐ plators (not want to quit). According to these categories, smokers with no motivation to quit, just received information about the benefits of quitting (control group). Smokers moti‐ vated to quit received information to increase their self-efficacy from discussing how they could deal with barriers to quitting, and additionally, information on nicotine replacement therapies according to the level of nicotine dependence was given. After six months, the ex‐ perimental group showed more attempts to quit smoking (44.9% vs. 36.5%), and quit smok‐

In a second study Wilson, Fitzsimons, Bradbury and Stuart [27] conducted a randomized controlled trial to evaluate the effectiveness of interventions to quit smoking based on the brief advise or brief advise accompanied by nurse support, either individually or in group; smoking status was biochemically validated at 2, 3, 6, 9 and 12 months. The sam‐ ple consisted of 91 smokers with COPD from an Ireland hospital. All patients received a brief smoking cessation intervention from the physician (5-10 minutes); afterwards, smok‐ ers were randomly assigned to one of three groups: usual care, individual, or group sup‐ port. The usual care group (n = 35) or control group, received only the brief advise; the intervention groups, individual support (n = 27) or group support (n = 29), underwent five weeks of support after the intervention; the nurses with approximately 6 hours of training carried out the interventions. Individual interventions consisted in providing selfhelp material and information about the personal benefits of quit smoking, discussion about the benefits of nicotine replacement therapy, to encourage setting a date to stop smoking and inform friends and family about the intent. In group interventions, patients were classified according to the phase of change [26] in order that nurses could adapt the intervention to the phase of each patient. In addition, the positive and negative aspects of smoking, particularly on health were discussed, as well as the previous efforts to quit smoking with the intention of to identify those factors that contributed to relapse. In addi‐ tion, role play in risk situations was performed to increase their confidence to quitting smoking. Both interventions consisted of five sessions of 60 minutes, in which nicotine re‐ placement therapy was given upon request. Twelve months after the intervention, no sig‐ nificant differences between the groups were observed. Abstinence in patients was of 6%

COPD (see table 1).

ing compared with control group (16.0% vs. 8.8%).

#### **4. Method**

To perform this bibliographic review it was searched in the Cochrane Tobacco Addiction Group, as well as the databases MEDLINE, EMBASE, PsycINFO, PubMed, and SCOPUS from 2000 to 2012. We used the following keywords: smoking cessation, tobacco cessation, hospital, patient, medical setting, and chronic obstructive pulmonary disease. The inclusion criteria used in this review were: a) studies involving interventions for smoking cessation in smokers who have COPD, b) that smokers had 18 or more years; and c) that smokers submit voluntarily to interventions for smoking cessation.

#### **5. Results**

Ten reports were identified and grouped in three categories:


### **6. Psychological interventions for smoking cessation in patients with COPD**

In a review on the effectiveness of interventions for smoking cessation, Lancaster and Stead [23] point out that psychological interventions that use behavioral and cognitive behavioral techniques are effective (e.g. control of stimuli, self-management, coping skills). In the same way, van der Meer et al. [24] reviewed the effectiveness of interventions for smoking cessa‐ tion in people with COPD. The five studies included show the effectiveness of psychosocial interventions combined with pharmacological intervention: psychosocial interventions com‐ bined with NRT and a bronchodilator respect to any treatment with a 5-year follow-up (RD = 0.16, RR = 4, 0); psychosocial interventions combined with NRT and placebo with respect to any treatment with a 5-year follow-up (RD = 0.17, RR = 4.19). In addition the results dem‐ onstrated the effectiveness of various combinations of psychosocial and pharmacological in‐ terventions in the follow up at 6 months (RD = 0.07, RR = 1.74). The limitation of this review is that none of the included studies compared psychosocial interventions without pharma‐ cotherapy. It is concluded that a combination of psychosocial and pharmacological interven‐ tions are superior to none apply any treatment. However, few studies have evaluated in the context of clinical trials the effectiveness of psychological interventions in patients with COPD (see table 1).

duct a review of the effectiveness of the treatments to quit smoking in patients with COPD, identifying the main difficulties to maintain abstinence and propose strategies to eliminate

To perform this bibliographic review it was searched in the Cochrane Tobacco Addiction Group, as well as the databases MEDLINE, EMBASE, PsycINFO, PubMed, and SCOPUS from 2000 to 2012. We used the following keywords: smoking cessation, tobacco cessation, hospital, patient, medical setting, and chronic obstructive pulmonary disease. The inclusion criteria used in this review were: a) studies involving interventions for smoking cessation in smokers who have COPD, b) that smokers had 18 or more years; and c) that smokers submit

such obstacles.

234 Respiratory Disease and Infection - A New Insight

**4. Method**

**5. Results**

(6), and

**COPD**

voluntarily to interventions for smoking cessation.

Ten reports were identified and grouped in three categories:

**a.** Studies with an experimental group with a psychological intervention (2)

**c.** Studies in which the intervention was done in hospitalized patients (2).

**b.** Studies with an experimental group in which the treatment consisted of the combina‐ tion of any pharmacological treatment with psychological intervention to quit smoking

**6. Psychological interventions for smoking cessation in patients with**

In a review on the effectiveness of interventions for smoking cessation, Lancaster and Stead [23] point out that psychological interventions that use behavioral and cognitive behavioral techniques are effective (e.g. control of stimuli, self-management, coping skills). In the same way, van der Meer et al. [24] reviewed the effectiveness of interventions for smoking cessa‐ tion in people with COPD. The five studies included show the effectiveness of psychosocial interventions combined with pharmacological intervention: psychosocial interventions com‐ bined with NRT and a bronchodilator respect to any treatment with a 5-year follow-up (RD = 0.16, RR = 4, 0); psychosocial interventions combined with NRT and placebo with respect to any treatment with a 5-year follow-up (RD = 0.17, RR = 4.19). In addition the results dem‐ onstrated the effectiveness of various combinations of psychosocial and pharmacological in‐ terventions in the follow up at 6 months (RD = 0.07, RR = 1.74). The limitation of this review is that none of the included studies compared psychosocial interventions without pharma‐ Hilberink, Jacobs, Bottema of Vries, and Grol [25] conducted in the Netherlands a random‐ ized controlled trial in patients with COPD (392 smokers) to evaluate the type of interven‐ tion on smoking cessation rates, the interventions were a minimal intensity intervention for smoking cessation or the usual care. The intervention was educational and included patient support by health professionals. In the first visit, it was spoken of symptoms, health status and treatment, smoking behavior and motivational state for smoking cessation; also a bro‐ chure and a video designed for people with COPD were given. Based on the stages of change [26], the patients were assigned in three categories: 1) preparers (wanted to quit in the next month), 2) contemplators (wanted to quit in the next 6 months), and 3) precontem‐ plators (not want to quit). According to these categories, smokers with no motivation to quit, just received information about the benefits of quitting (control group). Smokers moti‐ vated to quit received information to increase their self-efficacy from discussing how they could deal with barriers to quitting, and additionally, information on nicotine replacement therapies according to the level of nicotine dependence was given. After six months, the ex‐ perimental group showed more attempts to quit smoking (44.9% vs. 36.5%), and quit smok‐ ing compared with control group (16.0% vs. 8.8%).

