**Meet the editor**

Medical Doctor (MD), Universidad Central de Venezuela, Caracas, Venezuela. Master of Sciences in Protozoology (MSc), Universidad de Los Andes, Trujillo, Venezuela, scholar 2004-2005. Diploma in Tropical Medicine & Hygiene (DTM&H), Universidad Peruana Cayetano Heredia, Lima, Peru, and University of Alabama at Birmingham, Alabama, United States of America. Fellow

of the Royal Society for Tropical Medicine & Hygiene (FRSTMH), London, United Kingdom. Fellow of the Faculty of Travel Medicine (FFTM) of the Royal College of Physicians and Surgeons of Glasgow (RCPSG), Glasgow, Scotland, United Kingdom. Candidate to Doctor in Parasitology (PhD), Universidad Central de Venezuela, scholar 2005-2008. President, Travel Medicine Committee, Pan American ID Association. Senior Researcher, Colciencias (Results 693 of 2014). Professor, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia.

## Contents

#### **Preface XI**



Chapter 7 **Echinococcosis — Rare Locations and Uncommon Clinical Manifestations 119** Antonio Di Cataldo, Giuseppe Petrillo, Claudia Trombatore, Stefano Palmucci, Antonella Agodi, Martina Barchitta, Annalisa Quattrocchi, Nunzio Crimi, Silvia Fichera, Stefano Puleo, Amy Giarrizzo, Martina Calabrini, Rosalia Latino and Rosanna Portale


## Preface

Chapter 8 **Radiological Characteristics of Pulmonary Hydatid Cysts 137**

Chapter 9 **Immunotherapy Can Enhance Anthelmintic Efficacy in Alveolar**

**Section 3 Treatment and Prevention 157**

**VI** Contents

**Echinococcosis 159** Emília Dvorožňáková

YuRong Yang

Chapter 10 **Hydatidosis and Intervention Strategies 195**

Dilek Emlik, Kemal Ödev, Necdet Poyraz and Hasan Emin Kaya

Echinococcosis remains an important cause of morbidity and mortality in certain areas of the world, tropical and non-tropical, particularly in rural settings. Although in many coun‐ tries significant improvements have been made to considerably reduce this disease, its zoo‐ notic character remains a challenge in terms of control and prevention. For these reasons, research on this parasitic disease is of utmost relevance particularly in endemic settings, most importantly in developing countries. However, today, exposure to parasitic cycles as a consequence of travel to endemic areas in the context of globalization is increasing. As other zoonoses, Echinococcosis deserves more research and multiple new approaches for its study (Cascio et al., 2011). More approaches with the vision of "One Health" also are clearly neces‐ sary (Rodriguez-Morales & Schlagenhauf, 2014), as also more interaction between medical doctors and doctors of veterinary medicine. A holistic control approach requires the in‐ volvement of both, even more because most of the burden related to echinococcosis and oth‐ er neglected zoonotic diseases would be avoidable (Benitez et al., 2008).

With these conceptions in mind, this book includes different topics with regard to the epi‐ demiology, biology, clinical manifestations, treatment and prevention of the wide spectrum of diseases caused by the different species of Echinococcus involved in human and animal infection, with an aim to update the most significant research in many of them as well as to offer a multinational perspective on different aspects. This book has been organized into three major sections: (I) Epidemiology; (II) Biological and Clinical Aspects; and (III) Treat‐ ment and Prevention. Section I includes topics covering epidemiological studies in Colom‐ bia, Chile, Mexico and Tunisia, including molecular biology approaches to the study of parasite species. Section II includes topics covering the biology of some Echinococcus spe‐ cies affecting mainly animals, as also the human clinical manifestations in the central nerv‐ ous system (CNS), genitourinary tract and other organic typical and atypical locations, as well as radiological manifestations of pulmonary disease. Section III includes topics on the usefulness of immunotherapy for antihelmintic treatment and intervention strategies.

The commissioning of this book also gave me the opportunity to get back and examine the epidemiology of echinococcosis in Colombia, including commencing new studies, mapping diseases, characterizing their patterns and offering new information, particularly epidemio‐ logical, not available in bibliographical databases yet, such as Medline, Scopus and Science Citation, reporting estimates of morbidity due to the different forms of echinococcosis found in the country.

Following the same philosophy as in my two previous books with InTech, *Current Topics in Public Health* (Rodriguez-Morales AJ, 2013) and *Current Topics in Tropical Medicine* (Rodri‐ guez-Morales AJ, 2012), this book does not intend to be an exhaustive compilation. This first edition has included not just multiple topics but also a wide geographical participation from many countries in different regions of the world. Its online availability through the website of InTech, as well as the possibility to upload the complete book or its chapters on personal websites and institutions repositories, allows it to reach a global audience. Continuing on the series of "Current Topics", we are planning to develop in the future other projects such as *Current Topics in Infectious Diseases* and *Current Topics in Travel Medicine*, so if you are in‐ terested in participating in this endeavor as an author of one or more chapters, please con‐ tact us.

I would like to give a very special thanks to InTech (for the third time), and particularly to Ana Pantar (Publishing Process Manager) as well as to Ivona Lovric, for the opportunity to edit this interesting and important book, as also for their constant support. The implementa‐ tion and use of CrossCheck™, powered by iThenticate, during the review process of chap‐ ters of this book allows efficient management and prevention of professional plagiarism and other forms of scholarly misconduct, ultimately improving the quality of most chapters sig‐ nificantly.

I want to take this opportunity, as I have done before, to dedicate this book to my family (Aurora, Alfonso José, Alejandro, and Andrea the neurologist) and particularly to my lovely wife, Diana. She is the highest blessing in my life, my soul mate, and my strongest support in any journey I undertake; she provides everything in my life, from day to day. Also, thanks to my friends and my undergraduate and postgraduate students of health sciences in Colombia, Venezuela and around Latin America. It is time to say thanks to my colleagues at the Working Group on Zoonoses, International Society for Chemotherapy, and the Commit‐ tee on Zoonoses and Haemorrhagic Fevers of the Colombian Infectious Diseases Society (ACIN). Also, thanks to Dr. Juliana Buitrago-Jaramillo, Dean of the Faculty of Health Scien‐ ces, Universidad Tecnológica de Pereira, who is supporting most of the efforts of our re‐ search group, Public Health and Infection, directed by my friend and colleague Dr. Guillermo J. Lagos-Grisales, MD, MPH. Members of this research group and incubator con‐ sist of young and enthusiastic medical students and some veterinary medical students as well as young medical doctors who are pursuing significant improvements in the under‐ standing of the epidemiology of zoonotic, vector-borne, parasitic and, in general, infectious diseases in our country with international projection. Year 2015 is the year of publication of this book but also of the consolidation of this recognized group, which is positioned as a leader in infectious diseases epidemiology research in the coffee-triangle region and very soon in the entire country. Doctors Faccini and Mondragón also helped in the critical review of the editor's chapter, and I thank them.

Finally, I hope our readers enjoy reading the chapters of Current Topics in Echinococcosis as much as I did.

#### **References**

Benitez JA, Rodriguez-Morales AJ, Vivas P, Plaz J. 2008. Burden of zoonotic diseases in Ven‐ ezuela during 2004 and 2005. Ann N Y Acad Sci 1149:315-7.

Cascio A, Bosilkovski M, Rodriguez-Morales AJ, Pappas G. 2011. The socio-ecology of zoo‐ notic infections. Clin Microbiol Infect 17:336-42.

Rodriguez-Morales AJ. 2012 (Editor). Current Topics in Tropical Medicine. ISBN 978-953-51-0274-8. InTech, Croatia.

edition has included not just multiple topics but also a wide geographical participation from many countries in different regions of the world. Its online availability through the website of InTech, as well as the possibility to upload the complete book or its chapters on personal websites and institutions repositories, allows it to reach a global audience. Continuing on the series of "Current Topics", we are planning to develop in the future other projects such as *Current Topics in Infectious Diseases* and *Current Topics in Travel Medicine*, so if you are in‐ terested in participating in this endeavor as an author of one or more chapters, please con‐

I would like to give a very special thanks to InTech (for the third time), and particularly to Ana Pantar (Publishing Process Manager) as well as to Ivona Lovric, for the opportunity to edit this interesting and important book, as also for their constant support. The implementa‐ tion and use of CrossCheck™, powered by iThenticate, during the review process of chap‐ ters of this book allows efficient management and prevention of professional plagiarism and other forms of scholarly misconduct, ultimately improving the quality of most chapters sig‐

I want to take this opportunity, as I have done before, to dedicate this book to my family (Aurora, Alfonso José, Alejandro, and Andrea the neurologist) and particularly to my lovely wife, Diana. She is the highest blessing in my life, my soul mate, and my strongest support in any journey I undertake; she provides everything in my life, from day to day. Also, thanks to my friends and my undergraduate and postgraduate students of health sciences in Colombia, Venezuela and around Latin America. It is time to say thanks to my colleagues at the Working Group on Zoonoses, International Society for Chemotherapy, and the Commit‐ tee on Zoonoses and Haemorrhagic Fevers of the Colombian Infectious Diseases Society (ACIN). Also, thanks to Dr. Juliana Buitrago-Jaramillo, Dean of the Faculty of Health Scien‐ ces, Universidad Tecnológica de Pereira, who is supporting most of the efforts of our re‐ search group, Public Health and Infection, directed by my friend and colleague Dr. Guillermo J. Lagos-Grisales, MD, MPH. Members of this research group and incubator con‐ sist of young and enthusiastic medical students and some veterinary medical students as well as young medical doctors who are pursuing significant improvements in the under‐ standing of the epidemiology of zoonotic, vector-borne, parasitic and, in general, infectious diseases in our country with international projection. Year 2015 is the year of publication of this book but also of the consolidation of this recognized group, which is positioned as a leader in infectious diseases epidemiology research in the coffee-triangle region and very soon in the entire country. Doctors Faccini and Mondragón also helped in the critical review

Finally, I hope our readers enjoy reading the chapters of Current Topics in Echinococcosis as

Benitez JA, Rodriguez-Morales AJ, Vivas P, Plaz J. 2008. Burden of zoonotic diseases in Ven‐

Cascio A, Bosilkovski M, Rodriguez-Morales AJ, Pappas G. 2011. The socio-ecology of zoo‐

tact us.

VIII Preface

nificantly.

of the editor's chapter, and I thank them.

ezuela during 2004 and 2005. Ann N Y Acad Sci 1149:315-7.

notic infections. Clin Microbiol Infect 17:336-42.

much as I did. **References**

Rodriguez-Morales AJ. 2013 (Editor). Current Topics in Public Health. ISBN 978-953-51-1121-4. InTech, Croatia.

Rodriguez-Morales AJ, Schlagenhauf P. 2014. Zoonoses and travel medicine: "one world one health". Travel Med Infect Dis 12:555-6.

#### **Prof. Alfonso J. Rodriguez-Morales**

MD, MSc, DTM&H, FRSTMH (Lon), FFTM RCPS (Glasg), PhD (c). Public Health and Infection Research Group, School of Medicine and School of Veterinary Medicine and Zootechnics, Faculty of Health Sciences, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia Committee on Zoonoses and Haemorrhagic Fevers, Asociación Colombiana de Infectología, Bogotá, DC, Colombia Working Group on Zoonoses, International Society for Chemotherapy,

Aberdeen, United Kingdom

**Section 1**

## **Epidemiology**

#### **Chapter 1**

## **Echinococcosis in Colombia — A Neglected Zoonosis?**

Alfonso J. Rodriguez-Morales, Lauren Sofia Calvo-Betancourt, Camila Alarcón-Olave and Adrián Bolívar-Mejía

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60731

#### **Abstract**

Echinococcosis or hydatid disease is a near-cosmopolitan parasitic zoonosis caused by the larval stages of cestodes of the genus *Echinococcus*, whose spectrum of clinical manifestations can vary from asymptomatic infection to death, the liver being the most frequently affected organ. In the absence of an adequate treatment, case fatality rate of affected patients may reach 100%, so clinical suspicion should always be accom‐ panied by a complete workup and a prompt treatment. Like other zoonotic parasitic diseases, echinococcosis is associated with low socioeconomic and cultural levels, especially affecting rural areas of developing countries, not only by the morbidity and mortality it produces, but also by the economic impact associated to health care costs and productivity losses. Despite this, in Colombia and in other South American countries, epidemiological studies of this disease are limited, so it is not known with certainty the incidence, the prevalence, and the burden of the disease. The objective of this chapter is to summarize and describe epidemiological and clinical updated aspects of the disease, focusing on evidences and published research articles on echinococcosis in Colombia, finally questioning if this cestode zoonotic disease has been largely neglected.

