**1. Introduction**

Infectious diseases are responsible for a considerable number of deaths in infectious in entire world. Infectious diseases are human diseases caused by viruses, bacteria, parasites, fungi and other microorganisms. Most of them have been controlled by vaccines or antimicrobials. However, some of them still represent global public health problems and are being monitored by the WHO and Center for Disease Control and Prevention. This chapter provides an overview of the applications of molecular methods for infectious diseases caused by viruses (intracellular obligate parasites) of global impact such as Dengue virus, Hepatitis B virus or influenza A virus. The infectious diseases not only represent a potential danger to the life of all human beings but also a significant investment in its detection, treatment and control of their spread. The increase in opportunities of infection by globalization, high rates of mobility among most countries around the world, the patient susceptibility to diseases due to genetic variation in populations [1], the ability of the microorganisms to evade the host immune response has forced the World Health Organization (WHO) to establish better methods of detection, prevention and control of infectious diseases caused by viruses as influenza A virus, coronaviruses, dengue virus, among others [2]. On the other hand, some types of cancer are the result of chronic viral infections caused by human papillomavirus, hepatitis B and C virus. Other infectious diseases are related to the development of neurological disorders caused by the measles virus, or human immunodeficiency virus [3]. In the determination of the etiology

© 2014 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.

of such diseases has made extensive use of clinical procedures internationally validated as methods based on viral cultures and serological assays. However, it is increasing the use of nucleic acid tests in the diagnosis of infectious disease of viral etiology, considering that a critical step to proper treatment and control of any virus infection is a correct diagnosis. Diagnostic tests based on nucleic acid (NAT, nucleic acid tests) more used are the nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR) or real-time PCR for virus detection, genotyping and quantification. In addition, the automation of these techniques decrease in test time, low contamination risk, ease of performance, speed and have lower detection limits [4] show the relevance of their use. The detection of the infectious agent can be done by detection of the genomic DNA, genomic ARN and the viral messenger RNA (mRNA) using the follow techniques: Nucleic acids hybridization (Solid-phase, Liquid-phase or *in situ* hybridization*)*, amplification of the signal of nucleic acids (branched-DNA assays and Hybrid capture assays), nucleic acid amplification (PCR, Real-Time PCR, Nested PCR, Multiplex PCR, Transcriptional-based amplification methods coupled to qPCR or NASBA, Strand displacement amplification), Microarrays (DNA microarrays and Multiplexed micro‐ sphere-base array) [5]. Currently there are some variants of the above techniques aimed to screening for detection or simultaneous discrimination of various etiological agents using multiplex PCR techniques [6], MassTag PCR, a PCR platform coupled to a mass spectrometer which allows simultaneous detection of >20 different pathogens [7] or microarrays pathogen detection (Virochip) [8]. The routine use of molecular techniques for the fast differential diagnosis of viral infections is vital for a high quality care of the patient with an infectious disease, directs the best therapeutic scheme, thus reducing the likelihood of complications, the proper choice of antiviral drug or the best strategy for control of viral replication, reduces resistance to antivirals and prevent the worsening of the clinical picture, the spread of the disease and the death of the patient [9]. This chapter will present molecular techniques applied to the diagnosis of infectious disease of viral etiology and incidence worldwide. A critical first step to proper treatment and control of any virus infection is a correct diagnosis. Conventional diagnostic tests for viruses it based on amplification of conserved portions of the viral genome, detection of antibodies against to viral proteins, or replication of the virus in cell cultures.

ture intracellular virions contain prM, which is processed proteolytically during maturation to yield M protein. The envelope of the virus contains the viral surface proteins E and M [12,13].