In a second study Wilson, Fitzsimons, Bradbury and Stuart [27] conducted a randomized controlled trial to evaluate the effectiveness of interventions to quit smoking based on the brief advise or brief advise accompanied by nurse support, either individually or in group; smoking status was biochemically validated at 2, 3, 6, 9 and 12 months. The sam‐ ple consisted of 91 smokers with COPD from an Ireland hospital. All patients received a brief smoking cessation intervention from the physician (5-10 minutes); afterwards, smok‐ ers were randomly assigned to one of three groups: usual care, individual, or group sup‐ port. The usual care group (n = 35) or control group, received only the brief advise; the intervention groups, individual support (n = 27) or group support (n = 29), underwent five weeks of support after the intervention; the nurses with approximately 6 hours of training carried out the interventions. Individual interventions consisted in providing selfhelp material and information about the personal benefits of quit smoking, discussion about the benefits of nicotine replacement therapy, to encourage setting a date to stop smoking and inform friends and family about the intent. In group interventions, patients were classified according to the phase of change [26] in order that nurses could adapt the intervention to the phase of each patient. In addition, the positive and negative aspects of smoking, particularly on health were discussed, as well as the previous efforts to quit smoking with the intention of to identify those factors that contributed to relapse. In addi‐ tion, role play in risk situations was performed to increase their confidence to quitting smoking. Both interventions consisted of five sessions of 60 minutes, in which nicotine re‐ placement therapy was given upon request. Twelve months after the intervention, no sig‐ nificant differences between the groups were observed. Abstinence in patients was of 6% in control group and of 10% in intervention group, a significant reduction of their addic‐ tion to nicotine was observed in all groups. These data lead to the authors to conclude that patients with COPD were unable to quit smoking, regardless of the type of support they receive. Therefore, the reduction of tobacco consumption may be an alternative goal for these smokers.

(minimum 10 tablets and maximum 40 tablets per day) for over 16 cigarettes; 1 tablet per hour (6 to 30 tablets per day) for 10 to 15 cigarettes and 1 tablet per hour (3 to 10 tablets per day) those who smoked 6 to 9 cigarettes. Abstinence rates were statistically significantly su‐ perior with nicotine sublingual tablets (low support 14% vs. high support 14%) compared to

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

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237

No statistical differences were found between the different intensity of behavioral support. The main findings were sustained abstinence rates with nicotine sublingual tablet compared with placebo in a group of patients with mild, moderate and severe COPD. The authors point out the efficacy of NRT in combination with a smoking cessation program and suggest that NRT should be used for smoking cessation in smokers with COPD, regardless of the

In the second report [33] the objective was to identify the factors detected in the initial as‐ sessment to predict abstinence in COPD patients when two different interventions were ap‐ plied. The patients (N=225) with moderate to severe COPD were assigned to one of two interventions: 1) Minimal Intervention Strategy for Lung Patients (LMIS) of 180 min which consisted of individual counseling and telephone contacts and the use of pharmacological treatment was recommended if patients required or 2) Intensive Intervention [Smoke Stop Therapy (SST)] with 595 min of duration which consisted of group and individual counsel‐ ing, telephone calls and support for the use of bupropion (available for the patients). In a one-year follow-up, the continuous abstinence rates (validated with cotinine in saliva) were 9% for LMIS and 19% for SST [RR = 2.22, 95% CI: 1.06-4.65]. Regarding variables identifica‐ tion to predict abstinence, the SST was no predictor for success; while for LMIS the attitude toward smoking cessation (OR: 11.8, 95% CI: 1.7-8.15, p =.013) and cotinine level (OR: 2.1, 95% CI: 1.08-3.93, p =.028) were significant predictors, 31% the variance in continuous absti‐ nence was explained by these variables (p =.003). It is concluded that LMIS is suitable for COPD patients with a positive attitude to smoking cessation. SST may be an alternative for

Tashkin et al. [19] conducted a study to investigate the effect of bupropion in promoting ab‐ stinence from smoking in patients with COPD. Smokers (N=404) with mild or moderate COPD, who smoked 15 or more cigarettes per day, were assigned randomly to two groups receiving one of two treatments for 12 weeks: intervention group with bupropion (150 mg, twice daily) or placebo control. All patients received smoking cessation counseling by a nurse or doctor; each patient received telephone counseling to quit smoking for 3 days after discharge of the hospital and followed personally in each visit to the hospital. Medication was taking one week before patients attempted to quit. The objective was to obtain complete and continuous abstinence from week 4 until the end of week 7; there was a follow-up at 6 months. The abstinence rates were significantly higher in participants with bupropion com‐ pared with placebo (28% vs. 16%, p = 0.003). The abstinence rate between weeks 4 to 12 (18% vs. 10%) and between weeks 4 to 26 (16% vs. 9%) also were higher in participants receiving bupropion (p <0.05). The authors concluded that bupropion along with the counselling was

placebo (low support 5% vs. high support 6%).

patients without such features.

severity of the disease and the number of cigarettes smoked.

an effective aid to smoke cessation in patients with COPD.

#### **7. Combination of pharmacological and psychological interventions for smoking cessation in patients with COPD**

Pharmacologic therapies have as main function the relief of withdrawal symptoms pro‐ duced by suppressing the consumption of tobacco and consequently help people quit smok‐ ing. Pharmacological therapies are classified in two categories, the nicotinic therapies such as nicotine gum, nicotine transdermal patch and inhaler, and non-nicotinic therapies such as bupropion, nortriptyline and varenicline [28]. A review of clinical trials to assess the effec‐ tiveness of nicotinic therapies to smokers without chronic disease, describes that all these types of treatments increase the probability of abstinence and is higher than placebo [29]. While with non-nicotinic drug therapy in smokers without the diagnosis of a chronic dis‐ ease, varenicline and bupropion are most effective because the rate abstinence is higher than placebo or with any other treatment to stop smoking.

Several clinical trials had evaluated whether the efficacy of drug therapy increases when is combined with psychological intervention in patients with chronic diseases. For exam‐ ple, Molyneux et al. [30] compared NRT with placebo NRT or with no NRT. All subjects received a counseling intervention for smoking cessation. They found that counseling without NRT was equally effective. Another trial compared the effect of incorporating in‐ tensive cognitive-behavioral intervention or minimal counseling intervention combined with NRT [31]. The results showed that smoking cessation did not improve long-term quit rates with NRT interventions. The results are conflicting, some studies show that there is no increase in efficiency and others mention that it is indispensable the inclusion of a psy‐ chological intervention [24]. In the present review, we identified six studies (table 1) com‐ bining a psychological intervention with pharmacological therapy (bupropion, nortriptyline, varenicline or nicotine sublingual tablet).

In the first report [32] examined the effect of nicotine replacement therapy (NRT) in pa‐ tients with COPD. In a double-blind, multicenter, placebo-controlled trial involving 370 patients the efficacy of sublingual tablets or placebo combined with two levels of behavio‐ ral support for smoking cessation in COPD patients who smoked on average 19.6 ciga‐ rettes per day was evaluated, 6 and 12 months after treatment.

Participants were assigned to one of four experimental conditions: 1) nicotine sublingual tablet with low behavioral support, 2) nicotine sublingual tablet with high behavioral sup‐ port, 3) placebo sublingual tablet with low behavioral support or 4) placebo sublingual tab‐ let with high behavioral support. The instructions given by nurses were to use nicotine tablets according to the number of cigarettes smoked per day. One or 2 tablets per hour (minimum 10 tablets and maximum 40 tablets per day) for over 16 cigarettes; 1 tablet per hour (6 to 30 tablets per day) for 10 to 15 cigarettes and 1 tablet per hour (3 to 10 tablets per day) those who smoked 6 to 9 cigarettes. Abstinence rates were statistically significantly su‐ perior with nicotine sublingual tablets (low support 14% vs. high support 14%) compared to placebo (low support 5% vs. high support 6%).

in control group and of 10% in intervention group, a significant reduction of their addic‐ tion to nicotine was observed in all groups. These data lead to the authors to conclude that patients with COPD were unable to quit smoking, regardless of the type of support they receive. Therefore, the reduction of tobacco consumption may be an alternative goal