**Keywords:** Echinococcosis, *Echinococcus*, Colombia, neglected diseases, zoonotic in‐ fectious diseases

© 2015 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.

#### **1. Introduction**

Echinococcosis or hydatid disease is a near-cosmopolitan parasitic zoonosis caused by the larval stages (paratenic for humans) of cestodes of the genus *Echinococcus* (Figure 1) [1, 2]. This helminthiasis causes an important economic loss over agricultural and pastoral regions of limited resources, particularly in developing countries [3]. In addition, it is also important to notice that this is one of the important infectious disease groups caused by larval stages of cestodes, which also include cysticercosis (caused by larval stages of *Taenia solium*), coenuriasis (caused by larval stages of *Taenia multiceps gaigeri*), and sparganosis (caused by larval stages of *Spirometra*), among others.

Biologically and taxonomically, although in the past it was considered as part of the genus *Taenia*, *Echinococcus* genus belongs to the family *Taeniidae* (order Cyclophyllidea, subclass Eucestoda, class Cestoda, phylum Platyhelminthes). Family *Taeniidae* also includes the genus *Taenia*. Order Cyclophyllidea has two important families that infect humans: *Hymenolepidi‐ dae* (*Hymenolepis*) and *Dipylidiidae* (*Dipylidium*). Subclass Eucestoda includes the order Diphyl‐ lobothriidea that belongs to family *Diphyllobothriidae* and genus *Diphyllobothrium*.

Although, 12 species have been recognized, only four are of public health concern and produce human pathology: *Echinococcus granulosus* (cystic echinococcosis), *E. multilocularis* (alveolar echinococcosis), *E. vogeli*, and *E. oligarthrus* (both causing polycystic echinococcosis). The first two species are etiological agents of life-threatening diseases, having high fatality rate and poor prognosis if careful clinical management is not given [2]. *E. vogeli* and *E. oligarthrus* are responsible for polycystic echinococcosis in Central and South America [2]. Majority of the cases reported in these regions, till 2008, have corresponded to *E. vogeli* (54 of 172; 31.4%) and *E.* cf. *vogeli* (*confer*, *species affinis*) (114 of 172; 66.3%) [3]. Only three cases (1.7%) have been identified that have corresponded to *E. oligarthrus* and one has corresponded to *E. granulosus* (0.6%) [3]. In the past, it was believed that *E. multilocularis* was not present in Central and South America.

Living or having lived in rural areas where farming and animal husbandry are common is an important risk factor for this disease, in some areas particularly where people frequently come in contact with dogs that accompany paca hunters as well as where wild animal hunting for food is common practice (e.g., hunters and aboriginal people) [4]. In addition, new population migration patterns over the past four decades have caused a change in the profile of this disease, allowing its worldwide distribution [4]. In addition, in general, now it is considered that there is a risk of getting echinococcosis infection, as well other cestodiasis, while traveling to endemic zones.

This disease is related with extensive livestock or poor health infrastructures; usually associ‐ ated with a low socioeconomic level and lack of education. Its public health importance is related not only to the high fatality rate in humans but also with reduced work performance, costs of hospitalization, interventions, and disabilities [5].

Although echinococcosis has been recognized for centuries (at the 17th century, Francesco Redi illustrated that the hydatid cysts of echinococcosis were of "animal" origin), in some countries

of the neotropic region, such as Colombia, this disease was discovered during 20th century). Studies in this context in this country, as well as in other countries of South America, are still limited. Therefore, the objective of this chapter is to summarize and describe epidemiological and clinical updated aspects of this disease, focusing on evidences and published research articles on echinococcosis in Colombia, finally questioning if this cestode zoonotic disease has been largely neglected [1-84]. 5 This disease is related with extensive livestock or poor health infrastructures; usually associated with a low 6 socioeconomic level and lack of education. Its public health importance is related not only to the high fatality rate in 7 humans but also with reduced work performance, costs of hospitalization, interventions, and disabilities [5]. 9 Although echinococcosis has been recognized for centuries (at the 17th century, Francesco Redi illustrated that the 10 hydatid cysts of echinococcosis were of "animal" origin), in some countries of the neotropic region, such as Colombia, 11 this disease was discovered during 20th century). Studies in this context in this country, as well as in other countries of 12 South America, are still limited. Therefore, the objective of this chapter is to summarize and describe epidemiological 13 and clinical updated aspects of this disease, focusing on evidences and published research articles on echinococcosis in

3 echinococcosis infection, as well other cestodiasis, while traveling to endemic zones.

14 Colombia, finally questioning if this cestode zoonotic disease has been largely neglected [1-84].

18 <FIG>Figure 1. Scolex and hooks of *Echinococcus granulosus* (pictures by A. J. Rodriguez-Morales). **Figure 1.** Scolex and hooks of *Echinococcus granulosus* (pictures by A. J. Rodriguez-Morales).

#### 23 (Cystic echinococcosis) 25 *E. granulosus* is a cestode whose life cycle involves dogs (*Canis lupus familiaris*) and other canids (coyote, dingo, red fox, **2. Organisms causing human disease**

#### 26 among others) as definitive hosts for the intestinal tapeworm, which are infected by ingestion of food containing 27 hydatid cysts with viable protoscoleces, and domestic and wild ungulates as intermediate hosts for the tissue invading **2.1.** *Echinococcus granulosus*

<sup>20</sup> **<H1>2. Organisms causing human disease** <sup>21</sup>

#### 28 metacestode (larval) stage (Figure 1) [1]. The metacestode (or hydatid cyst) consists of two parasite-derived layers: an 29 inner nucleated germinal layer, and an outer acellular laminated layer surrounded by a host-derived fibrous capsule [2]. 30 Intermediate host species depends on the infecting strain of *E. granulosus*, regional or local differences in the availability (Cystic echinococcosis)

22 *Echinococcus granulosus* 

4

8

15

16 17

19

24

43

**1. Introduction**

4 Current Topics in Echinococcosis

of *Spirometra*), among others.

America.

to endemic zones.

Echinococcosis or hydatid disease is a near-cosmopolitan parasitic zoonosis caused by the larval stages (paratenic for humans) of cestodes of the genus *Echinococcus* (Figure 1) [1, 2]. This helminthiasis causes an important economic loss over agricultural and pastoral regions of limited resources, particularly in developing countries [3]. In addition, it is also important to notice that this is one of the important infectious disease groups caused by larval stages of cestodes, which also include cysticercosis (caused by larval stages of *Taenia solium*), coenuriasis (caused by larval stages of *Taenia multiceps gaigeri*), and sparganosis (caused by larval stages

Biologically and taxonomically, although in the past it was considered as part of the genus *Taenia*, *Echinococcus* genus belongs to the family *Taeniidae* (order Cyclophyllidea, subclass Eucestoda, class Cestoda, phylum Platyhelminthes). Family *Taeniidae* also includes the genus *Taenia*. Order Cyclophyllidea has two important families that infect humans: *Hymenolepidi‐ dae* (*Hymenolepis*) and *Dipylidiidae* (*Dipylidium*). Subclass Eucestoda includes the order Diphyl‐

Although, 12 species have been recognized, only four are of public health concern and produce human pathology: *Echinococcus granulosus* (cystic echinococcosis), *E. multilocularis* (alveolar echinococcosis), *E. vogeli*, and *E. oligarthrus* (both causing polycystic echinococcosis). The first two species are etiological agents of life-threatening diseases, having high fatality rate and poor prognosis if careful clinical management is not given [2]. *E. vogeli* and *E. oligarthrus* are responsible for polycystic echinococcosis in Central and South America [2]. Majority of the cases reported in these regions, till 2008, have corresponded to *E. vogeli* (54 of 172; 31.4%) and *E.* cf. *vogeli* (*confer*, *species affinis*) (114 of 172; 66.3%) [3]. Only three cases (1.7%) have been identified that have corresponded to *E. oligarthrus* and one has corresponded to *E. granulosus* (0.6%) [3]. In the past, it was believed that *E. multilocularis* was not present in Central and South

Living or having lived in rural areas where farming and animal husbandry are common is an important risk factor for this disease, in some areas particularly where people frequently come in contact with dogs that accompany paca hunters as well as where wild animal hunting for food is common practice (e.g., hunters and aboriginal people) [4]. In addition, new population migration patterns over the past four decades have caused a change in the profile of this disease, allowing its worldwide distribution [4]. In addition, in general, now it is considered that there is a risk of getting echinococcosis infection, as well other cestodiasis, while traveling

This disease is related with extensive livestock or poor health infrastructures; usually associ‐ ated with a low socioeconomic level and lack of education. Its public health importance is related not only to the high fatality rate in humans but also with reduced work performance,

Although echinococcosis has been recognized for centuries (at the 17th century, Francesco Redi illustrated that the hydatid cysts of echinococcosis were of "animal" origin), in some countries

costs of hospitalization, interventions, and disabilities [5].

lobothriidea that belongs to family *Diphyllobothriidae* and genus *Diphyllobothrium*.

31 of the various intermediate host species, and other factors [2], but would include primarily ungulates, also marsupials 32 [which for the case of Colombia would make think the possibility of occurrence in the country given the wide presence 33 of them, such as *Didelphis marsupialis* (locally called "chucha")]. Ten strains of *E. granulosus* have been identified so far 34 (G1–G10) with different host affinities: G1–G2 being sheep strains, G3 and G5 bovid strains, G4 a horse strain, G6 a 35 camelid strain, G7 a pig strain, G8 a cervid strain, G9 has been described in swine in Poland, and G10 in reindeer in 36 Eurasia [1, 81]. The sheep strain (G1) is most commonly associated with human infections [1]. The presence of diverse 37 strains of *E. granulosus* has important implications for public health. The shortened maturation time of the adult form of 38 the parasite in the intestine of dogs suggests that the interval period for administering anti-parasite drugs to infected 39 dogs will have to be shortened in those areas where the G2, G5, and G6 strains are found [1]. The adult *E. granulosus* is 40 2.0 to 7.0 mm in length. It has three proglottids (range 4–6). It is supposed that *E. granulosus* is found worldwide; 41 however, in many countries (including Colombia and other countries in South America), its presence has not been 42 confirmed both in humans and in animals. Hydatid cysts are predominantly found in liver and lungs (Figure 1). *E. granulosus* is a cestode whose life cycle involves dogs (*Canis lupus familiaris*) and other canids (coyote, dingo, red fox, among others) as definitive hosts for the intestinal tapeworm, which are infected by ingestion of food containing hydatid cysts with viable protoscoleces, and domestic and wild ungulates as intermediate hosts for the tissue invading metacestode (larval) stage (Figure 1) [1]. The metacestode (or hydatid cyst) consists of two parasite-derived layers:an inner nucleated germinal layer, and an outer acellular laminated layer surrounded by a host-derived fibrous capsule [2]. Intermediate host species depends on the infecting strain of *E. granulosus*, regional or local differences in the availability of the various intermediate host species, and other factors [2], but would include primarily ungulates, also marsupials [which for the case of Colombia would make think the possibility of occurrence in the country given the wide presence of them, such as *Didelphis marsupialis* (locally called "chucha")]. Ten strains of *E. granulosus* have been identified so far (G1–G10) with different host affinities: G1–G2 being sheep strains, G3 and G5 bovid strains, G4 a horse strain, G6 a camelid strain, G7 a pig strain, G8 a cervid strain, G9 has been described in swine in Poland, and G10 in reindeer in Eurasia [1, 81]. The sheep strain (G1) is most commonly associated with human infections [1]. The presence of diverse strains of *E. granulosus* has important implications for public health. The shortened maturation time of the adult form of the parasite in the intestine of dogs suggests that the interval period for administering anti-parasite drugs to infected dogs will have to be shortened in those areas where the G2, G5, and G6 strains are found [1]. The adult *E. granulo‐ sus* is 2.0 to 7.0 mm in length. It has three proglottids (range 4–6). It is supposed that *E. granulosus* is found worldwide; however, in many countries (including Colombia and other countries in South America), its presence has not been confirmed both in humans and in animals. Hydatid cysts are predominantly found in liver and lungs (Figure 1).