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**Figure 1.** Schematic diagram of dengue virus genome. The DENV genome of positive-sense RNA and single-stranded, comprises approximately 10,600 nucleotides. Any region of genome can be used for genome detection by molecular

There are 4 antigenically and genetically distinct serotypes (DENV-1,-2,-3 and-4), being the "Asian" genotypes of DEN-2 and DEN-3 the most frequently associated with severe disease accompanying secondary dengue infections [14]. In human, the virus can cause a spectrum of illness, 75% of DENV infections are asymptomatic. But in persons with symptomatic DENV infection (dengue), the illness occurs in three phases. Acute phase, with 2–7 days of fever or self-limiting influenza-like illness (dengue fever or DF), accompanied by headache, retroorbital eye pain, joint pain, muscle and/or bone pain, rash, mild bleeding manifestations and low white cell count. The critical phase of dengue which marks a 24 to 48 hours, period in which can occur the named severe dengue associated with vascular leakage, hemorrhage (dengue hemorrhagic fever or DHF), potentially leading to vascular shock (dengue shock syndrome or DSS), without appropriate treatment, patients with severe dengue are at risk of death. The convalescent phase of dengue lasts for 4-7 days [15-18]. During the past five decades, the incidence of dengue has increased 30-fold. In 2012, dengue was the most important mosquito-borne viral disease in the world. The emergence and spread of all four dengue viruses ("serotypes") from Asia to the Americas, Africa and the Eastern Mediterranean regions represent a global pandemic threat. Because epidemics of dengue result in human suffering, strained health services and massive economic losses, an international effort to reduce morbidity and mortality is long overdue coordinated by WHO named Global Strategy for Dengue Prevention and Control 2012-2020 [19]. The goal of the global strategy is to reduce the burden of dengue by to reduce mortality and morbidity from dengue by 2020 by at least 50% and 25% respectively. The laboratory diagnosis of dengue can detect severe cases, case confirmation and differential diagnosis with other infectious diseases. Diagnosis of dengue is made by detecting the infective virus, virus genome, dengue antigen or by analyzing, the serological responses (IgM or IgG) present after infection. Serology is currently the most widely applied in routine diagnosis [20] (Table 1). After the mosquito bites occurs an incubation period of 4–10 days, resulting in an asymptomatic or symptomatic dengue infection. In this period, the virus replicates and an antibody response is developed (Figure 2). The development of IgM antibody is coincident with the disappearance of fever and viraemia. In a primary infection (when an individual is infected for the first time with a flavivirus), viraemia develops from 1– 2 days before the onset of fever until 4–5 days after and anti-dengue IgM specific antibodies can be detected 3−6 days after fever onset. Low levels of IgM are still detectable around one to

techniques.

#### **2. Dengue virus**

Dengue virus (DENV) infection is the most common arthropodborne viral disease of humans; Aedes mosquitoes, principally *Aedes aegypti*, transmit this disease. According World Health Organization DENV is an emerging infectious agent that infects with an estimated 50–100 million clinical infections occurring annually worldwide [10]. DENV belongs to the family Flaviviridae, genus Flavivirus. DENV is a small are spherical and enveloped virus that contain a positive strand RNA genome of approximately 10,600 nucleotides coding for three structural proteins (capsid C, membrane, M, and the envelope, E) and seven non-structural proteins (NS1, xlinkA, xlinkB, NS3, NS4A, NS4B, NS5) [11], (Figure 1). The envelope protein (E) plays a key role in several important processes including receptor binding, blood cell hemagglutination, and induction of a protective immune response, membrane fusion and virion assembly. Two types of virions are recognized: mature extracellular virions contain M protein, while imma‐ ture intracellular virions contain prM, which is processed proteolytically during maturation to yield M protein. The envelope of the virus contains the viral surface proteins E and M [12,13].