**7. Combination of pharmacological and psychological interventions for**

Pharmacologic therapies have as main function the relief of withdrawal symptoms pro‐ duced by suppressing the consumption of tobacco and consequently help people quit smok‐ ing. Pharmacological therapies are classified in two categories, the nicotinic therapies such as nicotine gum, nicotine transdermal patch and inhaler, and non-nicotinic therapies such as bupropion, nortriptyline and varenicline [28]. A review of clinical trials to assess the effec‐ tiveness of nicotinic therapies to smokers without chronic disease, describes that all these types of treatments increase the probability of abstinence and is higher than placebo [29]. While with non-nicotinic drug therapy in smokers without the diagnosis of a chronic dis‐ ease, varenicline and bupropion are most effective because the rate abstinence is higher than

Several clinical trials had evaluated whether the efficacy of drug therapy increases when is combined with psychological intervention in patients with chronic diseases. For exam‐ ple, Molyneux et al. [30] compared NRT with placebo NRT or with no NRT. All subjects received a counseling intervention for smoking cessation. They found that counseling without NRT was equally effective. Another trial compared the effect of incorporating in‐ tensive cognitive-behavioral intervention or minimal counseling intervention combined with NRT [31]. The results showed that smoking cessation did not improve long-term quit rates with NRT interventions. The results are conflicting, some studies show that there is no increase in efficiency and others mention that it is indispensable the inclusion of a psy‐ chological intervention [24]. In the present review, we identified six studies (table 1) com‐ bining a psychological intervention with pharmacological therapy (bupropion,

In the first report [32] examined the effect of nicotine replacement therapy (NRT) in pa‐ tients with COPD. In a double-blind, multicenter, placebo-controlled trial involving 370 patients the efficacy of sublingual tablets or placebo combined with two levels of behavio‐ ral support for smoking cessation in COPD patients who smoked on average 19.6 ciga‐

Participants were assigned to one of four experimental conditions: 1) nicotine sublingual tablet with low behavioral support, 2) nicotine sublingual tablet with high behavioral sup‐ port, 3) placebo sublingual tablet with low behavioral support or 4) placebo sublingual tab‐ let with high behavioral support. The instructions given by nurses were to use nicotine tablets according to the number of cigarettes smoked per day. One or 2 tablets per hour

**smoking cessation in patients with COPD**

placebo or with any other treatment to stop smoking.

nortriptyline, varenicline or nicotine sublingual tablet).

rettes per day was evaluated, 6 and 12 months after treatment.

for these smokers.

236 Respiratory Disease and Infection - A New Insight

No statistical differences were found between the different intensity of behavioral support. The main findings were sustained abstinence rates with nicotine sublingual tablet compared with placebo in a group of patients with mild, moderate and severe COPD. The authors point out the efficacy of NRT in combination with a smoking cessation program and suggest that NRT should be used for smoking cessation in smokers with COPD, regardless of the severity of the disease and the number of cigarettes smoked.

In the second report [33] the objective was to identify the factors detected in the initial as‐ sessment to predict abstinence in COPD patients when two different interventions were ap‐ plied. The patients (N=225) with moderate to severe COPD were assigned to one of two interventions: 1) Minimal Intervention Strategy for Lung Patients (LMIS) of 180 min which consisted of individual counseling and telephone contacts and the use of pharmacological treatment was recommended if patients required or 2) Intensive Intervention [Smoke Stop Therapy (SST)] with 595 min of duration which consisted of group and individual counsel‐ ing, telephone calls and support for the use of bupropion (available for the patients). In a one-year follow-up, the continuous abstinence rates (validated with cotinine in saliva) were 9% for LMIS and 19% for SST [RR = 2.22, 95% CI: 1.06-4.65]. Regarding variables identifica‐ tion to predict abstinence, the SST was no predictor for success; while for LMIS the attitude toward smoking cessation (OR: 11.8, 95% CI: 1.7-8.15, p =.013) and cotinine level (OR: 2.1, 95% CI: 1.08-3.93, p =.028) were significant predictors, 31% the variance in continuous absti‐ nence was explained by these variables (p =.003). It is concluded that LMIS is suitable for COPD patients with a positive attitude to smoking cessation. SST may be an alternative for patients without such features.

Tashkin et al. [19] conducted a study to investigate the effect of bupropion in promoting ab‐ stinence from smoking in patients with COPD. Smokers (N=404) with mild or moderate COPD, who smoked 15 or more cigarettes per day, were assigned randomly to two groups receiving one of two treatments for 12 weeks: intervention group with bupropion (150 mg, twice daily) or placebo control. All patients received smoking cessation counseling by a nurse or doctor; each patient received telephone counseling to quit smoking for 3 days after discharge of the hospital and followed personally in each visit to the hospital. Medication was taking one week before patients attempted to quit. The objective was to obtain complete and continuous abstinence from week 4 until the end of week 7; there was a follow-up at 6 months. The abstinence rates were significantly higher in participants with bupropion com‐ pared with placebo (28% vs. 16%, p = 0.003). The abstinence rate between weeks 4 to 12 (18% vs. 10%) and between weeks 4 to 26 (16% vs. 9%) also were higher in participants receiving bupropion (p <0.05). The authors concluded that bupropion along with the counselling was an effective aid to smoke cessation in patients with COPD.

Wagena, Knipschild, Huibers, Wouters and van Schayck [34] explored the efficacy of bu‐ propion and nortriptyline in smokers at risk of COPD versus smokers with COPD. In a randomized placebo-controlled, double blind trial, 255 adults at risk for COPD or with COPD were assigned to one of three groups that received different smoking cessation in‐ tervention: a) bupropion (15 mg twice daily), b) nortriptyline (75 mg once daily) for 12 weeks and c) placebo bupropion. All patients received advice to quit smoking. The main indicator of outcome was prolonged abstinence from smoking from 4 to 26 weeks after beginning the date of withdrawal. The results showed that bupropion (27.9%) and nor‐ triptyline (25%) had high rates of prolonged abstinence at 26 weeks follow-up compared with placebo (14.6%), significant differences between bupropion and placebo were detect‐ ed (p = 0.03), 13.1% [95% CI: -1.2% to 25.1%], and no differences for nortriptyline 10.2% [95% CI: 1.7% to 22.2%). In patients with COPD, bupropion (27.3%) and nortriptyline (21.2%) were equally effective in prolonged abstinence rates (differences with placebo 18.9% [95% CI: 3.6% -34.2%], for bupropion and 12.9% [95% CI: -0.8% to 26.4%] for nor‐ triptyline). In subjects with COPD risk, no statistically significant differences were detect‐ ed compared with placebo in prolonged abstinence rates (bupropion= 28.6% vs. nortriptyline= 32.1% vs. placebo = 22%). The authors conclude that bupropion combined with smoking cessation counselling is an effective treatment for smoking cessation in pa‐ tients with COPD and nortriptyline is a useful alternative.

sults showed that the rate of continuous abstinence from week 9-12 was significantly higher for patients in the varenicline group (42.3%) than for placebo patients (8.8%) (OR: 8.40, 95% CI: 4.99 -14.14, p <.0001). The continuous abstinence rate in patients treated with varenicline remained significantly higher than for those treated with placebo through week 9 to 52 (18.6% vs. 5.6%) (OR: 4.04, 95% CI: 2.13-7.67; p <.0001). Side effects commonly reported by patients in the vareni‐ cline group were nausea, nightmares, respiratory infection, and insomnia. The authors con‐ clude that varenicline was more effective than placebo for smoking cessation in patients with

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

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239

mild and moderate COPD, and showed consistent with that observed in previous trials.

**8. Interventions for smoking cessation in hospitalized patients with**

tion for smoking cessation in patients hospitalized for COPD.

tion group at one year were 52% and 38% at follow-up to three years.

and smoking advice should be improved.