#### **2.2.** *Echinococcus multilocularis*

#### (Alveolar echinococcosis)

*E. multilocularis* is the etiological agent of the alveolar echinococcosis, its infection is perpetu‐ ated in a sylvatic cycle, with wild carnivores—mainly red (*Vulpes vulpes*) and arctic (*Alopex lagopis*) foxes—regarded as the most important definitive hosts. Domestic dogs and cats may be involved in a synanthropic cycle, and also raccoon dog and coyotes [2]. Small mammals (usually microtine and arvicolid rodents) act as intermediate hosts. The metacestode of *E. multilocularis* is a tumor-like, infiltrating structure consisting of many small vesicles embedded in stroma of connective tissue. The metacestode mass usually contains a semisolid matrix rather than fluid. The adult tapeworm is different in size than adult *E. granulosus* (1.2–4.5 mm) and its eggs are indistinguishable by light microscopy. The hydatid cysts do not form hyaline membrane and the scolices, which are abundant in rodents but absent in humans. People become exposed to *E. multilocularis* by handling of infected hosts, or by ingestion of food contaminated with eggs of the parasite [2].

It is endemic in North America, Alaska, Europe, Central Russia, China, Turkey, Japan, Uruguay, and Argentina. In humans, the most frequently affected organs are the liver (80%), lungs (10%), brain (5%), and other organs such as kidney and pancreas (5%) [6]. *E. multilocu‐ laris* has five proglottids (range 2–6). Hydatid cysts are primarily hepatic, with metastases in lungs, brain, and bones among other organs.

#### **2.3.** *Echinococcus oligarthrus*

#### (Polycystic echinococcosis)

*E. oligarthrus* is the smallest species of the genus, it only measures, in its adult form, 2.2 to 2.9 mm. Definitive hosts are different species of wild felidae (pampas cats, Geoffrey's cat, ocelot, jaguar, cougar, jaguarandi, puma, boat cats) and intermediate hosts are rodents like rats (spiny rat), agoutis, and pacas (both, very highly distributed in the whole territory of Colombia and in most of the tropical areas of South America, including Brazil, Paraguay, Bolivia, Peru, Ecuador, Venezuela, Guyana, Suriname, and French Guyana) [6]. Hydatid cysts are located in an extrahepatic location, mostly in the intermuscular tissue, pleural, and peritoneal cavity [6]. *E. oligarthrus* has three proglottids. Larval parasite of this species can be also located in eye and heart.

#### **2.4.** *Echinococcus vogeli*

G8 a cervid strain, G9 has been described in swine in Poland, and G10 in reindeer in Eurasia [1, 81]. The sheep strain (G1) is most commonly associated with human infections [1]. The presence of diverse strains of *E. granulosus* has important implications for public health. The shortened maturation time of the adult form of the parasite in the intestine of dogs suggests that the interval period for administering anti-parasite drugs to infected dogs will have to be shortened in those areas where the G2, G5, and G6 strains are found [1]. The adult *E. granulo‐ sus* is 2.0 to 7.0 mm in length. It has three proglottids (range 4–6). It is supposed that *E. granulosus* is found worldwide; however, in many countries (including Colombia and other countries in South America), its presence has not been confirmed both in humans and in

*E. multilocularis* is the etiological agent of the alveolar echinococcosis, its infection is perpetu‐ ated in a sylvatic cycle, with wild carnivores—mainly red (*Vulpes vulpes*) and arctic (*Alopex lagopis*) foxes—regarded as the most important definitive hosts. Domestic dogs and cats may be involved in a synanthropic cycle, and also raccoon dog and coyotes [2]. Small mammals (usually microtine and arvicolid rodents) act as intermediate hosts. The metacestode of *E. multilocularis* is a tumor-like, infiltrating structure consisting of many small vesicles embedded in stroma of connective tissue. The metacestode mass usually contains a semisolid matrix rather than fluid. The adult tapeworm is different in size than adult *E. granulosus* (1.2–4.5 mm) and its eggs are indistinguishable by light microscopy. The hydatid cysts do not form hyaline membrane and the scolices, which are abundant in rodents but absent in humans. People become exposed to *E. multilocularis* by handling of infected hosts, or by ingestion of food

It is endemic in North America, Alaska, Europe, Central Russia, China, Turkey, Japan, Uruguay, and Argentina. In humans, the most frequently affected organs are the liver (80%), lungs (10%), brain (5%), and other organs such as kidney and pancreas (5%) [6]. *E. multilocu‐ laris* has five proglottids (range 2–6). Hydatid cysts are primarily hepatic, with metastases in

*E. oligarthrus* is the smallest species of the genus, it only measures, in its adult form, 2.2 to 2.9 mm. Definitive hosts are different species of wild felidae (pampas cats, Geoffrey's cat, ocelot, jaguar, cougar, jaguarandi, puma, boat cats) and intermediate hosts are rodents like rats (spiny rat), agoutis, and pacas (both, very highly distributed in the whole territory of Colombia and in most of the tropical areas of South America, including Brazil, Paraguay, Bolivia, Peru, Ecuador, Venezuela, Guyana, Suriname, and French Guyana) [6]. Hydatid cysts are located in an extrahepatic location, mostly in the intermuscular tissue, pleural, and peritoneal cavity [6].

animals. Hydatid cysts are predominantly found in liver and lungs (Figure 1).

**2.2.** *Echinococcus multilocularis*

contaminated with eggs of the parasite [2].

lungs, brain, and bones among other organs.

**2.3.** *Echinococcus oligarthrus*

(Polycystic echinococcosis)

(Alveolar echinococcosis)

6 Current Topics in Echinococcosis

#### (Polycystic echinococcosis)

*E. vogeli* also causes polycystic echinococcosis and has been reported only in Central and South America. However, not every country in this region has reported cases of *E. vogeli*. Brazil (98 cases) and Colombia (30 cases) have been the countries with the highest number of reported cases, till 2010 (including *E.* cf. *vogeli*) [6]. The adult tapeworm measures 5.6 mm. The eggs are similar to that of the *Taenia* genus. Definitive hosts are domestic and wild dogs (bush dog and domestic dog). The bush dog, *Speothos venaticus*, is widely distributed in South America, particularly not only in Brazil (>80% of the territory) but also in most parts of Venezuela, Guyana, Suriname, and French Guyana, as well as in some areas of Colombia, Panama, Ecuador, Peru, Paraguay, Argentina, and Uruguay. Intermediate hosts are rodents, but mainly the agoutis and pacas. In this species, the hydatid cyst is of endogenous proliferation and it forms bags or folds inside the primary bladder [6]. While in rodent the larvae is limited, in humans it disseminates, due to exogenous vesiculation and due to the amount of scolice inside the cyst, which can cause reinfection when it breaks spontaneously or by medical manipulation [6]. The adult *E. vogeli* is 3.9 to 5.6 mm in length. *E. vogeli* has three proglottids.

The results of a survey (carried out to detect *Echinococcus* infections) involving more than 4,000 Colombian mammals showed interesting findings regarding *E. vogeli* and *E. oligarthrus*. Adult worms were found in 5 of 121 carnivores: *E. oligarthrus* in 1 of 11 ocelots, 2 of 9 jaguarundi cats, and single puma; and *E. vogeli* in 1 of 15 domestic dogs. Although bush dogs were present, none could be examined. Polycystic larvae were found in 96 of 325 pacas (29.5%) and in 6 of 1,168 (0.5%) spiny rats, *Proechimys spp*. None of the 118 agouti showed hydatid cysts but an infected heart was provided by hunters. The paca's infection rate increased with age but was not related to gender or geographic region. In 73 of 96 pacas, the infection was due to *E. vogeli*, and the cysts were located in the liver. In three cases, it was due to *E. oligarthrus* and the hydatid cysts were extrahepatic, mainly attached to muscles. In the remaining 20, the species involved could not be determined. The parasites in two of the spiny rats, and in the agouti heart were *E. oligarthrus* [48]. Although most of the infected animals were collected in the eastern plains, other records and verbal information indicate that, at least in Colombia where man has not exterminated pacas, agoutis, wild canids, and felids, one still can find enzootic neotropical *Echinococcus* infection. The cycle of *E. vogeli* involves the bush dog and paca as hosts, and that of *E. oligarthrus*, the paca, agouti, spiny rat, and several species of wild felids [48].

Other species in the genus *Echinococcus* have also been discovered and identified. Currently, there are 12 species, including those four causing human pathology, six identified in animals: *E. canadensis* (main intermediate host: cervids; known definite hosts: wolves, dog), *E. equinus* (main intermediate host: horses; known definite host: dogs), *E. felidis* (main intermediate host: warthogs; known definite host: lions), *E. intermedius* (main intermediate hosts: camels, pigs, sheep; known definite hosts lions), *E. ortleppi* (main intermediate host: cattle; known definite hosts dogs), and *E. shiquicus* (main intermediate host: pika; known definite host: Tibetan fox); and two to be assigned to name species (*Echinococcus sp*. GL-2010 and *Echinococcus sp*. GL-2012) (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=6209) [46].

Some authors have recently exposed that all these species would be infectious for hu‐ mans, probably with the exception of *E. equinus*, *E. felidis*, and *E. shiquicus*, and also the two to be assigned to name species [46]. Probably, with more studies in molecular epidemiolo‐ gy in the future, their role will become clear.. However, it is interesting to note that at the GenBank (http://www.ncbi.nlm.nih.gov/gene/?term=echinococcus), there are 271 genes identified from different species of *Echinococcus*: *E. oligarthrus* (36 genes), *E. vogeli* (36 genes), *E. shiquicus* (36 genes), *E. canadensis* (36 genes), *E. equinus (36* genes), *E. felidis* (36 genes), *E. ortleppi* (14 genes), *E. multilocularis* (12 genes), and *E. granulosus* (12 genes). At Gen‐ Bank, there are also 43,971 sequences of proteins of the genus *Echinococcus* (http:// www.ncbi.nlm.nih.gov/protein/?term=echinococcus): *E. granulosus* (24,310 sequences), *E. multilocularis* (18,670 sequences), *E. canadensis* (476 sequences), *E. shiquicus* (111 sequen‐ ces), *E. equinus* (77 sequences), *E. ortleppi* (66 sequences), *E. vogeli* (51 sequences), *E. oligarthrus* (47 sequences), and *E. felidis* (34 sequences).

More importantly, a draft genomic sequence for *E. granulosus* comprising 151.6 Mb encoding 11,325 genes has been achieved [47], including the evaluation of the regulation of genes in the adult, oncosphere, hydatid cyst, and protoscoleces, which would be of great significance for further studies in diagnostic as well in the immunology and vaccine development, among other things.

#### **3. Life cycle**

The adult *E. granulosus* lives in the small intestine of the definitive hosts (dogs or other canids) (Figure 2). Gravid proglottids release eggs that are passed in the feces of these animals (which would come in close contact with humans). After ingestion by a suitable intermediate host (sheep, goat, swine, cattle, horses, camel, under usual natural conditions), embryonated eggs are hatched in the small intestine of the intermediate host and release the oncosphere which penetrates the intestinal wall and migrates through the circulatory system into various organs, especially the liver and lungs (Figure 2).

In these organs, the oncosphere develops into a cyst that enlarges gradually, producing protoscolices and daughter cysts that fill the cyst interior (Figure 2). The definitive host becomes infected by ingesting the cyst-containing organs of the infected intermediate host (Figure 2). After ingestion, the protoscolices evaginate, attach to the intestinal mucosa, and develop into adult stages in approximately 32 to 80 days (Figure 2). Humans can also get infected by ingesting eggs from feces of definitive hosts infected, then resulting with the release of oncospheres in the intestine and the development of cysts in different organs (mainly liver and lungs) (Figure 2).

The same life cycle occurs with *E. multilocularis*, *E. vogeli*, and *E. oligarthrus*, with the differences in the definitive or main hosts and the intermediate hosts involved for these species (Figure 2).