of such diseases has made extensive use of clinical procedures internationally validated as methods based on viral cultures and serological assays. However, it is increasing the use of nucleic acid tests in the diagnosis of infectious disease of viral etiology, considering that a critical step to proper treatment and control of any virus infection is a correct diagnosis. Diagnostic tests based on nucleic acid (NAT, nucleic acid tests) more used are the nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR) or real-time PCR for virus detection, genotyping and quantification. In addition, the automation of these techniques decrease in test time, low contamination risk, ease of performance, speed and have lower detection limits [4] show the relevance of their use. The detection of the infectious agent can be done by detection of the genomic DNA, genomic ARN and the viral messenger RNA (mRNA) using the follow techniques: Nucleic acids hybridization (Solid-phase, Liquid-phase or *in situ* hybridization*)*, amplification of the signal of nucleic acids (branched-DNA assays and Hybrid capture assays), nucleic acid amplification (PCR, Real-Time PCR, Nested PCR, Multiplex PCR, Transcriptional-based amplification methods coupled to qPCR or NASBA, Strand displacement amplification), Microarrays (DNA microarrays and Multiplexed micro‐ sphere-base array) [5]. Currently there are some variants of the above techniques aimed to screening for detection or simultaneous discrimination of various etiological agents using multiplex PCR techniques [6], MassTag PCR, a PCR platform coupled to a mass spectrometer which allows simultaneous detection of >20 different pathogens [7] or microarrays pathogen detection (Virochip) [8]. The routine use of molecular techniques for the fast differential diagnosis of viral infections is vital for a high quality care of the patient with an infectious disease, directs the best therapeutic scheme, thus reducing the likelihood of complications, the proper choice of antiviral drug or the best strategy for control of viral replication, reduces resistance to antivirals and prevent the worsening of the clinical picture, the spread of the disease and the death of the patient [9]. This chapter will present molecular techniques applied to the diagnosis of infectious disease of viral etiology and incidence worldwide. A critical first step to proper treatment and control of any virus infection is a correct diagnosis. Conventional diagnostic tests for viruses it based on amplification of conserved portions of the viral genome, detection of antibodies against to viral proteins, or replication of the virus in cell cultures.

Dengue virus (DENV) infection is the most common arthropodborne viral disease of humans; Aedes mosquitoes, principally *Aedes aegypti*, transmit this disease. According World Health Organization DENV is an emerging infectious agent that infects with an estimated 50–100 million clinical infections occurring annually worldwide [10]. DENV belongs to the family Flaviviridae, genus Flavivirus. DENV is a small are spherical and enveloped virus that contain a positive strand RNA genome of approximately 10,600 nucleotides coding for three structural proteins (capsid C, membrane, M, and the envelope, E) and seven non-structural proteins (NS1, xlinkA, xlinkB, NS3, NS4A, NS4B, NS5) [11], (Figure 1). The envelope protein (E) plays a key role in several important processes including receptor binding, blood cell hemagglutination, and induction of a protective immune response, membrane fusion and virion assembly. Two types of virions are recognized: mature extracellular virions contain M protein, while imma‐

**2. Dengue virus**

90 Trends in Infectious Diseases

**Figure 1.** Schematic diagram of dengue virus genome. The DENV genome of positive-sense RNA and single-stranded, comprises approximately 10,600 nucleotides. Any region of genome can be used for genome detection by molecular techniques.