An additional component in treatments for smoking cessation in patients with COPD is hospi‐ talization, which provides an excellent opportunity to help to stop smoking. Given that in this condition the perceived vulnerability of patients increase, as well the receptivity to the messages for the abandonment of smoking. In addition, access to health care allows patients to have direct contact with health professionals who can provide messages or interventions for smoking cessa‐ tion, and smokers may find it easier to quit smoking in an environment smoke-free [37]. Despite this, in practice few hospitals provide this kind of help to their patients. Table 1 shows interven‐

Regarding the above, a study [38] measured in successive discharged patients of the de‐ partment of neumology of a university hospital, the percentage of smoking history and smoking cessation medical advice contained in 100 medical reports. They detected that most of the main diagnoses were smoking related diseases. From all reports, only 48% of patients had history related to tobacco, 14 were smokers and 11 had in the report a writ‐ ten advice to quit smoking. Only 36.7% of smokers received smoking advice. Of the pa‐ tients who had no history, 16 were smokers and had not received advice. In addition, the first hospital admissions were 5.9% more likely to count with clinical history than read‐ missions (77.8% in first admission compared to 35.6% in readmissions). An interesting finding was that physicians who smoked where less likely record the smoking history than non-smoking physicians. The authors conclude that the interview related to tobacco

Sundblad, Larsson and Nathell [39] designed a smoking cessation program in which patients were hospitalized for 11 days to develop their motivation to quit smoking through informa‐ tion, exercise the option to make a nicotine replace therapy, learning coping strategies and per‐ sonal support. The results on smoking were evaluated after 1 and 3 years. The abstinence was compared in the patients with COPD who participated in the smoking cessation program (N = 247) and the ones receiving usual care (N = 231). Abstinence rates obtained in smoking cessa‐

**COPD**

van Schayck, et al. [35] conducted a randomized, double-blind placebo-controlled trial to evaluate the efficacy of bupropion and nortriptyline in combination with behavioral cogni‐ tive intervention in smokers with COPD risk in Netherlands. Smokers (n=255) with COPD risk between 30-70 years, were counseled to quit smoking (three sessions of 20 minutes and 6 calls of 5 minutes). They were randomly allocated to one of three groups: bupropion, nor‐ triptyline or placebo for 12 weeks. The results showed prolonged abstinence rate (defined by report of participants have not smoked in week 2 to 52 after the beginning of abstinence) for bupropion (20.9%), nortriptyline (20%) and placebo (13.5%). Significant differences were ob‐ tained between bupropion and placebo [relative risk (RR) = 1.6, 95%, confidence interval (CI) 0.8-3.0], in contrast, the differences between nortriptyline and placebo were not signifi‐ cant. The severity of the airway obstruction did not influence the significance of abstinence. The social costs were € 1368 with bupropion, € 1906 with nortriptyline and € 1212 with pla‐ cebo. The authors concluded that bupropion and nortriptyline are equally effective, but bu‐ propion was more cost effective compared with placebo and nortriptyline. One possible reason for the high cost of nortriptyline may be that participants who used nortriptyline ex‐ perienced more side effects from treatment.

Finally, one study evaluated varenicline combined with psychological intervention. In 27 cen‐ ters a randomized, controlled, double blind, trial in patients (N=504) with mild to moderate COPD the efficacy of varenicline was evaluated [36]. The intervention consisted in 12 weeks of varenicline with 40 weeks follow-up. All participants received an educational booklet on smoking cessation information and brief counseling sessions (10 minutes) at each telephone call or visit at the clinic. The primary endpoint to confirm the continuous abstinence rate with the carbon monoxide level was week 9 to 12. The secondary endpoint was week 9 to 52. The re‐ sults showed that the rate of continuous abstinence from week 9-12 was significantly higher for patients in the varenicline group (42.3%) than for placebo patients (8.8%) (OR: 8.40, 95% CI: 4.99 -14.14, p <.0001). The continuous abstinence rate in patients treated with varenicline remained significantly higher than for those treated with placebo through week 9 to 52 (18.6% vs. 5.6%) (OR: 4.04, 95% CI: 2.13-7.67; p <.0001). Side effects commonly reported by patients in the vareni‐ cline group were nausea, nightmares, respiratory infection, and insomnia. The authors con‐ clude that varenicline was more effective than placebo for smoking cessation in patients with mild and moderate COPD, and showed consistent with that observed in previous trials.

Wagena, Knipschild, Huibers, Wouters and van Schayck [34] explored the efficacy of bu‐ propion and nortriptyline in smokers at risk of COPD versus smokers with COPD. In a randomized placebo-controlled, double blind trial, 255 adults at risk for COPD or with COPD were assigned to one of three groups that received different smoking cessation in‐ tervention: a) bupropion (15 mg twice daily), b) nortriptyline (75 mg once daily) for 12 weeks and c) placebo bupropion. All patients received advice to quit smoking. The main indicator of outcome was prolonged abstinence from smoking from 4 to 26 weeks after beginning the date of withdrawal. The results showed that bupropion (27.9%) and nor‐ triptyline (25%) had high rates of prolonged abstinence at 26 weeks follow-up compared with placebo (14.6%), significant differences between bupropion and placebo were detect‐ ed (p = 0.03), 13.1% [95% CI: -1.2% to 25.1%], and no differences for nortriptyline 10.2% [95% CI: 1.7% to 22.2%). In patients with COPD, bupropion (27.3%) and nortriptyline (21.2%) were equally effective in prolonged abstinence rates (differences with placebo 18.9% [95% CI: 3.6% -34.2%], for bupropion and 12.9% [95% CI: -0.8% to 26.4%] for nor‐ triptyline). In subjects with COPD risk, no statistically significant differences were detect‐ ed compared with placebo in prolonged abstinence rates (bupropion= 28.6% vs. nortriptyline= 32.1% vs. placebo = 22%). The authors conclude that bupropion combined with smoking cessation counselling is an effective treatment for smoking cessation in pa‐

van Schayck, et al. [35] conducted a randomized, double-blind placebo-controlled trial to evaluate the efficacy of bupropion and nortriptyline in combination with behavioral cogni‐ tive intervention in smokers with COPD risk in Netherlands. Smokers (n=255) with COPD risk between 30-70 years, were counseled to quit smoking (three sessions of 20 minutes and 6 calls of 5 minutes). They were randomly allocated to one of three groups: bupropion, nor‐ triptyline or placebo for 12 weeks. The results showed prolonged abstinence rate (defined by report of participants have not smoked in week 2 to 52 after the beginning of abstinence) for bupropion (20.9%), nortriptyline (20%) and placebo (13.5%). Significant differences were ob‐ tained between bupropion and placebo [relative risk (RR) = 1.6, 95%, confidence interval (CI) 0.8-3.0], in contrast, the differences between nortriptyline and placebo were not signifi‐ cant. The severity of the airway obstruction did not influence the significance of abstinence. The social costs were € 1368 with bupropion, € 1906 with nortriptyline and € 1212 with pla‐ cebo. The authors concluded that bupropion and nortriptyline are equally effective, but bu‐ propion was more cost effective compared with placebo and nortriptyline. One possible reason for the high cost of nortriptyline may be that participants who used nortriptyline ex‐

Finally, one study evaluated varenicline combined with psychological intervention. In 27 cen‐ ters a randomized, controlled, double blind, trial in patients (N=504) with mild to moderate COPD the efficacy of varenicline was evaluated [36]. The intervention consisted in 12 weeks of varenicline with 40 weeks follow-up. All participants received an educational booklet on smoking cessation information and brief counseling sessions (10 minutes) at each telephone call or visit at the clinic. The primary endpoint to confirm the continuous abstinence rate with the carbon monoxide level was week 9 to 12. The secondary endpoint was week 9 to 52. The re‐

tients with COPD and nortriptyline is a useful alternative.