**Figure 2.** Life cycle of echinococcosis (pictures by A. J. Rodriguez-Morales).

#### **4. Geographical distribution**

and two to be assigned to name species (*Echinococcus sp*. GL-2010 and *Echinococcus sp*. GL-2012)

Some authors have recently exposed that all these species would be infectious for hu‐ mans, probably with the exception of *E. equinus*, *E. felidis*, and *E. shiquicus*, and also the two to be assigned to name species [46]. Probably, with more studies in molecular epidemiolo‐ gy in the future, their role will become clear.. However, it is interesting to note that at the GenBank (http://www.ncbi.nlm.nih.gov/gene/?term=echinococcus), there are 271 genes identified from different species of *Echinococcus*: *E. oligarthrus* (36 genes), *E. vogeli* (36 genes), *E. shiquicus* (36 genes), *E. canadensis* (36 genes), *E. equinus (36* genes), *E. felidis* (36 genes), *E. ortleppi* (14 genes), *E. multilocularis* (12 genes), and *E. granulosus* (12 genes). At Gen‐ Bank, there are also 43,971 sequences of proteins of the genus *Echinococcus* (http:// www.ncbi.nlm.nih.gov/protein/?term=echinococcus): *E. granulosus* (24,310 sequences), *E. multilocularis* (18,670 sequences), *E. canadensis* (476 sequences), *E. shiquicus* (111 sequen‐ ces), *E. equinus* (77 sequences), *E. ortleppi* (66 sequences), *E. vogeli* (51 sequences), *E.*

More importantly, a draft genomic sequence for *E. granulosus* comprising 151.6 Mb encoding 11,325 genes has been achieved [47], including the evaluation of the regulation of genes in the adult, oncosphere, hydatid cyst, and protoscoleces, which would be of great significance for further studies in diagnostic as well in the immunology and vaccine development, among

The adult *E. granulosus* lives in the small intestine of the definitive hosts (dogs or other canids) (Figure 2). Gravid proglottids release eggs that are passed in the feces of these animals (which would come in close contact with humans). After ingestion by a suitable intermediate host (sheep, goat, swine, cattle, horses, camel, under usual natural conditions), embryonated eggs are hatched in the small intestine of the intermediate host and release the oncosphere which penetrates the intestinal wall and migrates through the circulatory system into various organs,

In these organs, the oncosphere develops into a cyst that enlarges gradually, producing protoscolices and daughter cysts that fill the cyst interior (Figure 2). The definitive host becomes infected by ingesting the cyst-containing organs of the infected intermediate host (Figure 2). After ingestion, the protoscolices evaginate, attach to the intestinal mucosa, and develop into adult stages in approximately 32 to 80 days (Figure 2). Humans can also get infected by ingesting eggs from feces of definitive hosts infected, then resulting with the release of oncospheres in the intestine and the development of cysts in different organs (mainly liver

The same life cycle occurs with *E. multilocularis*, *E. vogeli*, and *E. oligarthrus*, with the differences in the definitive or main hosts and the intermediate hosts involved for these species (Figure 2).

(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=6209) [46].

*oligarthrus* (47 sequences), and *E. felidis* (34 sequences).

other things.

8 Current Topics in Echinococcosis

**3. Life cycle**

and lungs) (Figure 2).

especially the liver and lungs (Figure 2).

Cystic echinococcosis or cystic hydatidosis (caused by *E. granulosus*) is an important public health problem in South America, the Middle East and Eastern Mediterranean, some sub-Saharan African countries, China, the former Soviet Union, Russia, and the new related countries (Figure 3) [7, 8]. The overall prevalence of echinococcal infection is underestimated in many countries because systematic population surveys are not performed in all endemic areas, and this disease is not under surveillance neither in humans nor in animals in most of these countries [9]. Even more, in some countries, such as Colombia, there are reports from animal health authorities claiming that echinococcosis as a disease has "never" been registered in the country [49]. In endemic areas, its incidence has been estimated to be around 50 cases per 100,000 population per year [10].

In South America, although there is underreporting, it has been estimated that each year there would be occurring 2,000 cases [3], but these are not the exact figures. Uruguay has reported 9.2 cases per 100,000 population in 1995, and Chile has reported between 6.6 and 8.4 new cases per 100,000 population per year. Besides this, echinococcosis is also a health problem in Argentina, Bolivia, Brazil, and Peru [10, 82]. In the Peruvian Andes, observed prevalence rates in one study among humans, canines, and sheep were 9%, 46%, and 32%, respectively [9].

World Health Organization (WHO) had developed a global map of the distribution of *E. granulosus* and cystic echinococcosis (hydatidosis, 2009); however, there is no recent official WHO map for distribution of other *Echinococcus* species (Figure 3) [50]. However, in 2001, WHO and World Organization for Animal Health also developed a map of the approxi‐

**Figure 3.** Distribution of *Echinococcus granulosus* and cystic echinococcosis (hydatidosis), 2009 (2010) [50].

mate geographical distribution of *E. multilocularis* and alveolar echinococcosis (1999) (Figure 4) [51, 52].

*E. multilocularis* is limited to the northern hemisphere (Figure 4). It occurs in Central and Eastern Europe, the former Soviet Union (now Russia), Turkey, Iraq, northern India, central China, some Japanese islands, some provinces of Canada, Alaska, and some north central states of the United States [9] (Figure 4).

Distribution of *E. oligarthrus* and *E. vogeli* is limited to South and Central America, but there is no precise map regarding their presence in the countries of these regions.

Since definitive host of *E. vogeli* exists only from Panama to the North of Argentina, cases of polycystic hydatidosis outside of this region of the world are probably imported by *E. oligarthrus* [9]. However, there are no precise estimations of *E. oligarthrus*. An attempt to map the distribution of *E. vogeli* was presented in 2005 by Colombian authors (Figure 5).

Normally, infections from species of the genus *Echinococcus* involve wild and rural hosts; however, due to human actions (temporary by recreation and work, but also definitive) which have permanently used dogs in work grazing and fed them with viscera of sacrificed animals, this cycle has been altered [6]. Since all types of echinococcosis or hydatidosis are acquired by ingestion of water or food contaminated with the eggs in the feces shed by the carnivores, it

**Figure 4.** Approximate geographical distribution of *Echinococcus multilocularis* and alveolar echinococcosis (1999) [51, 52].

mate geographical distribution of *E. multilocularis* and alveolar echinococcosis (1999) (Figure

**Figure 3.** Distribution of *Echinococcus granulosus* and cystic echinococcosis (hydatidosis), 2009 (2010) [50].

*E. multilocularis* is limited to the northern hemisphere (Figure 4). It occurs in Central and Eastern Europe, the former Soviet Union (now Russia), Turkey, Iraq, northern India, central China, some Japanese islands, some provinces of Canada, Alaska, and some north central states

Distribution of *E. oligarthrus* and *E. vogeli* is limited to South and Central America, but there is

Since definitive host of *E. vogeli* exists only from Panama to the North of Argentina, cases of polycystic hydatidosis outside of this region of the world are probably imported by *E. oligarthrus* [9]. However, there are no precise estimations of *E. oligarthrus*. An attempt to map

Normally, infections from species of the genus *Echinococcus* involve wild and rural hosts; however, due to human actions (temporary by recreation and work, but also definitive) which have permanently used dogs in work grazing and fed them with viscera of sacrificed animals, this cycle has been altered [6]. Since all types of echinococcosis or hydatidosis are acquired by ingestion of water or food contaminated with the eggs in the feces shed by the carnivores, it

the distribution of *E. vogeli* was presented in 2005 by Colombian authors (Figure 5).

no precise map regarding their presence in the countries of these regions.

4) [51, 52].

10 Current Topics in Echinococcosis

of the United States [9] (Figure 4).

**Figure 5.** Drawing illustrates the geographic distribution of hydatid disease from *Echinococcus granulosus* (red dots), *E. multilocularis* (black dots), and *E. vogeli* (green dots). *E. granulosus* is the most common of the *Echinococcus* species (1999) [53]. This is not precise and requires more specific information from the countries regarding the distribution of cases by the territories included in the map.

is advisable to take special care with the feces of these animals, especially when considering that these eggs resist partial desiccation for several months [6].

### **5. Echinococcosis studies in Colombia**

In Colombia, echinococcosis does not seem to be a major problem if we base such assumptions from human and animal health authorities [49]. But, as has been evidenced, cases of infection with the larval forms of *E. granulosus* have been reported [11]. However, prevalence and incidence of this entity have not been determined. Echinococcosis is not under surveillance and there is no obligation to report cases related to this infection to health authorities in the country; then known cases have been just those reported in national and international biomedical literature (Figure 6).

**Figure 6.** Departments of Colombia where cases of echinococcosis have been reported. (First date reported is the year of the case, then the location, year of publication in parenthesis, and main author.)

The first case was reported in 1941 [12], but since 1957, more cases had been reported [54] (Figure 6). Two decades later, D'Alessandro began studies in the country. This author found more than a dozen of human cases of polycystic echinococcosis produced by the larval stage of *E. vogeli* whose definitive hosts were wild and domestic dogs and intermediate host was paca ("guagua," *Cuniculus paca*) [3, 55], after suspecting the presence of disease due to four cases of calcified hydatid cysts [55].

is advisable to take special care with the feces of these animals, especially when considering

In Colombia, echinococcosis does not seem to be a major problem if we base such assumptions from human and animal health authorities [49]. But, as has been evidenced, cases of infection with the larval forms of *E. granulosus* have been reported [11]. However, prevalence and incidence of this entity have not been determined. Echinococcosis is not under surveillance and there is no obligation to report cases related to this infection to health authorities in the country; then known cases have been just those reported in national and international

**Figure 6.** Departments of Colombia where cases of echinococcosis have been reported. (First date reported is the year

The first case was reported in 1941 [12], but since 1957, more cases had been reported [54] (Figure 6). Two decades later, D'Alessandro began studies in the country. This author found

of the case, then the location, year of publication in parenthesis, and main author.)

that these eggs resist partial desiccation for several months [6].

**5. Echinococcosis studies in Colombia**

biomedical literature (Figure 6).

12 Current Topics in Echinococcosis

Most of the infected people were native from Colombia who acquired the disease by ingesting food or water contaminated with dog feces and by eating raw viscera of "guagua." Botero et al. describe three more unpublished cases: two from the coast of the Pacific Ocean and one from Santa Marta [13, 14]. D'Alessandro's studies demonstrated that in Colombia, the guagua is the main intermediate host and the wild dogs known as gouache fox is the definitive host. The guatin, which is also an edible animal for Colombian indigenous tribes, is a more specific host for *E. oligarthrus* [6].

Till 2008, the country in South America with the highest number of cases reported was Brazil, with 99 cases, followed by Colombia with 29 (Figure 6) [3, 50-60]. In Colombia, some authors believe that there are more cases not reported, in fact due to the lack of records. This also probes that occurrence of echinococcosis is more evidently reported, when people in the rural and jungle areas receive health services and physicians are more aware of the disease [60].

Two years later, in 2010, another and the last case till March 2015, has been reported [61]. A case of cardiac echinococcosis, detected in echocardiographic finding in an 18-year-old woman diagnosed in Medellin, Antioquia [61]. Fortunately in this case, physicians suspected of this cestode infection; however, more awareness among physicians regarding the compromise of cardiovascular system in many parasitic diseases is still necessary, particularly in endemic areas [63-67].

Based on this review of cases, including information from the departments of the country where this disease has been reported, a map for Colombia has been proposed (Figure 6). This map shows the departments of the country (including locations of origin of one or more cases), where echinococcosis have been reported (Figure 6). At least 10 departments (out of 32 in total) have reported cases of echinococcosis (Figure 6) [14, 54, 59-61, 72].

Beyond this, with an increasing number of published reports concerning this disease, medical personnel in tropical areas, as in Colombia and more recently in Brazil, have been keenly interested in undertaking epidemiological surveys [3, 68, 69]. Even more, there is a significant zoonotic potential, given the fact that the investigations in Colombia demonstrated a large enzootic areas of transmission in the oriental plains of that country [3, 58], where multiple species and a high number of animals have been found positive in the evaluations for *Echinococcus*, particularly *E. oligarthrus*.

As in other zoonotic diseases, parasitic and of other etiologies, there is a clear need of more studies, as well as more interaction between physicians and veterinarians. More research is necessary; currently, there are just 10 reports available in Medline for Colombia (using the terms "Colombia" AND "Echinococc\*" [means *Echinococcus* and/or echinococcosis]) [53, 55-60, 62, 70, 71]. There is no significantly different information available at Scopus. At SciELO, there is one additional case, a patient with hepatic hydatidosis who appeared as an eosinophilic pleural effusion serologically confirmed, treated with combined therapy with albendazole and the percutaneous aspiration-injection-reaspiration drainage technique [72].

Finally, an undergoing research of our group is analyzing a new source of information for communicable diseases, which are not under surveillance and require compulsory notifica‐ tion. In Colombia, there is a service, which is now available for analyses, the so-called personal health records (*Registro Individual de Prestación de Servicios*, RIPS), where diagnoses are classified according to WHO cause groups International Classification of Diseases version 10 (ICD-10). At this system, we retrieved the cases of echinococcosis between years 2009 and 2013, finding 303 cases (Figure 7).

**Figure 7.** Cases of echinococcosis (ICD-10 codes B67.0 to B67.9) reported to the personal health records of Colombia (RIPS) during 2009–2013.