There are 4 antigenically and genetically distinct serotypes (DENV-1,-2,-3 and-4), being the "Asian" genotypes of DEN-2 and DEN-3 the most frequently associated with severe disease accompanying secondary dengue infections [14]. In human, the virus can cause a spectrum of illness, 75% of DENV infections are asymptomatic. But in persons with symptomatic DENV infection (dengue), the illness occurs in three phases. Acute phase, with 2–7 days of fever or self-limiting influenza-like illness (dengue fever or DF), accompanied by headache, retroorbital eye pain, joint pain, muscle and/or bone pain, rash, mild bleeding manifestations and low white cell count. The critical phase of dengue which marks a 24 to 48 hours, period in which can occur the named severe dengue associated with vascular leakage, hemorrhage (dengue hemorrhagic fever or DHF), potentially leading to vascular shock (dengue shock syndrome or DSS), without appropriate treatment, patients with severe dengue are at risk of death. The convalescent phase of dengue lasts for 4-7 days [15-18]. During the past five decades, the incidence of dengue has increased 30-fold. In 2012, dengue was the most important mosquito-borne viral disease in the world. The emergence and spread of all four dengue viruses ("serotypes") from Asia to the Americas, Africa and the Eastern Mediterranean regions represent a global pandemic threat. Because epidemics of dengue result in human suffering, strained health services and massive economic losses, an international effort to reduce morbidity and mortality is long overdue coordinated by WHO named Global Strategy for Dengue Prevention and Control 2012-2020 [19]. The goal of the global strategy is to reduce the burden of dengue by to reduce mortality and morbidity from dengue by 2020 by at least 50% and 25% respectively. The laboratory diagnosis of dengue can detect severe cases, case confirmation and differential diagnosis with other infectious diseases. Diagnosis of dengue is made by detecting the infective virus, virus genome, dengue antigen or by analyzing, the serological responses (IgM or IgG) present after infection. Serology is currently the most widely applied in routine diagnosis [20] (Table 1). After the mosquito bites occurs an incubation period of 4–10 days, resulting in an asymptomatic or symptomatic dengue infection. In this period, the virus replicates and an antibody response is developed (Figure 2). The development of IgM antibody is coincident with the disappearance of fever and viraemia. In a primary infection (when an individual is infected for the first time with a flavivirus), viraemia develops from 1– 2 days before the onset of fever until 4–5 days after and anti-dengue IgM specific antibodies can be detected 3−6 days after fever onset. Low levels of IgM are still detectable around one to three months after fever. In addition, the primary infection is characterized by slowly increas‐ ing but low levels of dengue-specific IgG, becoming elevated at days 9−10. Low IgG levels persist for decades, an indication of a past dengue infection. In a secondary infection, there is a rapid and higher increase of anti-dengue specific IgG antibodies. High IgG levels remain for 30–40 days [21]. The serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immu‐ noglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. High cross-reactivity was observed with these tests. MAC-ELISA and rapid tests are the most frequent methods for IgM detection and in the detection of IgG are used HIA or ELISA [22-25]. Dengue non-structural protein 1 (NS1) is secreted from infected cells and produces a very strong humoral response. NS1 is a useful early serum marker for primary and secondary dengue infections, and is typically present between days 1–9 after onset of clinical signs [23]. In febrile patients, the early diagnosis of dengue virus infection on based on NS1 viral antigen [26-27]. At the end of the acute phase of infection, serology is the method of choice for diagnosis by detection anti-DENV IgM and IgG antibodies using MAC-ELISA. High sensitivity for NS1 antigen detection was observed by the association of MAC-ELISA with a commercial anti-DENV IgM/IgG rapid test (PanbioH Dengue Early Rapid test) [28]. A DENV NS1 capture assay using a test NS1 Ag Strip (BioRad Laboratories) is a valuable tool to postmortem dengue confirmation [29]. There are several commercial kits to dengue diagnostic such as the SD Bioline Dengue Duo device (Standard Diagnostic Inc., Korea) this test is composed of 2 tests to detect DENV NS1 antigen (first test) and anti-DENV IgM/IgG (second test) in serum, plasma or whole blood. With this test, still after the onset of illness, the virus can detected in serum, plasma, circulating blood cells and other tissues for 4-5 days.