238 Respiratory Disease and Infection - A New Insight

perienced more side effects from treatment.

### **8. Interventions for smoking cessation in hospitalized patients with COPD**

An additional component in treatments for smoking cessation in patients with COPD is hospi‐ talization, which provides an excellent opportunity to help to stop smoking. Given that in this condition the perceived vulnerability of patients increase, as well the receptivity to the messages for the abandonment of smoking. In addition, access to health care allows patients to have direct contact with health professionals who can provide messages or interventions for smoking cessa‐ tion, and smokers may find it easier to quit smoking in an environment smoke-free [37]. Despite this, in practice few hospitals provide this kind of help to their patients. Table 1 shows interven‐ tion for smoking cessation in patients hospitalized for COPD.

Regarding the above, a study [38] measured in successive discharged patients of the de‐ partment of neumology of a university hospital, the percentage of smoking history and smoking cessation medical advice contained in 100 medical reports. They detected that most of the main diagnoses were smoking related diseases. From all reports, only 48% of patients had history related to tobacco, 14 were smokers and 11 had in the report a writ‐ ten advice to quit smoking. Only 36.7% of smokers received smoking advice. Of the pa‐ tients who had no history, 16 were smokers and had not received advice. In addition, the first hospital admissions were 5.9% more likely to count with clinical history than read‐ missions (77.8% in first admission compared to 35.6% in readmissions). An interesting finding was that physicians who smoked where less likely record the smoking history than non-smoking physicians. The authors conclude that the interview related to tobacco and smoking advice should be improved.

Sundblad, Larsson and Nathell [39] designed a smoking cessation program in which patients were hospitalized for 11 days to develop their motivation to quit smoking through informa‐ tion, exercise the option to make a nicotine replace therapy, learning coping strategies and per‐ sonal support. The results on smoking were evaluated after 1 and 3 years. The abstinence was compared in the patients with COPD who participated in the smoking cessation program (N = 247) and the ones receiving usual care (N = 231). Abstinence rates obtained in smoking cessa‐ tion group at one year were 52% and 38% at follow-up to three years.


van Schayck et al.(2009) Netherlands

Tashkin et al. (2011)

Sundblad, Larsson y Nathell (2008)

Borglykke, Pisinger, Jørgensen & Ibsen (2008) To evaluate the efficacy of bupropion and nortriptyline compared with placebo in smokers with COPD risk

To evaluate the efficacy of varenicline in patients with

To evaluate a smoking cessation program

To evaluate the effect of a smoking cessation group in hospitalized patients with

COPD

COPD

255 COPD risk IG 1: Bupropion-12 weeks

counseling

counseling

478 COPD patients IG: hospitalization for 11 days were

CG: usual care

when necessary CG: usual care

**Table 1.** Interventions for smoking cessation in patients with chronic obstructive pulmonary disease (COPD).

tion showed positive impact in hospitalization in survival and reducing phlegm.

Also, Borglykke, Pisinger, Jorgensen and Ibsen [40] evaluated the effect of a smoking cessa‐ tion group in hospitalized patients with COPD. Patients were assigned to a control group (n = 102) or an intervention group (n = 121). In the first two sessions of the intervention, the group received information on smoking cessation and had to set a date to start the with‐ drawal that was supported NRT when necessary. At 1 year follow-up 36 (30%) patients in the intervention group remained abstinent compared with 13 (13%) patients in the control group [odds ratio (95% confidence interval): 2.83 (1.40 -5.74). A significant difference was observed between the intervention group and control group with respect to the self report‐ ing of the phlegm production however no significant improvement was observed in terms of survival benefit at 3 years follow up period (intervention group 86% vs. control group 85%).Therefore, the authors conclude that this study showed that a group intervention for chronic patients made it possible to get high withdrawal rates. Furthermore, this interven‐

**INTERVENTIONS FOR SMOKING CESSATION IN PATIENTS HOSPITALIZED FOR COPD Author Objetive Patients Interventions Results Validation**

504 patients with mild and moderate

223 hospitalized patients with COPD

IG: Intervention Group; CG: Control Group; NRT: Nicotine Replace Therapy

COPD

IG 2: nortriptyline -12 weeks CG: Placebo 12 weeks

IG: Varenicline + educational booklet on smoking cessation information and brief sessions (10 minutes) telephone

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

CG: placebo + educational booklet on smoking cessation information and brief sessions (10 minutes) telephone

used to develop the motivation to quit smoking through information, exercise the option to take a nicotine replacement therapy, learning coping strategies and given personal support

IG: Participants attended group intervention sessions (two hours weekly for five weeks) NRT was used Prolonged Abstinence: IG1:20.9%, IG2: 20%, G.C:13.5% Significant differences between bupropion and placebo [RR=1.6, Cotinine in urine Self-reports 241

http://dx.doi.org/10.5772/53265

Self-reports Carbon monoxide in expired air

Self-reports Carbon monoxide in expired air

Self-reports Carbon monoxide in expired air

95% IC=0.8-3.0]

IG:42.3% CG:8.8% p<.0001

Continuous abstinence

Abstinence rates at 12 months IG: 52% vs. CG: 7% Abstinence rates at 3 years IG: 38% vs. CG: 10%

Abstinence rates at 12 months

IG:30% CG:13%

#### Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary... http://dx.doi.org/10.5772/53265 241


**PSYCHOLOGICAL INTERVENTIONS Author Objetive Patients Interventions Results Validation**

> help material) CG: usual care (counsel)

weeks).

support

support

IG: brief intervention (counsel+ self-

IG 1: individual support (briefings, selfhelp materials and telephone support 5

IG 2: group support by stage of change (group information sessions + 5 followup sessions after discharge). IG 3: usual care (brief counsel).

IG1: sublingual tablet + low behavioral

IG2: sublingual tablet + high behavioral

CG1: sublingual tablet + placebo with

CG2: placebo sublingual tablet + high

CG: moderately intensive intervention: individual counseling + telephone contacts Total time: 180 minutes. IG: intensive intervention , group and individual counseling and support +phone calls + bupropion. Total time:

IG: bupropion (150 mg twice daily) + counseling for smoking cessation CG: placebo for 12 weeks + counsel

IG1:bupropion (15 mg twice daily) IG2: nortriptyline (75 mg once/day X

low behavioral support

behavioral support

595 minutes.

12 weeks)

CG: bupropion placebo

**PHARMACOLOGICAL TREATMENT COMBINED WITH A PSYCHOLOGICAL INTERVENTION FOR SMOKING CESSATION Author Objetive Patients Interventions Results Validation**

Attempts to stop the smoking at

Self-reports

Self-reports Carbon monoxide in expired air Cotinine in saliva

Self-reports Carbon monoxide in expired air

Cotinine in saliva Selfreports

Self-reports Carbon monoxide in expired air

Self-reports Carbon monoxide in expired air

IG: 44.9% vs. CG: 36.5%. Abstinence at 6 months: IG: 16.0% vs. CG: 8.8%.

Abstinence at 12 months: IG 2: 10% vs CG: 6%. There were no significant differences between groups (p =

Prolonged abstinence IG1: 14% vs.IG2: 14% CG1: 5% vs.CG2: 6% 12 months point prevalence IG1: 17% vs.IG2: 18% CG1: 6% vs.CG2: 13%

Abstinence rates CG:9% IG:19%

Abstinence at 6 months: IG:28% vs. CG:16%. Abstinence between weeks 4

Prolonged Abstinence 26 weeks: IG1:27.9% vs. IG2:25% vs. CG:14.6 %(p=0.03)

and 12: IG:18% vs. CG:10%. Abstinence between weeks 4

and 26: IG:16% vs. CG:9%.

6 months:

0.7).