Although these cases corresponded in 18.2% to "Echinococcosis, unspecified, of liver" (B67.8) and 21.5% to "Echinococcosis, other and unspecified" (B67.9), there are 60.4% (n = 183) of them classified as infection due to *E. granulosus* (47.5%) and *E. multilocularis* (12.9%) in different organs (Figure 7), mainly lung for *E. granulosus* (17.8%) (Figure 7). Although there are limita‐ tions to this source, there is useful information that should be further analyzed. Epidemiolog‐ ical factors (geographic distribution, age groups, gender) involved in these cases would be of high interest in describing the situation of echinococcosis in the country.

In these cases, looked retrospectively, the limitation in the approach is that there is no information regarding the diagnostic techniques implemented in each case, and how these cases were therapeutically managed. Then, based on this information, cross-sectional and prospective studies should be implemented in the countries, and there should also be an increase in the awareness about the existence and occurrence of such diseases in different areas of the country, and particularly in those that had previously reported case reports and case series since early decades (Figure 6).

#### **6. Economic burden of echinococcosis**

pleural effusion serologically confirmed, treated with combined therapy with albendazole and

Finally, an undergoing research of our group is analyzing a new source of information for communicable diseases, which are not under surveillance and require compulsory notifica‐ tion. In Colombia, there is a service, which is now available for analyses, the so-called personal health records (*Registro Individual de Prestación de Servicios*, RIPS), where diagnoses are classified according to WHO cause groups International Classification of Diseases version 10 (ICD-10). At this system, we retrieved the cases of echinococcosis between years 2009 and 2013,

**Figure 7.** Cases of echinococcosis (ICD-10 codes B67.0 to B67.9) reported to the personal health records of Colombia

Although these cases corresponded in 18.2% to "Echinococcosis, unspecified, of liver" (B67.8) and 21.5% to "Echinococcosis, other and unspecified" (B67.9), there are 60.4% (n = 183) of them classified as infection due to *E. granulosus* (47.5%) and *E. multilocularis* (12.9%) in different organs (Figure 7), mainly lung for *E. granulosus* (17.8%) (Figure 7). Although there are limita‐ tions to this source, there is useful information that should be further analyzed. Epidemiolog‐ ical factors (geographic distribution, age groups, gender) involved in these cases would be of

high interest in describing the situation of echinococcosis in the country.

the percutaneous aspiration-injection-reaspiration drainage technique [72].

finding 303 cases (Figure 7).

14 Current Topics in Echinococcosis

(RIPS) during 2009–2013.

According to the World Bank, economic losses due to this disease stand at the fourth position after other diseases and trauma [3].

Echinococcosis also affects the economy of the endemic regions. In humans, losses are due to costs of hospitalization, treatment, disability, etc. However, in livestock, losses are due to lost productivity, losses in quality of the wool and meat, and decreased milk production and fertility [15]. Estimated annual economic losses in Peru are \$ 178,705 [10]. In Colombia, estimation of the economic burden of this disease has not been reported, but given the increase in the number of economic evaluations of infectious diseases in the country and in the region [73, 74], there would be more clear details once the number of cases reported would be defined.

#### **7. Clinical manifestations**

Regardless of the etiology, a new concept of organ compromise was proposed in 2014, when two or more organs are affected by echinococcosis at the same time in a patient. This has been defined as multivisceral echinococcosis, simultaneous localization of hydatid cysts in more than one organ [83]. Multivisceral echinococcosis should be differentiated from multiple echinococcosis [83]. The latter is the localization of multiple cysts in one organ [83]. Even in paired organs (lungs, kidneys) if both are compromised, it will be a case of multivisceral echinococcosis, given the implications not just in diagnosis, but particularly in surgical management.

#### **7.1.** *Echinococcus granulosus*

Echinococcosis is a larval infection caused in humans by *Echinococcus* species, a metacestode with the ability to create a large spectrum of clinical manifestations, including the asympto‐ matic infection, and a severe and fatal disease. There are four species of echinococcosis that have been recognized that cause various forms of the disease, specifically the *E. granulosus* species which is responsible for cystic echinococcosis [16].

#### *7.1.1. Cystic echinococcosis*

As a general rule, the phase of primary infection is always asymptomatic because the cysts are located in different organs, characterized by their small size, which is less than 5 cm and are encapsulated. This first phase is not been associated with major pathology and might remain asymptomatic for a long period or permanently [16, 17]. The incubation period is undefined but it could be in around months or even years, before the infection becomes symptomatic. There have been reported cases of spontaneous cure due to cyst calcification and collapse and on the other hand, there are cases of early symptomatology because of cyst rupture [16, 18].

#### *7.1.2. Patient characteristics*

Cystic echinococcosis is seen in all ages, including patients aged below 1 year and above 75 years, with a majority between 6 and 15 years, 21 and 30, and 30 and 40 years. The most risky profession involves farm labors [16].

#### *7.1.3. Symptoms*

There is a huge spectrum of clinical manifestations that are variable among the patients and none of these are considered as pathognomonic of echinococcosis [16]. All the symptoms might depend on different characteristics such as the host-parasite interaction, the different organs involved, cyst size, and the presence of different complications such as cyst rupture or trauma [16, 18]. The most commonly compromised organs are the liver, followed by lung, kidney, spleen, and less common organs like muscles, skin, abdominal cavity, and pelvic cavity. The symptoms may vary in accordance with the organ involved [16-18].

Liver infections include symptoms such as hepatomegaly, cholecystitis, jaundice, liver abscess, or secondary biliary cirrhosis. Lung infections include symptoms like chest pain, expectora‐ tion, hemoptysis, and dyspnea [19]. Other severe manifestations include cyst getting rupture into biliary tree causing biliary colic, jaundice and can be confused with pancreatitis or cholangitis [16, 19].

#### *7.1.4. Liver involvement*

The liver is the most commonly involved organ, representing around two-thirds of the cases. The right lobe is compromised in the majority of the cases (60% to 80%) but it frequently does not produce symptoms and when these once occur, it is estimated that the cyst size is around 10 cm [20, 21]. Once a cyst develops a larger size, hepatomegaly might occur and other symptoms like right upper quadrant pain, nausea, and vomiting can also result. Due to the mass defect of the cyst compression of the portal and hepatic veins, it can result in Budd-Chiari syndrome [19, 20].

#### *7.1.5. Lung involvement*

The most common symptom of lung infection is the presence of cough in around 53% to 62% of the patients, followed by chest pain, dyspnea, and hemoptysis. But generally, most of the patients with lung infection are asymptomatic [21, 22].

#### *7.1.6. Other organs*

*7.1.1. Cystic echinococcosis*

16 Current Topics in Echinococcosis

*7.1.2. Patient characteristics*

*7.1.3. Symptoms*

cholangitis [16, 19].

syndrome [19, 20].

*7.1.5. Lung involvement*

patients with lung infection are asymptomatic [21, 22].

*7.1.4. Liver involvement*

profession involves farm labors [16].

As a general rule, the phase of primary infection is always asymptomatic because the cysts are located in different organs, characterized by their small size, which is less than 5 cm and are encapsulated. This first phase is not been associated with major pathology and might remain asymptomatic for a long period or permanently [16, 17]. The incubation period is undefined but it could be in around months or even years, before the infection becomes symptomatic. There have been reported cases of spontaneous cure due to cyst calcification and collapse and on the other hand, there are cases of early symptomatology because of cyst rupture [16, 18].

Cystic echinococcosis is seen in all ages, including patients aged below 1 year and above 75 years, with a majority between 6 and 15 years, 21 and 30, and 30 and 40 years. The most risky

There is a huge spectrum of clinical manifestations that are variable among the patients and none of these are considered as pathognomonic of echinococcosis [16]. All the symptoms might depend on different characteristics such as the host-parasite interaction, the different organs involved, cyst size, and the presence of different complications such as cyst rupture or trauma [16, 18]. The most commonly compromised organs are the liver, followed by lung, kidney, spleen, and less common organs like muscles, skin, abdominal cavity, and pelvic cavity. The

Liver infections include symptoms such as hepatomegaly, cholecystitis, jaundice, liver abscess, or secondary biliary cirrhosis. Lung infections include symptoms like chest pain, expectora‐ tion, hemoptysis, and dyspnea [19]. Other severe manifestations include cyst getting rupture into biliary tree causing biliary colic, jaundice and can be confused with pancreatitis or

The liver is the most commonly involved organ, representing around two-thirds of the cases. The right lobe is compromised in the majority of the cases (60% to 80%) but it frequently does not produce symptoms and when these once occur, it is estimated that the cyst size is around 10 cm [20, 21]. Once a cyst develops a larger size, hepatomegaly might occur and other symptoms like right upper quadrant pain, nausea, and vomiting can also result. Due to the mass defect of the cyst compression of the portal and hepatic veins, it can result in Budd-Chiari

The most common symptom of lung infection is the presence of cough in around 53% to 62% of the patients, followed by chest pain, dyspnea, and hemoptysis. But generally, most of the

symptoms may vary in accordance with the organ involved [16-18].

Heart infection might generate rupture with pericardial tamponade, cardiac insufficiency, embolism or pericardial effusion [23-25, 60, 64, 66]. The central nervous system can lead to seizures or symptoms due to an increase in the intracranial pressure [26]. Kidney disease can cause hematuria, and even glomerulonephritis and nephrotic syndrome have been described. Bones are mostly asymptomatic and only develop manifestations when a pathological fracture occurs [23].

#### *7.1.7. Outcome*

Surgeries are performed in around 15% of the patients after the initial diagnosis. And in patients without surgery, majority would remain asymptomatic through many years [18].

#### **7.2.** *Echinococcus multilocularis*

The most common presenting complaints include malaise, weight loss, and right upper quadrant discomfort due to hepatomegaly. Cholestatic jaundice, cholangitis, portal hyperten‐ sion, and the Budd-Chiari syndrome can also occur. The clinical presentation may mimic that of hepatocellular carcinoma [18].

Extrahepatic primary disease is very rare (1% of cases). Multiorgan disease was described in 13% of cases in one series in which metacestodes involved the lungs, spleen, or brain in addition to the liver [2]. If left untreated, more than 90% of patients will die within 10 years of the onset of clinical symptoms, and virtually 100% will die by 15 years [18].

#### **7.3.** *Echinococcus oligarthus* **and** *Echinococcus vogeli*

These species cause polycystic echinococcosis, but they represent a very rare cause of echino‐ coccosis disease, found in muscles and skin but it has been only described in a few cases in Brazil, Venezuela, and Surinam [16, 18, 28, 29].

#### **8. Diagnosis**

For an optimal diagnosis, both imaging and serological tests are required.

#### **8.1. Imaging**

The cysts may be visualized by ultrasonography, computed tomography (CT), or magnetic resonance imaging (MRI). However ultrasonography is the most commonly employed technique due to its easy performance and low cost. The sensitivity of ultrasonography for the evaluation of echinococcosis is 90% to 95% and the appearance of a cyst in the ultrasound is as an anechoic and smooth cyst. Another important aspect of the ultrasonography is that it helps in the classification of the disease, according to the WHO echinococcosis classification based on their ultrasound appearance. CT has an overall sensitivity of 95% to 100% and is the preferred imaging test to describe the size, location, and number of cysts. MRI has the same sensitivity as that of CT and has no major advantages over CT, but it is better for the determi‐ nation of intra- and extra-hepatic changes; however, it is not usually a routine examination technique in the patients [18, 27].

#### **8.2. Serological tests**

They are used in the diagnosis and follow-up of the patients. Some of the laboratory tests include complement fixation, latex agglutination, and indirect immunofluorescence [27, 84]. However, there are two important antigens of the *E. granulosus* disease [18]:


#### **8.3. Other laboratory tests for diagnosis**

Nonspecific leukopenia or thrombocytopenia, mild eosinophilia, and nonspecific liver function may be detected but are not diagnostic. Hypergammaglobulinemia and elevated serum IgE levels are present in more than 50% of cases.

Imaging: On ultrasonography or CT, the lesions usually have an irregular contour with no well-defined wall, central necrosis, and irregular intralesional and wall calcifications.

#### **8.4. Comparing serologies**

Serologic tests are more reliable for diagnosis of *E. multilocularis* infection than for *E. granulo‐ sus* infection; sensitivity and specificity rates are 95% to 100%. A specific *E. multilocularis* antigen such as the affinity purified Em2 antigen from alveolar echinococcosis metacestodes is often used; the Em2-ELISA can discriminate between *E. granulosus* and *E. multilocularis* in 95% of cases. Serology usually remains positive indefinitely; following complete surgical resection, serology may normalize within a few years [18].