Dengue diagnosis also relies on viral cell culture methods [30,31]. Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia (usually before day 5). Virus may be recovery from serum, plasma and peripheral blood mononuclear cells. Dengue virus is heat-labile; specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. Cell culture is the most widely used method for dengue virus isolation usually takes several days. Four methods of viral isolation have been routinely used for dengue viruses: intracerebral inoculation of newborn mice, inoculation on mammalian cell cultures, intrathoracic inoculation of adult mosquitoes, and inoculation on mosquito cell cultures [32]. The mosquito cell line C6/36 (cloned from *Ae. albopictus*) or AP61 (cell line from *Ae. pseudoscutellaris*) are the host cells of choice for isolation of virus. Indirect fluorescent antibody staining of the infected cell culture is often regarded as the "gold standard" in dengue diagnostics. However, it is tedious, time-consuming, and requires cell culture. All these studies have shown that this can be a valuable approach, especially in the early phase of infection; however, in some cases, they do not identify the viral serotype and these assays may not be as sensitive as the detection of viral RNA by nucleic acid amplification tests (NAAT) using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR may assume a very important role in dengue diagnosis at confirms an acute dengue infection [33, 20, 21]. Molecular diagnosis methods are usually rapid, sensitive, and simple when correctly standardized and can be used for serotype identification and quantification of genome copies in human clinical samples, biopsies, autopsy tissues, or mosquitoes. NAAT assays may identify viral RNA within 24–48 hours. Although, also be able to detect dengue viruses up to the 10th day after the onset of the symptoms. RNA extraction from clinical samples can be performed with the QIAamp Viral RNA kit [34], VERSANT Molecular System SP (Siemens) or HighPureViral Nucleic Acid Kit (Roche). Several RT-PCR procedures to detect and identify dengue serotypes in clinical specimens have been reported such as one-step, twostep, nested RT–PCR or real time RT–PCR [35]. These PCR assays vary in the amplified gene regions of the genome, in the detection method of RT-PCR products, and the virus typing methods. Real-time RT-PCR assays "singleplex" or "multiplex" can identify all four serotypes from a single sample, in a single reaction and is useful to determine viral titer in a clinical sample. Real-Time-PCR detection and typing of DENV usually addresses to the partially conserved 3'-UTR region of the genome. Due to the typical RNA-viral sequence variations it is difficult to identify one particular probe to be strictly related to one type or which could be

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Many laboratories utilize a nested RT-PCR assay, using universal dengue primers targeting the C/prM region of the genome for an initial reverse transcription and amplification step, followed by a nested PCR amplification that is serotype-specific. The analysis of the amplifi‐ cation efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, between the region protein-5 (NS5) versus the capsid/pre-membrane region (C-prM) and the 3' noncoding region (3'NC) showed that the non-structural conserved genomic region the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples [37]. A fragment located at the 5'-UTR region of the virus genome was successfully used to identify and quantify distinct dengue virus strains and serotypes in clinical samples, in sera from patients infected with dengue virus, and in the mosquito *Aedes*

used for a melting curve based analysis [36].


ELISA (Enzyme-linked immunosorbent assay). HIA (Haemagglutination inhibition assay). IgG (Immunoglubulin G). IgM (Immunoglobulin M). NS1 Ag (Non-structural protein 1). RT-PCR (Reverse transcriptase polymerase chain reaction). In‐ formation based on Chapter 4. Laboratory Diagnosis and Diagnostic Tests. Dengue Guidelines for Diagnosis, Treat‐ ment, Prevention and Control. Geneva. TDR/World Health Organization. 2012 [21].