Hilberink et al. (2005)

Wilson et al. (2006)

Tonnesen, Mikkelsen & Bremann (2006)

Christenhusz , Pieterse, Seydel & van der Palen (2007)

Tashkin et al. (2001)

Wagena et al.(2005)

To compare smoke cessation rates depending on the type

240 Respiratory Disease and Infection - A New Insight

Evaluate the effectiveness of interventions for smoking cessation based on brief counsel alone or with support from nurses (individual or group).

To identify predictors of

To evaluate the efficacy of sublingual tablets or placebo combined with two levels of behavioral support for smoking cessation in COPD patients after 6 and 12 months.

To investigate the effect of bupropion promoting abstinence from smoking in patients with COPD

Explore the efficacy of bupropion and nortriptyline in smokers with COPD risk compared with smokers with COPD.

abstinence

392 smokers with COPD

91 smokers with COPD IG 1=27 IG 2= 29 IG 3= 35

370 patients with COPD who smoke on average 19.6 cigarettes per day

225 patients with moderate to severe

404 smokers with mild or moderate COPD who smoked 15 or more cigarettes / day.

255 adults at risk for COPD or with COPD

COPD

of intervention.

**Table 1.** Interventions for smoking cessation in patients with chronic obstructive pulmonary disease (COPD).

Also, Borglykke, Pisinger, Jorgensen and Ibsen [40] evaluated the effect of a smoking cessa‐ tion group in hospitalized patients with COPD. Patients were assigned to a control group (n = 102) or an intervention group (n = 121). In the first two sessions of the intervention, the group received information on smoking cessation and had to set a date to start the with‐ drawal that was supported NRT when necessary. At 1 year follow-up 36 (30%) patients in the intervention group remained abstinent compared with 13 (13%) patients in the control group [odds ratio (95% confidence interval): 2.83 (1.40 -5.74). A significant difference was observed between the intervention group and control group with respect to the self report‐ ing of the phlegm production however no significant improvement was observed in terms of survival benefit at 3 years follow up period (intervention group 86% vs. control group 85%).Therefore, the authors conclude that this study showed that a group intervention for chronic patients made it possible to get high withdrawal rates. Furthermore, this interven‐ tion showed positive impact in hospitalization in survival and reducing phlegm.

### **9. Training of professionals who carry out interventions for smoking cessation**

training [41, 43-44]. Moreover, according to a survey, the majorities of patients are not of‐ fered help to quit smoking or are advised to make a follow-up with the physician or con‐

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

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243

In addition, in the available reports, the interventions are very diverse both in relation to their content and in relation to their intensity. There are also differences in the professionals that conduct the interventions: physicians, nurses, psychologists, counselors or any other health personnel; this makes very difficult to compare results of different interventions. An important aspect to consider when analyzing the data obtained in present review is the dif‐ ference in health staff that carried out the smoking cessation interventions, since their level of training was not considered; therefore, this could influence the results and the effective‐ ness of interventions. In some studies, nurses with little training to perform this task carried out the interventions [27]. It would be interesting to compare the results obtained in inter‐ vention carried by different professionals with no specialized training with those obtained

In the studies reviewed, depending on the research involved, in some cases to calculate rates of abstinence only self-reports were used [e.g. 25], while in others to corroborate self-report‐ ed abstinence different biochemical test were used. This is another important aspect to con‐

Another problem that limits replication is that many studies do not describe the procedure followed in the psychological interventions, which could be central in the analysis of the re‐ sults. That is, in most reports only the use of behavioral cognitive interventions is reported, but the techniques are not explained; therefore it is indispensable to specify and describe all

A different area identified is the lack of assessment of psychological factors associated with relapse in the evaluation of different treatments, only side effects are evaluated with respect to the use of drug treatments and only evaluates both the level of depression with the "Beck Depression Inventory" and the level of motivation. Some reports suggest that depression and anxiety are present in patients with COPD, but do not mention if it is consequence of the disease or for quitting. It is known that lack of social support, depression, anxiety, anxi‐ ety sensitivity, negative affect, and deficiency of coping skills are factors associated with the ineffectiveness of interventions in smokers without the diagnosis of a disease, but little is known about the factors associated with relapse in the context of smoking cessation inter‐

On the other hand, in terms of difficulties for smoking cessation in patients with COPD the

**•** Many patients with COPD continue to smoke after diagnosis and those who manage to quit smoking have a high rate of relapse, for that reason many patients do not try quit

by professionals who specializes in treating addictive behaviors.

the techniques used for patients who want to quit smoking and COPD.

sider when analyze and interpreting present findings.

ventions in patients with COPD.

review allowed to identify some factors:

again because their expectative is low.

tact with a trained professional [43].

Ballbé et al. [41] assessed if the lack of promotion on the cessation of smoking in hospitals is due to deficits in professional training. In this study, knowledge, attitudes and action re‐ garding the smoking behavior of 66 health professionals before and after training on brief intervention was evaluated. The performance of these professionals with 170 patients was compared before and another 170 training (patient's report). It was found was that the inter‐ vention training for smoking cessation increases knowledge of psychological skills by 23.3% and 27.1% of pharmacological resources. However no changes were observed with respect to the question of whether to smoke (30.8% before vs. 38.2% after training), to records the smoking status of patient medical history (73.4% vs. 65.9%), whether the patient want to quit (25% vs. 12.5%) or with respect to anti-smoking advice (21.9 % vs. 20.8%). This study shows that health care activity in hospitals tends to focus on treating specific diseases for which pa‐ tients attend, and leave behind preventive interventions, even when the professionals have the training.

In a same line, Efraimsson, Fossum, Ehrenberg, Larson and Klang [42] tried to assess wheth‐ er with a four-day training in motivational interviewing, a group of nurses will assume the communication style of motivational interventions (MI) for smoking cessation in primary care conducted with smokers with COPD. The nurses in their practice did not take the con‐ tents of the training they have received, which indicates that a course in motivational inter‐ viewing a few days was insufficient. It is concluded that training in communication methods should be integrated into the nursing curriculum, since the management of MI is a complex skill that requires a great workout.

In addition, sometimes personnel not sufficiently trained carry out the interventions. For ex‐ ample, Wilson et al. [27] obtained no differences between the interventions applied. The au‐ thors attribute the lack of success to patients and we must not forget that nurses who had received only 6 hours of training applied such interventions. It would be interesting to eval‐ uate the results when intervention is performed by a psychologists with sufficient training in smoking cessation.

#### **10. Discussion**

The aim of this study was to conduct a review of the effectiveness of the treatments to quit smoking in patients with COPD, identifying the main difficulties to maintain absti‐ nence and to propose strategies to eliminate such obstacles. As a result of the revision, we can say that although it is known how smoking affects COPD, the research attempting to demonstrate the benefits of quitting smoking on health in smokers diagnosed with COPD is insufficient. In addition, smoking history and the advice or counsel is not always a pri‐ ority in hospitals. A large percentage of health professionals do not ask to patients if they smoke, therefore do not advise to quit, even though many of them have received formal training [41, 43-44]. Moreover, according to a survey, the majorities of patients are not of‐ fered help to quit smoking or are advised to make a follow-up with the physician or con‐ tact with a trained professional [43].