Cyst aspiration or biopsy: In the absence of a positive serologic test, percutaneous aspiration or biopsy may be required to confirm the diagnosis by demonstrating the presence of proto‐ scolices, hooklets, or hydatid membranes.

Polymerase chain reaction: Polymerase chain reaction techniques are limited to research settings but may play a diagnostic role in the future. DNA probes using Southern hybridization tests are also being developed.

#### **9. Treatment**

Echinococcosis treatment includes a multidisciplinary conduct and a general overview of the patient condition in order to provide the best management that includes surgical treatment, pharmacological treatment, the "watch and wait," and both of them, depending on the specific characteristics of the disease and most importantly the type of Echinococcosis that the patient develops. The overall approach depends on the different species that infect humans, particu‐ larly two of them: *E. granulosus* and *E. multilocularis*, causing cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively [85].

#### **9.1. Cystic echinococcosis**

preferred imaging test to describe the size, location, and number of cysts. MRI has the same sensitivity as that of CT and has no major advantages over CT, but it is better for the determi‐ nation of intra- and extra-hepatic changes; however, it is not usually a routine examination

They are used in the diagnosis and follow-up of the patients. Some of the laboratory tests include complement fixation, latex agglutination, and indirect immunofluorescence [27, 84].

**•** Antigen 5 which is a parasite antigen; however, the studies have shown low specificity of

**•** Antigen B is a lipoprotein and is related with a superior specificity in comparison with the

Nonspecific leukopenia or thrombocytopenia, mild eosinophilia, and nonspecific liver function may be detected but are not diagnostic. Hypergammaglobulinemia and elevated

Imaging: On ultrasonography or CT, the lesions usually have an irregular contour with no

Serologic tests are more reliable for diagnosis of *E. multilocularis* infection than for *E. granulo‐ sus* infection; sensitivity and specificity rates are 95% to 100%. A specific *E. multilocularis* antigen such as the affinity purified Em2 antigen from alveolar echinococcosis metacestodes is often used; the Em2-ELISA can discriminate between *E. granulosus* and *E. multilocularis* in 95% of cases. Serology usually remains positive indefinitely; following complete surgical

Cyst aspiration or biopsy: In the absence of a positive serologic test, percutaneous aspiration or biopsy may be required to confirm the diagnosis by demonstrating the presence of proto‐

Polymerase chain reaction: Polymerase chain reaction techniques are limited to research settings but may play a diagnostic role in the future. DNA probes using Southern hybridization

Echinococcosis treatment includes a multidisciplinary conduct and a general overview of the patient condition in order to provide the best management that includes surgical treatment,

well-defined wall, central necrosis, and irregular intralesional and wall calcifications.

However, there are two important antigens of the *E. granulosus* disease [18]:

technique in the patients [18, 27].

its extensive use in the diagnosis [18].

**8.3. Other laboratory tests for diagnosis**

serum IgE levels are present in more than 50% of cases.

resection, serology may normalize within a few years [18].

scolices, hooklets, or hydatid membranes.

tests are also being developed.

**9. Treatment**

**8.2. Serological tests**

18 Current Topics in Echinococcosis

antigen 5 [18].

**8.4. Comparing serologies**

As mentioned earlier, the treatment is complex, and even these days there are no clinical trials and studies that compare the different modalities to say which one is superior to the other. However, it is accepted that the surgical treatment can potentially remove all the *E. granulo‐ sus* cysts for complete cure [30]. The different treatment strategies include surgery, percuta‐ neous management, pharmacological management, and observation [31].

Overall, the diagnosis approach depends on the WHO diagnostic classification, performed in 2003, when the Informal Working Group on echinococcosis established a standardized ultrasound classification based on the status of the cyst by its ultrasound appearance. This classification has the intention to promote uniform standards of diagnosis and treatment, and has important implications for the clinical decision.

There are three different stages, including CE1 and CE2 that are cyst-containing protoscolices. The CE3 stage is divided in CE3a, a detached endocyst, and CE3b, a solid cyst with daughter cysts [32]. Recommendations indicate that CE1 and CE3a stages, in which the cyst size is less than 5 cm, should be treated with albendazole only. Stages CE1 and CE3a with cyst size more than 5 cm may be treated with albendazole in combination with percutaneous treatment via PAIR (puncture, aspiration, injection, and reaspiration). The CE2 and CE3b stages are charac‐ terized by multiple cyst compartments that require individual puncture, and these patients usually require PAIR. However, the optimal choice among the different approaches (PAIR, surgery, or drug therapy) is uncertain. Finally, stages CE4 and CE5 involve inactive cysts that only require observation [33, 34].

#### **9.2. Treatment options**

#### *9.2.1. Surgery*

It is the first treatment option in cases of complicated cysts, including a cyst rupture or infection and hemorrhage. It is also an alternative treatment option for those cysts with many daughter vesicles that are not suitable for percutaneous treatment. Another indications are large liver cysts, cyst diameter more than 10cm, extrahepatic disease (lung, bone, or brain), cysts situated superficially with high risk of rupture, and cysts affecting the biliary tree [31, 35]. Contrain‐ dications are relative to each patient; for example, old-aged people, people suffering from concomitant diseases, pregnant women, or patients with small cysts [7]. As far as the compli‐ cations are concerned, secondary infection, intra-abdominal abscess, fistula, or sclerosing cholangitis have been reported. Recurrent echinococcosis may occur in e 2% to 25% of the cases, depending basically on the surgeon's experience [35].

Before choosing the surgical technique, it's important to administrate drug therapy to mini‐ mize the surgical risk of secondary echinococcosis from seeding of protoscolices in the abdominal cavity. Usually, albendazole is administered a week before surgery, and continued at least four weeks after the surgery. Praziquantel is another option, but there is no specific evidence regarding its efficacy [31, 36].

Surgical approach must be individualized, depending on each patient disease and its charac‐ teristics. There are two options; the first one is to remove the intact cyst, and another option is to open the cyst with protoscolicidal agents, such as hypertonic saline, followed by the evacuation of the cyst contents and removal of the cyst [31]. Other protoscolicidal used includes 70% to 95% ethanol and 0.5% cetrimide solution. The protoscolicidal agent must be in contact with the germinal layer for at least 15 minutes to make its effect. Albendazole, ivermectin, and praziquantel have been also used as protoscolicidal agents, but their efficacy and safety need more studies. Protoscolicidal agents should not be used in the presence of biliary communi‐ cation, because of the risk of sclerosing cholangitis or pancreatitis [31, 35].

There is also the possibility of laparoscopic surgery for the treatment of echinococcosis; however, there are no randomized trials done yet comparing the efficacy of laparoscopy with open procedures. Possible indications for laparoscopic treatment are the presence of anteriorly located hepatic cysts, although this procedure is related with associated risk of spillage because of the elevation of intra-abdominal pressures due to the pneumoperitoneum. Contraindica‐ tions include deep intraparenchymal cysts, the presence of calcified walls cyst, and cysts located next to the vena cava [31].

In a review article published in 2014 with the aim to study the world literature of the laparo‐ scopic treatment of liver hydatid cyst, a total of 914 patients were identified to evaluate the mortality, morbidity, and clinical outcomes of the laparoscopic treatment. It was found that the most common procedure was cystectomy in 60.39% of patients, followed by partial pericystectomy with 14.77% of patients, and finally pericystectomy with 8.21% of patients, and the rest of them were treated via segmentectomy. They also reported that conversion of laparoscopy to open laparotomy occurred in the 4.92% of the cases, due to anatomical limitations and inaccessible locations. Mortality was reported in 0.22% of the patients and morbidity was reported in 15.07% of the patients. There was no intraoperative deaths reported and the most reported complication was bile leakage. In general terms, they concluded that the laparoscopic procedure is safe, with acceptable ranges of mortality and morbidity and clinical outcomes comparable to open surgery [37].

#### *9.2.2. Percutaneous management*

There are two percutaneous techniques. The first one called PAIR (puncture, aspiration, injection of a protoscolicidal agent, and reaspiration) is usually effective in the treatment of cysts without daughter cysts. And the second one, that involves the evacuation of the cyst with a large bore catheter. This technique helps in cases in which the drain becomes difficult or tends to relapse after PAIR, like in the case of WHO stages CE2 and CE3b cysts [31, 38]. Percutaneous treatment is associated with anaphylaxis, happening according to a review in the 1.6% of the patients [39]. And as in surgery, albendazole must be administered for at least one month after the percutaneous treatment [31].

#### *9.2.3. PAIR procedure*

Before choosing the surgical technique, it's important to administrate drug therapy to mini‐ mize the surgical risk of secondary echinococcosis from seeding of protoscolices in the abdominal cavity. Usually, albendazole is administered a week before surgery, and continued at least four weeks after the surgery. Praziquantel is another option, but there is no specific

Surgical approach must be individualized, depending on each patient disease and its charac‐ teristics. There are two options; the first one is to remove the intact cyst, and another option is to open the cyst with protoscolicidal agents, such as hypertonic saline, followed by the evacuation of the cyst contents and removal of the cyst [31]. Other protoscolicidal used includes 70% to 95% ethanol and 0.5% cetrimide solution. The protoscolicidal agent must be in contact with the germinal layer for at least 15 minutes to make its effect. Albendazole, ivermectin, and praziquantel have been also used as protoscolicidal agents, but their efficacy and safety need more studies. Protoscolicidal agents should not be used in the presence of biliary communi‐

There is also the possibility of laparoscopic surgery for the treatment of echinococcosis; however, there are no randomized trials done yet comparing the efficacy of laparoscopy with open procedures. Possible indications for laparoscopic treatment are the presence of anteriorly located hepatic cysts, although this procedure is related with associated risk of spillage because of the elevation of intra-abdominal pressures due to the pneumoperitoneum. Contraindica‐ tions include deep intraparenchymal cysts, the presence of calcified walls cyst, and cysts

In a review article published in 2014 with the aim to study the world literature of the laparo‐ scopic treatment of liver hydatid cyst, a total of 914 patients were identified to evaluate the mortality, morbidity, and clinical outcomes of the laparoscopic treatment. It was found that the most common procedure was cystectomy in 60.39% of patients, followed by partial pericystectomy with 14.77% of patients, and finally pericystectomy with 8.21% of patients, and the rest of them were treated via segmentectomy. They also reported that conversion of laparoscopy to open laparotomy occurred in the 4.92% of the cases, due to anatomical limitations and inaccessible locations. Mortality was reported in 0.22% of the patients and morbidity was reported in 15.07% of the patients. There was no intraoperative deaths reported and the most reported complication was bile leakage. In general terms, they concluded that the laparoscopic procedure is safe, with acceptable ranges of mortality and morbidity and

There are two percutaneous techniques. The first one called PAIR (puncture, aspiration, injection of a protoscolicidal agent, and reaspiration) is usually effective in the treatment of cysts without daughter cysts. And the second one, that involves the evacuation of the cyst with a large bore catheter. This technique helps in cases in which the drain becomes difficult or tends to relapse after PAIR, like in the case of WHO stages CE2 and CE3b cysts [31, 38].

cation, because of the risk of sclerosing cholangitis or pancreatitis [31, 35].

evidence regarding its efficacy [31, 36].

20 Current Topics in Echinococcosis

located next to the vena cava [31].

*9.2.2. Percutaneous management*

clinical outcomes comparable to open surgery [37].

It was first introduced in the mid-1980s and includes the realization of percutaneous puncture of the cyst by ultrasonic guidance, aspiration of the cyst fluid, injection of a protoscolicidal substance, and finally reaspiration of the cyst fluid. PAIR has the advantages of being less invasive than surgery and can be a diagnostic and therapeutic procedure and it has a success rate of more than 95%. This procedure may be used in cases of echinococcosis of WHO stages CE1 and CE3a as primary treatment and for the relapse after medical therapy in stages CE1 and CE3a, and in case of relapse after surgery. PAIR also showed to be successful in the management of liver cysts and in those cysts located in other abdominal parts such as kidney, spleen, and in the abdominal cavity. There are controversies in the use of PAIR in pregnant women and in children, although the application of PAIR is indicated in symptomatic pregnant women [16, 31, 38].

The procedure is performed by ultrasound or by CT guidance. Then the cyst is punctured and the fluid aspiration is realized and a histopathological study must be done to evaluate the presence of protoscolices. After the administration of protoscolicidal agent, the reaspiration must be done for at least 10 to 15 minutes. Four hours before PAIR, adjunctive drug therapy with albendazole or mebendazole should be administrated. Recommendations indicate that albendazole should be continued after a month of the procedure and mebendazole should be continued for three months. The most important contraindications of the PAIR are the following: the presence of cysts without drainable solid material, superficial cyst with risk of rupture in the abdominal cavity, calcified cysts, and cyst with biliary communication [31, 40].