**Table 1.** Dengue diagnostic methods.

Dengue diagnosis also relies on viral cell culture methods [30,31]. Specimens for virus isolation should be collected early in the course of the infection, during the period of viraemia (usually before day 5). Virus may be recovery from serum, plasma and peripheral blood mononuclear cells. Dengue virus is heat-labile; specimens awaiting transport to the laboratory should be kept in a refrigerator or packed in wet ice. Cell culture is the most widely used method for dengue virus isolation usually takes several days. Four methods of viral isolation have been routinely used for dengue viruses: intracerebral inoculation of newborn mice, inoculation on mammalian cell cultures, intrathoracic inoculation of adult mosquitoes, and inoculation on mosquito cell cultures [32]. The mosquito cell line C6/36 (cloned from *Ae. albopictus*) or AP61 (cell line from *Ae. pseudoscutellaris*) are the host cells of choice for isolation of virus. Indirect fluorescent antibody staining of the infected cell culture is often regarded as the "gold standard" in dengue diagnostics. However, it is tedious, time-consuming, and requires cell culture. All these studies have shown that this can be a valuable approach, especially in the early phase of infection; however, in some cases, they do not identify the viral serotype and these assays may not be as sensitive as the detection of viral RNA by nucleic acid amplification tests (NAAT) using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR may assume a very important role in dengue diagnosis at confirms an acute dengue infection [33, 20, 21]. Molecular diagnosis methods are usually rapid, sensitive, and simple when correctly standardized and can be used for serotype identification and quantification of genome copies in human clinical samples, biopsies, autopsy tissues, or mosquitoes. NAAT assays may identify viral RNA within 24–48 hours. Although, also be able to detect dengue viruses up to the 10th day after the onset of the symptoms. RNA extraction from clinical samples can be performed with the QIAamp Viral RNA kit [34], VERSANT Molecular System SP (Siemens) or HighPureViral Nucleic Acid Kit (Roche). Several RT-PCR procedures to detect and identify dengue serotypes in clinical specimens have been reported such as one-step, twostep, nested RT–PCR or real time RT–PCR [35]. These PCR assays vary in the amplified gene regions of the genome, in the detection method of RT-PCR products, and the virus typing methods. Real-time RT-PCR assays "singleplex" or "multiplex" can identify all four serotypes from a single sample, in a single reaction and is useful to determine viral titer in a clinical sample. Real-Time-PCR detection and typing of DENV usually addresses to the partially conserved 3'-UTR region of the genome. Due to the typical RNA-viral sequence variations it is difficult to identify one particular probe to be strictly related to one type or which could be used for a melting curve based analysis [36].

three months after fever. In addition, the primary infection is characterized by slowly increas‐ ing but low levels of dengue-specific IgG, becoming elevated at days 9−10. Low IgG levels persist for decades, an indication of a past dengue infection. In a secondary infection, there is a rapid and higher increase of anti-dengue specific IgG antibodies. High IgG levels remain for 30–40 days [21]. The serological tests have been used for the diagnosis of dengue infection: hemagglutination-inhibition (HI), complement fixation (CF), neutralization test (NT), immu‐ noglobulin M (IgM) capture enzyme linked immunosorbent assay (MAC-ELISA) and indirect immunoglobulin G ELISA. High cross-reactivity was observed with these tests. MAC-ELISA and rapid tests are the most frequent methods for IgM detection and in the detection of IgG are used HIA or ELISA [22-25]. Dengue non-structural protein 1 (NS1) is secreted from infected cells and produces a very strong humoral response. NS1 is a useful early serum marker for primary and secondary dengue infections, and is typically present between days 1–9 after onset of clinical signs [23]. In febrile patients, the early diagnosis of dengue virus infection on based on NS1 viral antigen [26-27]. At the end of the acute phase of infection, serology is the method of choice for diagnosis by detection anti-DENV IgM and IgG antibodies using MAC-ELISA. High sensitivity for NS1 antigen detection was observed by the association of MAC-ELISA with a commercial anti-DENV IgM/IgG rapid test (PanbioH Dengue Early Rapid test) [28]. A DENV NS1 capture assay using a test NS1 Ag Strip (BioRad Laboratories) is a valuable tool to postmortem dengue confirmation [29]. There are several commercial kits to dengue diagnostic such as the SD Bioline Dengue Duo device (Standard Diagnostic Inc., Korea) this test is composed of 2 tests to detect DENV NS1 antigen (first test) and anti-DENV IgM/IgG (second test) in serum, plasma or whole blood. With this test, still after the onset of illness, the virus

can detected in serum, plasma, circulating blood cells and other tissues for 4-5 days.

IgM detection (recent

infection)

ment, Prevention and Control. Geneva. TDR/World Health Organization. 2012 [21].