**9. Training of professionals who carry out interventions for smoking**

Ballbé et al. [41] assessed if the lack of promotion on the cessation of smoking in hospitals is due to deficits in professional training. In this study, knowledge, attitudes and action re‐ garding the smoking behavior of 66 health professionals before and after training on brief intervention was evaluated. The performance of these professionals with 170 patients was compared before and another 170 training (patient's report). It was found was that the inter‐ vention training for smoking cessation increases knowledge of psychological skills by 23.3% and 27.1% of pharmacological resources. However no changes were observed with respect to the question of whether to smoke (30.8% before vs. 38.2% after training), to records the smoking status of patient medical history (73.4% vs. 65.9%), whether the patient want to quit (25% vs. 12.5%) or with respect to anti-smoking advice (21.9 % vs. 20.8%). This study shows that health care activity in hospitals tends to focus on treating specific diseases for which pa‐ tients attend, and leave behind preventive interventions, even when the professionals have

In a same line, Efraimsson, Fossum, Ehrenberg, Larson and Klang [42] tried to assess wheth‐ er with a four-day training in motivational interviewing, a group of nurses will assume the communication style of motivational interventions (MI) for smoking cessation in primary care conducted with smokers with COPD. The nurses in their practice did not take the con‐ tents of the training they have received, which indicates that a course in motivational inter‐ viewing a few days was insufficient. It is concluded that training in communication methods should be integrated into the nursing curriculum, since the management of MI is a

In addition, sometimes personnel not sufficiently trained carry out the interventions. For ex‐ ample, Wilson et al. [27] obtained no differences between the interventions applied. The au‐ thors attribute the lack of success to patients and we must not forget that nurses who had received only 6 hours of training applied such interventions. It would be interesting to eval‐ uate the results when intervention is performed by a psychologists with sufficient training

The aim of this study was to conduct a review of the effectiveness of the treatments to quit smoking in patients with COPD, identifying the main difficulties to maintain absti‐ nence and to propose strategies to eliminate such obstacles. As a result of the revision, we can say that although it is known how smoking affects COPD, the research attempting to demonstrate the benefits of quitting smoking on health in smokers diagnosed with COPD is insufficient. In addition, smoking history and the advice or counsel is not always a pri‐ ority in hospitals. A large percentage of health professionals do not ask to patients if they smoke, therefore do not advise to quit, even though many of them have received formal

**cessation**

242 Respiratory Disease and Infection - A New Insight

the training.

in smoking cessation.

**10. Discussion**

complex skill that requires a great workout.

In addition, in the available reports, the interventions are very diverse both in relation to their content and in relation to their intensity. There are also differences in the professionals that conduct the interventions: physicians, nurses, psychologists, counselors or any other health personnel; this makes very difficult to compare results of different interventions. An important aspect to consider when analyzing the data obtained in present review is the dif‐ ference in health staff that carried out the smoking cessation interventions, since their level of training was not considered; therefore, this could influence the results and the effective‐ ness of interventions. In some studies, nurses with little training to perform this task carried out the interventions [27]. It would be interesting to compare the results obtained in inter‐ vention carried by different professionals with no specialized training with those obtained by professionals who specializes in treating addictive behaviors.

In the studies reviewed, depending on the research involved, in some cases to calculate rates of abstinence only self-reports were used [e.g. 25], while in others to corroborate self-report‐ ed abstinence different biochemical test were used. This is another important aspect to con‐ sider when analyze and interpreting present findings.

Another problem that limits replication is that many studies do not describe the procedure followed in the psychological interventions, which could be central in the analysis of the re‐ sults. That is, in most reports only the use of behavioral cognitive interventions is reported, but the techniques are not explained; therefore it is indispensable to specify and describe all the techniques used for patients who want to quit smoking and COPD.

A different area identified is the lack of assessment of psychological factors associated with relapse in the evaluation of different treatments, only side effects are evaluated with respect to the use of drug treatments and only evaluates both the level of depression with the "Beck Depression Inventory" and the level of motivation. Some reports suggest that depression and anxiety are present in patients with COPD, but do not mention if it is consequence of the disease or for quitting. It is known that lack of social support, depression, anxiety, anxi‐ ety sensitivity, negative affect, and deficiency of coping skills are factors associated with the ineffectiveness of interventions in smokers without the diagnosis of a disease, but little is known about the factors associated with relapse in the context of smoking cessation inter‐ ventions in patients with COPD.

On the other hand, in terms of difficulties for smoking cessation in patients with COPD the review allowed to identify some factors:

**•** Many patients with COPD continue to smoke after diagnosis and those who manage to quit smoking have a high rate of relapse, for that reason many patients do not try quit again because their expectative is low.

**•** In general, smokers with COPD have characteristics that make difficult to quit, for exam‐ ple the level of dependency is severe, the number of cigarettes is high [45]; they are older and therefore have smoked for many years resulting in high dependency [46-47].

propose alternatives to solve the problem make decisions and implement solutions to assess the effectiveness of the alternative. For patients with COPD the strategies can be applied not only to the problems associated with quitting smoking, but also to emerging situations by

Psychological Approaches to Increase Tobacco Abstinence in Patients with Chronic Obstructive Pulmonary...

http://dx.doi.org/10.5772/53265

245

Functional analysis of problem behavior consists in the identification of the context of occur‐ rence of the inadequate behavior and the consequences that maintain it. In treatments for smoking cessation implies the clear identification of the situation that triggers the consump‐ tion of cigarettes, and the positive and negative consequences of consumption, in order to

Self-monitoring is a technique in which the patient is asked to record the duration, frequen‐ cy and severity of the problem behavior. In the context of smoking cessation treatment, par‐ ticularly in patients with COPD, it may ask the patient to record each cigarette consumed, the place of consumption, how it feels when smoke each cigarette, and to register the pres‐

The proposal of a individualized plan for quitting would consist of two phases, one evalua‐ tion phase and the intervention phase that will include the techniques mentioned. In the evaluation phase, the goal would be to get specific information of psychological factors re‐ lated with the consumption of cigarettes by the patient with COPD, to consider the individ‐

Initially, it would be necessary to identify the stage of readiness to change [26], the pattern of consumption of cigarettes (daily average consumption per day, monthly consumption), identified the factors causing the consumption (moods, places, people or their combination) and the level of anxiety, depression, negative affection, social support, the level of depend‐

From the results of the evaluation would be conducted a personalized feedback and deci‐ sional balance would be applied using the techniques of motivational interviewing (empa‐

Based on the decisional balance the person must choose one of two strategies for reaching abstinence: 1) gradual reduction of the nicotine and tar consisting in the decline of 30 % of

To initiate strategies to abstinence from cigarettes consumption, start a record of a functional analysis of the behavior of smoking. This analysis include the negative effects of abstinence (nervousness, irritability, anxiety, depression, hunger, trouble sleeping) and the positive ef‐ fects of abstinence, i.e. the registration of the reduction of the problems of the COPD from

Also, teach patients coping strategies for anxiety, depression, negative affect, and problem solving strategies to apply during periods in which the patient is still in situations or in the

To summarize, the revisions point out that in COPD the smoking cessation programs should be intense, sustained over time and adapted to each patient individually. It also emphasizes

the presence of the disease, such as social, physical and economic problems.

plan and apply strategies that lead to abstinence from cigarettes [57].

thy, reflective listening, cognitive dissonance, without confrontation).

the initial consumption pattern every week until it reaches 0 %, or 2) abstinence.

the change in the pattern of consumption (phlegm, breathing problems, cough).

presence of factors (moods or people) that cause the consumption of cigarettes.

ence and severity of respiratory symptoms.

ency and the coping strategies of the patient.

ualized treatment.


#### **11. Conclusions**

From present review about the interventions for smoking cessation and the identification of barriers to achieve and maintain abstinence, we suggest the inclusion of different techniques to overcome these barriers, since providing information about harm caused by smoking is not enough. It is imperative that patients with COPD to realize how smoking is decreasing their quality of life and deteriorating their health.

The motivational interviewing techniques [52-53] could be an effective method to support smoke cessation [42], but require significant training [54]. It is suggested the inclusion of motivational interviewing in the treatment for smoking cessation in smokers with COPD and personalized feedback with the use of measures of spirometry, Fletcher curve and to ex‐ ploit the adverse events related to COPD to promote the decision of quitting [48]. En el pa‐ tient with COPD the motivation for quitting increases if patients perceive that the respiratory symptoms are due to use of tobacco [55].