#### *9.2.4. Drug therapy*

In most of the cases, it is useful as an adjunctive therapy to surgery and percutaneous treatment. The use of pharmacological approach as definitive management is indicated in few cases. The recommendations indicate that in cases of small cysts of WHO stages CE1 and CE3a with cyst size less than 5 cm, the initial management with only drug therapy is appropriate, with optimal duration of treatment depending on the clinical factors, but usually estimated in around one to six months. The effectiveness of pharmacological therapy decreases with the presence of a cyst size more than 5 cm, with multiple compartments of cysts [16, 31, 39].

As an adjunctive treatment, albendazole is used as perioperative drug therapy in surgery or in percutaneous treatment. It reduces the risk of recurrent disease by the inactivation of the protoscolices. There is no general consensus of the duration of the treatment, but generally it is initiated four days before surgery and continued at least one month after the surgery in case of albendazole and for three months in case of mebendazole [16, 31, 40, 42].

Albendazole is the first drug option and the primary antiparasitic agent for treatment of *E. granulosus*, and in cases of absence of albendazole, mebendazole may be used as an alternative, but it is less absorbed than albendazole. The primary action of albendazole is that it inhibits microtubules assembly, causing a glycogen depletion, followed by the degeneration of the mitochondria and the endoplasmic reticulum, finally causing the cell death. It is reported that albendazole helps cyst resolution in 30% of the patients, cyst size reduction in 30% to 50% patients, and in 20% to 40% of the patients, there are no changes in the cysts. However, the outcome of the treatment with albendazole depends on multiple factors like the clinical characteristic of the patient, the cyst size, and its location [31, 43].

However, albendazole has more advantages than mebendazole. For example, albendazole is well tolerated by patients and it takes a shorter duration of therapy. Dose of albendazole is usually 10 to 15 mg/kg per day divided in two doses and mebendazole dose is 40 to 50 mg/kg per day divided in three doses. Adverse effects include hepatotoxicity in 1% to 5% of the patients, cytopenia, and alopecia in less than 1% of the patients. Due to hepatotoxicity, there might be an increase in aminotransferase enzymes. Other rare side effect like agranulocytosis has also been reported. Contraindications of using albendazole include patients with liver disease or bone marrow suppression [31, 44]. Patients with pharmacological therapy must be monitored every two to three weeks for first three months and then monthly [31]. Praziquantel has protoscolicidal activity, but its efficacy in clinical studies is variable, but is considered as an alternative when the albendazole treatment cannot be administrated [44, 45].

Observation: Also known as the "watch and wait," indicated in the WHO stages CE4 and CE5, in patients with absence of complications or inactive liver cysts [40].

#### **9.3. Alverolar echinococcosis**

Unfortunately, the treatment of alveolar echinococcosis is less effective than the treatment of cystic echinococcosis. The general approach to the treatment is based on surgery. The main idea is to remove all the infected tissue and sometimes it is necessary to also remove part of the host tissue. Management with albendazole is recommended after surgery with an uncertain optimal duration. The benefit of preoperative albendazole administration has not been reported [31].

#### **10. Conclusions**

Echinococcosis has always been neglected worldwide and especially in Colombia. Particularly in this country, research was done and published only between 1950s and 1990s. There is no subsequent interest in the occurrence of this disease and all its implications.

Due to the impact of echinococcosis, the burden it creates, and the consequences in the patients, it becomes important to create strategies in order to improve and have a better control on this disease in our country. This probably should begin at medical schools, where more emphasis on teaching about this parasitic disease, would increase awareness about it. As has been mentioned earlier, there should be regular interaction among physicians, biologists and veterinarians to better understand the implications of this zoonotic disease, which at least from an enzootic point of view is clearly extended. As an additional reflection in the middle of the process and discussion about peace in Colombia, there are chances that once this would be reached, after the La Habana discussions, more people, not only missionary, health, and military corps, would be allowed to enter in the guerrillas-controlled areas, which are precisely located where enzootic evidences of echinococcosis have been reported, increasing the exposure to species of *Echinococcus* and probably making more visible the occurrence of this disease in the country [78].

Even more, it's not clear yet, if Colombia would be considered properly a risky area for travelers visiting the country in the view of acquisition of echinococcosis. But certainly in other highly endemic countries, echinococcosis is included in the list of infectious diseases that can be acquired during travel, and cases of echinococcosis, as well from other cestodiasis, have been reported increasingly during the last few years [75-78].

Therefore, it becomes crucial to create a clear and easy consensus to help and guide the physicians to make a better approach towards helping the patients in finding the opportune diagnosis and the best treatment option. The creation of a normative and the global vision of echinococcosis as a public health problem might help improve the control of this disease.

Echinococcosis, and other zoonotic diseases, can re-emerge just when they are considered to be under control or eliminated. They can go into hiding in the animal reservoirs, so there is no possibility for complacency [78]. Epidemiology of zoonoses such as echinococcosis is complex and dynamic, being influenced by varying parameters that can roughly be categorized as human-related, pathogen-related, and climate/environment-related [79, 80]. But certainly for echinococcosis, there is still much to understand.

#### **Acknowledgements**

Albendazole is the first drug option and the primary antiparasitic agent for treatment of *E. granulosus*, and in cases of absence of albendazole, mebendazole may be used as an alternative, but it is less absorbed than albendazole. The primary action of albendazole is that it inhibits microtubules assembly, causing a glycogen depletion, followed by the degeneration of the mitochondria and the endoplasmic reticulum, finally causing the cell death. It is reported that albendazole helps cyst resolution in 30% of the patients, cyst size reduction in 30% to 50% patients, and in 20% to 40% of the patients, there are no changes in the cysts. However, the outcome of the treatment with albendazole depends on multiple factors like the clinical

However, albendazole has more advantages than mebendazole. For example, albendazole is well tolerated by patients and it takes a shorter duration of therapy. Dose of albendazole is usually 10 to 15 mg/kg per day divided in two doses and mebendazole dose is 40 to 50 mg/kg per day divided in three doses. Adverse effects include hepatotoxicity in 1% to 5% of the patients, cytopenia, and alopecia in less than 1% of the patients. Due to hepatotoxicity, there might be an increase in aminotransferase enzymes. Other rare side effect like agranulocytosis has also been reported. Contraindications of using albendazole include patients with liver disease or bone marrow suppression [31, 44]. Patients with pharmacological therapy must be monitored every two to three weeks for first three months and then monthly [31]. Praziquantel has protoscolicidal activity, but its efficacy in clinical studies is variable, but is considered as

Observation: Also known as the "watch and wait," indicated in the WHO stages CE4 and CE5,

Unfortunately, the treatment of alveolar echinococcosis is less effective than the treatment of cystic echinococcosis. The general approach to the treatment is based on surgery. The main idea is to remove all the infected tissue and sometimes it is necessary to also remove part of the host tissue. Management with albendazole is recommended after surgery with an uncertain optimal duration. The benefit of preoperative albendazole administration has not been

Echinococcosis has always been neglected worldwide and especially in Colombia. Particularly in this country, research was done and published only between 1950s and 1990s. There is no

Due to the impact of echinococcosis, the burden it creates, and the consequences in the patients, it becomes important to create strategies in order to improve and have a better control on this disease in our country. This probably should begin at medical schools, where more emphasis

subsequent interest in the occurrence of this disease and all its implications.

an alternative when the albendazole treatment cannot be administrated [44, 45].

in patients with absence of complications or inactive liver cysts [40].

**9.3. Alverolar echinococcosis**

22 Current Topics in Echinococcosis

reported [31].

**10. Conclusions**

characteristic of the patient, the cyst size, and its location [31, 43].

Authors would like to thank the critical comments of anonymous reviewers. Also, Dr. Rodriguez-Morales would like to thank Dr. Juliana Buitrago-Jaramillo, Dean, Faculty of Health Sciences, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia, for her support to his research group during the activities in year 2014, including presentation and conferences at major international meetings (mainly in Peru, Mexico, and Cuba), and also in 2015 (Ecuador). The authors would also like to thank the Asociación Colombiana de Infectología and Interna‐ tional Society for Chemotherapy, for supporting the research and presentations of studies in zoonoses since 2011 (Italy, Japan, Brazil, Ecuador, Mexico, Peru, Cuba). Finally, the authors would like to thank the Ministry of Health of Colombia, for the access to SIVIGILA and RIPS databases through the system Cubo de datos del SISPRO.

## **Author details**

Alfonso J. Rodriguez-Morales1,2,3\*, Lauren Sofia Calvo-Betancourt4 , Camila Alarcón-Olave5 and Adrián Bolívar-Mejía6

\*Address all correspondence to: arodriguezm@utp.edu.co

1 Public Health and Infection Research Group, School of Medicine and School of Veterinary Medicine and Zootechnics, Faculty of Health Sciences, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia

2 Committee on Zoonoses and Haemorrhagic Fevers, Asociación Colombiana de Infectolo‐ gía, Bogotá, DC, Colombia

3 Working Group on Zoonoses, International Society for Chemotherapy, Aberdeen, United Kingdom

4 Fundación Cardiovascular de Colombia, Floridablanca, Santander, Colombia

5 Universidad Autónoma de Bucaramanga, Bucaramanga, Santander, Colombia

6 Clínica FOSCAL Internacional, Floridablanca, Santander, Colombia

#### **References**


[8] Romig T, Dinkel A, Mackenstedt U. The present situation of echinococcosis in Eu‐ rope. Parasitol Int. 2006;55:S187-S191.

**Author details**

24 Current Topics in Echinococcosis

Adrián Bolívar-Mejía6

Pereira, Risaralda, Colombia

gía, Bogotá, DC, Colombia

Kingdom

**References**

2008;21(2):380-401.

2004;15:173-183.

Alfonso J. Rodriguez-Morales1,2,3\*, Lauren Sofia Calvo-Betancourt4

1 Public Health and Infection Research Group, School of Medicine and School of Veterinary Medicine and Zootechnics, Faculty of Health Sciences, Universidad Tecnológica de Pereira,

2 Committee on Zoonoses and Haemorrhagic Fevers, Asociación Colombiana de Infectolo‐

3 Working Group on Zoonoses, International Society for Chemotherapy, Aberdeen, United

4 Fundación Cardiovascular de Colombia, Floridablanca, Santander, Colombia

5 Universidad Autónoma de Bucaramanga, Bucaramanga, Santander, Colombia

[1] Moro P, Schantz P. Echinococcosis: A review. Int J Infect Dis. 2009;13: 125-133.

[2] McManus D, Zhang W, Li J, Bartley P. Echinococcosis. Lancet. 2003;362:1295-1304.

[3] D'Alessandro A, Rausch RL. New aspects of neotropical polycystic (Echinococcus vogeli) and unicystic (Echinococcus oligarthrus) echinococcosis. Clin Microbiol Rev.

[4] Khanfar N. Hydatid disease: A review and update. Curr Anaesth & Crit Care.

[5] Soledad S, Rodríguez V, Candia M, Bonastre P. Hidatidosis Pulmonar. Revista de

[6] Bonilla C. Hidatidosis: Una zoonosis de múltiples Presentaciones clínicas. Revista In‐ vestigación y Desarrollo Social. Universidad Militar Nueva Granada. 2001;24:89-106.

[7] Jenkins DJ, Romig T, Thompson RC. Emergence/re-emergence of Echinococcus spp.

6 Clínica FOSCAL Internacional, Floridablanca, Santander, Colombia

Posgrado de la VIa Cátedra de Medicina. 2005;152:16-18.

A global update. Int J Parasitol. 2005;35:1205-1219.

\*Address all correspondence to: arodriguezm@utp.edu.co

, Camila Alarcón-Olave5 and


[40] Rinaldi F, De Silvestri A, Tamarozzi F, at al. Medical treatment versus "Watch and Wait" in the clinical management of CE3b echinococcal cysts of the liver. BMC Infect Dis. 2014;14:492.

[25] Díaz-Menéndez M, Pérez-Molina JA, Norman FF, et al. Management and outcome of cardiac and endovascular cystic echinococcosis. PLoS Negl Trop Dis. 2012;6:e1437.