**Serological response**

92 Trends in Infectious Diseases

**Virus detection and its components**

Paired sera (acute serum from 1-5 days and second serum 15-21

Serum after day 5 of

(1-5 days of fever) and necropsy tissues

days after)

Acute serum

fever

**Table 1.** Dengue diagnostic methods.

**SAMPLE DIAGNOSTIC METHOD METHODOLOGY**

HIA

ELISA Rapid tests

HIA

Viral isolation Mosquito cell culture inoculation Nucleic acid detection RT-PCR and real time RT-PCR

NS1 Ag ELISA

Immuno-histochemistry

Neutralization test

IgM or IgG seroconversion ELISA

IgG detection IgG ELISA

ELISA (Enzyme-linked immunosorbent assay). HIA (Haemagglutination inhibition assay). IgG (Immunoglubulin G). IgM (Immunoglobulin M). NS1 Ag (Non-structural protein 1). RT-PCR (Reverse transcriptase polymerase chain reaction). In‐ formation based on Chapter 4. Laboratory Diagnosis and Diagnostic Tests. Dengue Guidelines for Diagnosis, Treat‐

Antigen detection NS1 Ag rapid tests

Many laboratories utilize a nested RT-PCR assay, using universal dengue primers targeting the C/prM region of the genome for an initial reverse transcription and amplification step, followed by a nested PCR amplification that is serotype-specific. The analysis of the amplifi‐ cation efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, between the region protein-5 (NS5) versus the capsid/pre-membrane region (C-prM) and the 3' noncoding region (3'NC) showed that the non-structural conserved genomic region the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples [37]. A fragment located at the 5'-UTR region of the virus genome was successfully used to identify and quantify distinct dengue virus strains and serotypes in clinical samples, in sera from patients infected with dengue virus, and in the mosquito *Aedes*

approved by the US Food and Drug Administration (FDA) for the identification of dengue virus serotypes 1, 2, 3 or 4 from viral RNA in serum or plasma (sodium citrate) collected during the acute phase. The limit of detection (LoD) of assay was determined to be between 1 x 104

by real-time RT-PCR, a positive PCR result is a definite proof of current infection and it usually confirms the infecting serotype. However, a negative result is interpreted as "indeterminate". Patients receiving negative results before 5 days of illness are usually asked to submit a second serum sample for serological confirmation after the 5th day of illness. Also, it was used other options to diagnostics dengue infection. The first is Virochip is a pan-viral microarray platform; this assay can detect to virus of Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In other hand, for viral detection exists the option Deep sequencing and shotgun sequencing of human clinical samples by pyrosequencing using

Influenza is a highly contagious respiratory disease of humans, with propensity for seasonal epidemics and occasional pandemics. This disease constitutes a global health issue, leading to morbidity, mortality, and economic losses. During influenza season, influenza viruses circulate ubiquitously in the population. Global influenza surveillance forms the primary line of defense against the occurrence of influenza pandemics by identifying emerging influenza virus strains that pose a potential threat [45]. Influenza affects all age groups that result from its pulmonary complications. The virus initially infects the upper airways but can directly extend to the lower airways in severe cases, resulting in a viral pneumonia with significant morbidity and mortality [46, 47]. Influenza viruses belong to the Orthomyxoviridae family and are organize into types A, B and C. Influenza types A and B are responsible for epidemics of respiratory illness in humans and animals. The etiologic agent of influenza is the influenza virus with negative-strand, segmented RNA genome. Influenza type A and B viruses have 8 genes that code for 10 proteins. The virion has two surface glycoproteins (hemagglutinin (HA) and neuraminidase (NA) and the M2 protein protrude through envelop (Figure 3). Influenza virus binds to its sialic (neuraminic) acid receptor on respiratory epithelial cells by means of the HA protein [48]. The influenza type A viruses are sub classified into different subtypes according to HA and NA proteins, there are 16 HA subtypes and 9 NA subtypes, all of them have been identified and isolated from birds, humans and can affect a range of mammal species. The influenza A subtypes that circulated extensively in humans are A(H1N1); A(H1N2); A(H2N2); and seasonal influenza A (H3N2) [49]. Influenza A viruses (IAV) are the cause of pandemics, which are generated by the rearrangement (reassortment) of viral RNA