Problem-Solving Therapy helps patients to effectively deal with diverse problems from dif‐ ferent learning strategies [56]. The objective of this therapy is to clearly define the problem, propose alternatives to solve the problem make decisions and implement solutions to assess the effectiveness of the alternative. For patients with COPD the strategies can be applied not only to the problems associated with quitting smoking, but also to emerging situations by the presence of the disease, such as social, physical and economic problems.

**•** In general, smokers with COPD have characteristics that make difficult to quit, for exam‐ ple the level of dependency is severe, the number of cigarettes is high [45]; they are older

**•** A main barrier to quit smoking is the motivation. For example, in a study it was shown that patients with COPD stop smoking according the motivational state (precontempla‐ tion, contemplation, preparation, action), smokers in precontemplation associated signifi‐ cantly fewer advantages to quit smoking compared with smokers in contemplation or preparation stages. Smokers in preparation had significantly higher self-efficacy expecta‐ tions about quitting than other smokers. Patients in preparation for quitting complained more about the symptoms associated with COPD. Smokers in contemplation and prepa‐ ration to quit developed more plans to try to switch to action to stop smoking [48]. At this point, it is proposed to design an intervention for smokers with COPD motivated to quit

**•** The comorbidity with anxiety and depression is very high, particularly in women [49-50]. This difficulties to achieve the abstinence and justifies the use of psychological treatments

**•** Another factor that makes difficult to stop smoking is weight gain, particularly in women [45]. When patients stop smoking and gain weight, symptoms related with COPD are ex‐

**•** Social support between the moments the patients are diagnosed COPD and when decide to quit, is other factor. Some authors suggest that social support should be from a family member or someone close to the patient, instead of for example, another patient with

From present review about the interventions for smoking cessation and the identification of barriers to achieve and maintain abstinence, we suggest the inclusion of different techniques to overcome these barriers, since providing information about harm caused by smoking is not enough. It is imperative that patients with COPD to realize how smoking is decreasing

The motivational interviewing techniques [52-53] could be an effective method to support smoke cessation [42], but require significant training [54]. It is suggested the inclusion of motivational interviewing in the treatment for smoking cessation in smokers with COPD and personalized feedback with the use of measures of spirometry, Fletcher curve and to ex‐ ploit the adverse events related to COPD to promote the decision of quitting [48]. En el pa‐ tient with COPD the motivation for quitting increases if patients perceive that the

Problem-Solving Therapy helps patients to effectively deal with diverse problems from dif‐ ferent learning strategies [56]. The objective of this therapy is to clearly define the problem,

and therefore have smoked for many years resulting in high dependency [46-47].

and other unmotivated.

244 Respiratory Disease and Infection - A New Insight

acerbated and causes relapses.

their quality of life and deteriorating their health.

respiratory symptoms are due to use of tobacco [55].

COPD [46,51].

**11. Conclusions**

to improve outcomes of smoking cessation treatments.

Functional analysis of problem behavior consists in the identification of the context of occur‐ rence of the inadequate behavior and the consequences that maintain it. In treatments for smoking cessation implies the clear identification of the situation that triggers the consump‐ tion of cigarettes, and the positive and negative consequences of consumption, in order to plan and apply strategies that lead to abstinence from cigarettes [57].

Self-monitoring is a technique in which the patient is asked to record the duration, frequen‐ cy and severity of the problem behavior. In the context of smoking cessation treatment, par‐ ticularly in patients with COPD, it may ask the patient to record each cigarette consumed, the place of consumption, how it feels when smoke each cigarette, and to register the pres‐ ence and severity of respiratory symptoms.

The proposal of a individualized plan for quitting would consist of two phases, one evalua‐ tion phase and the intervention phase that will include the techniques mentioned. In the evaluation phase, the goal would be to get specific information of psychological factors re‐ lated with the consumption of cigarettes by the patient with COPD, to consider the individ‐ ualized treatment.

Initially, it would be necessary to identify the stage of readiness to change [26], the pattern of consumption of cigarettes (daily average consumption per day, monthly consumption), identified the factors causing the consumption (moods, places, people or their combination) and the level of anxiety, depression, negative affection, social support, the level of depend‐ ency and the coping strategies of the patient.

From the results of the evaluation would be conducted a personalized feedback and deci‐ sional balance would be applied using the techniques of motivational interviewing (empa‐ thy, reflective listening, cognitive dissonance, without confrontation).

Based on the decisional balance the person must choose one of two strategies for reaching abstinence: 1) gradual reduction of the nicotine and tar consisting in the decline of 30 % of the initial consumption pattern every week until it reaches 0 %, or 2) abstinence.

To initiate strategies to abstinence from cigarettes consumption, start a record of a functional analysis of the behavior of smoking. This analysis include the negative effects of abstinence (nervousness, irritability, anxiety, depression, hunger, trouble sleeping) and the positive ef‐ fects of abstinence, i.e. the registration of the reduction of the problems of the COPD from the change in the pattern of consumption (phlegm, breathing problems, cough).

Also, teach patients coping strategies for anxiety, depression, negative affect, and problem solving strategies to apply during periods in which the patient is still in situations or in the presence of factors (moods or people) that cause the consumption of cigarettes.

To summarize, the revisions point out that in COPD the smoking cessation programs should be intense, sustained over time and adapted to each patient individually. It also emphasizes the need of combining pharmacological and psychological treatments, particularly behavio‐ ral focused to the relapse prevention and stress management [58,59]. What is clear is that smoking intervention should be considered as a substantial part of the treatment of COPD. The psychological techniques mentioned previously, could help to eliminate barriers and provide personalized feedback to increase abstinence rates in patients with COPD.

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http://dx.doi.org/10.5772/53265

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#### **Author details**

Jennifer Lira-Mandujano1 , M. Carmen Míguez-Varela2 and Sara E. Cruz-Morales3

1 Facultad de Psicología, Universidad Michoacana de San Nicolás de Hidalgo, México

2 Facultad de Psicología, Universidad de Santiago de Compostela, Spain

3 Psicofarmacología, UNAM-FES-Iztacala, México

#### **References**


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the need of combining pharmacological and psychological treatments, particularly behavio‐ ral focused to the relapse prevention and stress management [58,59]. What is clear is that smoking intervention should be considered as a substantial part of the treatment of COPD. The psychological techniques mentioned previously, could help to eliminate barriers and

and Sara E. Cruz-Morales3

provide personalized feedback to increase abstinence rates in patients with COPD.

, M. Carmen Míguez-Varela2

2 Facultad de Psicología, Universidad de Santiago de Compostela, Spain

3 Psicofarmacología, UNAM-FES-Iztacala, México

www.goldcopd.org (accessed 22 June 2012).

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1 Facultad de Psicología, Universidad Michoacana de San Nicolás de Hidalgo, México

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**Author details**

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Jennifer Lira-Mandujano1

246 Respiratory Disease and Infection - A New Insight


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### *Edited by Bassam H. Mahboub*

Medicine is an ever-changing science. Every day we are encountered with the new developments and knowledge in the pathogenesis, mechanism of disease, newer diagnostic modalities, treatment options and new challenges in the management of the various diseases. The same holds true for respiratory diseases with the emergence of new respiratory pathogens having significant impact on the respiratory system. Respiratory Diseases are an important contributor to the morbidity and mortality of mankind since antiquity and its prevalence is on rise in with new disease are being recognized, however little importance has been given to the respiratory disease due to low level of awareness in physicians and general public. This book has been designed to deliver the detailed knowledge about the various respiratory infections including viral, bacterial, and helminthic infections.

Respiratory Disease and Infection - A New Insight

Respiratory Disease

and Infection

A New Insight

*Edited by Bassam H. Mahboub*

Photo by rudchenko / iStock