Pandemic viruses of influenza A virus including "Spanish influenza" (H1N1 in 1918) and A/ H1N1 or A(H1N1)pdm09 (H1N1 in 2009) [51] or rarely, a novel influenza A virus infection. For the diagnosis, the influenza tests that provide accurate and timely results are the most recommended. The appropriate respiratory samples for influenza testing are upper respiratory

segments in cells infected with two different viral strains [50].

GCE/mL for all serotypes in both formats in serum and plasma [43]. In all reactions

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and 1 x 103

the Illumina GAII platform [44].

**3. Influenza virus**

**Figure 2.** Immune response to virus dengue. In the infection course of dengue virus, the host's B cells produce IgM and IgG antibodies, which are released for recognize and neutralize the dengue virus and molecules such as the den‐ gue NS1 protein with the purpose of eliminates the virus. The figure shows the viremia can detect by the amplification of the NS1 gene by RT-PCR and the antibodies titers by ELISA. Information based on Guzman MG, et al., 2010 [25] and WHO, Geneva, 2009 [10].

*aegypti*, as well as to study virus replication in different cell lines using TaqMan probes [38]. A real time RT-PCR (qRT-PCR) for DENV using TaqMan Minor Groove Binding (MGB) probe approach was development for detection and quantitation of all four serotypes using a single probe primer set targeted against the 3'UTR of DENV. In this assay, the limit of detection was DENV-1 (0.98 UFP/ml), DENV-2 (0.99 UFP/ml), DENV-3 (0.99 UFP/ml) and DENV-4 (0.99 UFP/ml) [39]. Other option to diagnosis to provide serotype specificity is a multiplex rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome using molecular beacons. This assay was linear from 7.0 to 1.0 log10 cDNA equivalents/mL for each serotype. The limit of detection was calculated to be 0.3 cDNA equivalents/mL for DENV-1, 13.8 for DENV-2, 0.8 for DENV-3, and 12.4 for DENV-4 [40]. A technique for the multiplex detection and typing of all DENV serotypes from clinical samples is PCR-ligase detection reaction (LDR). In this technique, a serotype-specific PCR amplifies the regions of genes C and E simultaneously. Then, two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). The detection limit of the assay ranged from 0.004 to 0.7 equivalent PFU/reaction. The assay was 100 times more sensitive for DENV-2 and DENV-1 (LOD, 0.004 and 0.017 equivalent PFU, respectively) than for the others serotypes [41]. Detection of DENV in urine is other diagnostic method, a study by real-time RT-PCR, reported the detection of viral genome in urine between days 6 to 16[42]. Recently, CDC development a CDC DENV-1-4 Real-Time RT-PCR Assay, the first molecular test approved by the US Food and Drug Administration (FDA) for the identification of dengue virus serotypes 1, 2, 3 or 4 from viral RNA in serum or plasma (sodium citrate) collected during the acute phase. The limit of detection (LoD) of assay was determined to be between 1 x 104 and 1 x 103 GCE/mL for all serotypes in both formats in serum and plasma [43]. In all reactions by real-time RT-PCR, a positive PCR result is a definite proof of current infection and it usually confirms the infecting serotype. However, a negative result is interpreted as "indeterminate". Patients receiving negative results before 5 days of illness are usually asked to submit a second serum sample for serological confirmation after the 5th day of illness. Also, it was used other options to diagnostics dengue infection. The first is Virochip is a pan-viral microarray platform; this assay can detect to virus of Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In other hand, for viral detection exists the option Deep sequencing and shotgun sequencing of human clinical samples by pyrosequencing using the Illumina GAII platform [44].