[26] Nourbakhsh A, Vannemreddy P, Minagar A, et al. Hydatid disease of the central nervous system: A review of literature with an emphasis on Latin American coun‐

[27] Wuestenberg J, Gruener B, Oeztuerk S, at al. Diagnostics in cystic echinococcosis: Se‐

[28] Soares Mdo C, Rodrigues AL, Moreira Silva CA, et al. Anatomo-clinical and molecu‐ lar description of liver neotropical echinococcosis caused by Echinococcus oligarth‐

[29] Zimmerman DM, Douglass M, Reavill DR, Greiner EC. Echinococcus oligarthrus cystic hydatidosis in Brazilian agouti (Dasyprocta leporina). J Zoo Wildl Med.

[30] Brunetti E, Kern P, Vuitton DA. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop. 2010;114(1):1-16. [31] Moro P. Treatment of echinococcosis. UpToDate. Last updated March 06, 2013.

[32] Brunetti E, Filice C. Echinococcosis Hydatid Cyst Workup. Medscape. Last updated

[33] Rinaldi F, Brunetti E, Neumayr A, et al. Cystic echinococcosis of the liver: A primer

[34] Stojkovic M, Rosenberger K, Kauczor HU, Junghanss T, Hosch W. Diagnosing and staging of cystic echinococcosis: How do CT and MRI perform in comparison to ul‐

[35] McNanus D, Gray DJ, Zhang W, Yang Y. Diagnosis, treatment, and management of

[36] Bygott JM, Chiodini PL. Praziquantel: Neglected drug? Ineffective treatment? Or

[37] Tuxun T, Zhang JH, Zhao JM, et al. World review of laparoscopic treatment of liver

[38] Ormeci N. PAIR vs Örmeci technique for the treatment of hydatid cyst. Turk J Gas‐

[39] Neumayr A, Troia G, de Bernardis C, et al. Justified concern or exaggerated fear: the risk of anaphylaxis in percutaneous treatment of cystic echinococcosis-a systematic

therapeutic choice in cystic hydatid disease? Acta Trop. 2009;111:95.

cystic echinococcosis—914 patients. Int J Infect Dis. 2014;24:43-50.

rology versus ultrasonography. Turk J Gastroenterol. 2014;25(4):398-404.

rus in human host. Acta Trop. 2013;125(1):110-114.

for hepatologists. World J Hepatol. 2014;6(5):293-305.

trasound? PLoS Negl Trop Dis. 2012;6(10):e1880.

literature review. PLoS Negl Trop Dis. 2011;5:e1154.

Echinococcosis. BMJ. 2012;344:e3866.

troenterol. 2014;25(4):358-364.

tries. Neurol Res. 2010;32:245.

26 Current Topics in Echinococcosis

2009;40(3):551-558.

October 19, 2011.


[68] Pastore R, Vitali LH, Macedo Vde O, Prata A. A serological survey of the infection by Echinococcus sp. in the municipality of Sena Madureira, AC. Rev Soc Bras Med Trop. 2003;36(4):473-477.

[53] Martínez S, Restrepo CS, Carrillo JA, et al. Thoracic manifestations of tropical para‐

[54] Lichtember E. Equinococosis humana (quiste hidatídico) dos casos. Revista de la Fac‐

[55] D'Alessandro A, Lega J, Vera MA. Cystic calcifications of the liver in Colombia. Echi‐ nococcosis or calcified abscesses? Am J Trop Med Hyg. 1966;15(6):908-913.

[56] D'Alessandro A, Rausch RL, Morales GA, Collet S, Angel D. Echinococcus infections

[57] Rausch RL, D'Alessandro A, Rausch VR. Characteristics of the larval Echinococcus vogeli Rausch and Bernstein, 1972 in the natural intermediate host, the paca, Cunicu‐ lus paca L. (Rodentia: Dasyproctidae). Am J Trop Med Hyg. 1981;30(5):1043-1052.

[58] Wells EA, D'Alessandro A, Morales GA, Angel D. Mammalian wildlife diseases as hazards to man and livestock in an area of the Llanos Orientales of Colombia. J Wildl

[59] D'Alessandro A, Rausch RL, Cuello C, Aristizabal N. Echinococcus vogeli in man, with a review of polycystic hydatid disease in Colombia and neighboring countries.

[60] Cataño J. Cardiac echinococcosis, an unusual echocardiographic finding. Am J Trop

[61] Gómez G, Córdoba E, Córdoba A. Quiste hidatídico hepático. Rev Colomb Gastroen‐

[62] Morales GA, Guzman VH, Wells EA, Angel D. Polycystic echinococcosis in Colom‐ bia: The larval cestodes in infected rodents. J Wildl Dis. 1979;15(3):421-428.

[63] Bolívar-Mejía A, Rodríguez-Morales AJ, Paniz-Mondolfi AE, Delgado O. Cardiovas‐ cular manifestations of human toxocariasis. Arch Cardiol Mex. 2013;83(2):120-129.

[64] Hidron A, Vogenthaler N, Santos-Preciado JI, et al. Cardiac involvement with para‐

[65] Franco-Paredes C, Rouphael N, Méndez J, et al. Cardiac manifestations of parasitic infections. Part 1: Overview and immunopathogenesis. Clin Cardiol. 2007;30(4):

[66] Franco-Paredes C, Rouphael N, Méndez J, et al. Cardiac manifestations of parasitic infections. Part 2: Parasitic myocardial disease. Clin Cardiol. 2007;30(5):218-222.

[67] Franco-Paredes C, Rouphael N, Méndez J, et al. Cardiac manifestations of parasitic infections. Part 3: Pericardial and miscellaneous cardiopulmonary manifestations.

sitic infections. Clin Microbiol Rev. 2010;23(2):324-349.

sitic infections: A pictorial review. Radiographics. 2005;25(1):135-155.

in Colombian animals. Am J Trop Med Hyg. 1981;30(6):1263-1276.

ultad de Medicina (Bogotá). 1957; 25(3-4):119-127.

Dis. 1981;17(1):153-162.

28 Current Topics in Echinococcosis

Med Hyg. 2010;82(2):173.

terol. 2003;18:183-186.

195-199.

Clin Cardiol. 2007;30(6):277-280.

Am J Trop Med Hyg. 1979;28(2):303-317.


## **Echinococcosis in Mexico — A Story Worth Sharing**

Ana Flisser, Pablo Maravilla, Pilar Mata-Miranda and Fernando Martinez-Hernandez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60868

#### **Abstract**

[83] Grozavu C, Ilias M, Pantile D. Multivisceral echinococcosis: Concept, diagnosis,

[84] Wuestenberg J, Gruener B, Oeztuerk S, et al. Diagnostics in cystic echinococcosis: Se‐

[85] Gomez I, Gavara C, López-Andújar R, et al. Review of the treatment of liver hydatid

rology versus ultrasonography. Turk J Gastroenterol. 2014;25(4):398-404.

management. Chirurgia (Bucur). 2014;109(6):758-768.

30 Current Topics in Echinococcosis

cysts. World J Gastroenterol. 2015;21(1):124-131.

At the beginning of the XXI millennium, while working at a general hospital in Mexico City, a young lady arrived with a previous diagnosis of liver amebiasis given six years earlier. Different treatments in various clinical settings were provided. In the hospital, the first approach was an ultrasound study and unexpectedly an *Echinococcus granulosus* cyst was clearly identified. The patient received adequate treatment and accepted to participate as the index case in an epidemiological survey performed in her community. Inhabitants, dogs, cattle, pigs, and sheep were studied; cysts in humans, pigs, and sheep were searched by ultrasound and dogs by coproantigens, livestock, and dogs were negative for larval or adult stages respectively. The use of ultrasound allowed the detection of two cases (overall prevalence 0.95). DNA of the patient's cyst recovered by surgery was purified, amplified, sequenced, and multiple alignments were performed and analyzed, identifying to *Echinococcus ortleppi*. Subsequently, in a population genetics study focused to evaluate the presence and genetic variability of the intestinal tapeworm in dogs and of cystic echinococcosis in livestock in central areas from Mexico, *Echinococcus canadensis* G7 was identified and was found only in pigs. Based on a genetic network analysis, the following deductions were made: 1) *E. canadensis* G7 in Mexico is very diverse and was probably introduced from abroad several times from different sources and from different countries; 2) G7 haplotypes grouped in the North American wildlife cluster are placed far from Mexican isolates, thus they might be ruled out as sources of introduction to Mexico; and 3) the species status for G7, formally named *E. canadensis*, is still controversial, because biologically different strains (G6 to G10) are currently unified, though ecological and genetic data appear to indicate otherwise.

**Keywords:** Cystic echinococcosis, *Echinococcus canadensis*, *Echinococcus granulosus*, hydatid cyst, Mexico

© 2015 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.

#### **1. Introduction**

Echinococcosis, a well-known helminthic disease [1, 2] is practically absent in Mexico since there were only 10 clinical cases of this disease reported in PubMed since 1962. Between 1990 and 1998, only 33 hospitalized cases with postoperative diagnosis of echinococcosis were reported in the yearbook statistics of the Mexican Ministry of Health. Except for two, all cases were in adult men: 10 were reported in 1993 and 21 in 1996. Furthermore, cases might have been in immigrants or travelers but there is no information available [3].

On the other hand, taxonomy of the genus *Echinococcus* has been a matter of controversy and some confusion, particularly for *E. canadensis,* since ecological and geographical strains have been unified into a single species [4-8]. In Mexico, the pig-dog cycle is mainly present and maintains the pig strain of *E. granulosus* sensu lato (s.l.) or genotype (G)7. Some epidemiological studies performed from northern Mexico indicate the presence of the larval stage or cystic echinococcosis (CE) in livestock [9-11]. In addition, *E. oligarthra* has been recorded in a wildcat (*Lynx rufus texensis*) [12], *E. granulosus* sensu stricto (s.s.) in a rural pig [13], and *E. ortleppi* (*E. granulosus* s.l., G5) in a patient [14]. However, there is no evidence that these species are being maintained in Mexico, because only isolated cases have been documented [15, 16]. Here we review and discuss those studies conducted on *Echinococcus granulosus* s.l. in Mexico.

#### **2. The clinical case**

In the year 2000, a 38-year-old female from a community in the State of Mexico, two hours NW from Mexico City, attended the General Hospital "Dr. Manuel Gea Gonzalez". She had never lived or visited outside her community. She was treated six years before for cholecystectomy; the surgical finding was a normal bile vesicle, as commented by the patient. Her main complaint was pain in the right hypochondrium without migration that started two months before admission to the hospital, fever, malaise, fatigue, moderate pain with spontaneous exacerbation, and partial remission. Her family history had no importance for the present disease. Previous medical treatment included various antibiotics and metronidazole for the treatment of a hepatic amebic abscess. These drugs were provided simultaneously with painkillers, thus the patient partially improved. A private clinician performed an ultrasonog‐ raphy study (US) and found an undefined cyst with an internal calcification and referred her to the General Hospital "Dr. Manuel Gea Gonzalez".

One of the hospital clinicians undertook physical examination. No cardiopulmonary abnor‐ malities were seen and peristalsis was normal. On palpation, muscular resistance in the right hypochondria, hepatomegaly, and no signs of acute abdomen were found. Laboratory tests were normal. US showed a solitary cyst that measured around 6 cm and contained undulating hyperechogenic membranes floating inside the cyst fluid (Figure 1a). Computed tomography (CT) image showed a similar pattern (Figure 1b). Since both images resembled a hydatid cyst, an intradermal Casoni test was performed. A thickening of 4 cm at 15 minutes and at 24 hours was detected. Since this test cross-reacts with other helminthes [17], coproparasitological tests were performed for larva and eggs and the results were negative. Albendazole at 800 mg daily was given for two weeks.

**1. Introduction**

32 Current Topics in Echinococcosis

**2. The clinical case**

to the General Hospital "Dr. Manuel Gea Gonzalez".

Echinococcosis, a well-known helminthic disease [1, 2] is practically absent in Mexico since there were only 10 clinical cases of this disease reported in PubMed since 1962. Between 1990 and 1998, only 33 hospitalized cases with postoperative diagnosis of echinococcosis were reported in the yearbook statistics of the Mexican Ministry of Health. Except for two, all cases were in adult men: 10 were reported in 1993 and 21 in 1996. Furthermore, cases might have

On the other hand, taxonomy of the genus *Echinococcus* has been a matter of controversy and some confusion, particularly for *E. canadensis,* since ecological and geographical strains have been unified into a single species [4-8]. In Mexico, the pig-dog cycle is mainly present and maintains the pig strain of *E. granulosus* sensu lato (s.l.) or genotype (G)7. Some epidemiological studies performed from northern Mexico indicate the presence of the larval stage or cystic echinococcosis (CE) in livestock [9-11]. In addition, *E. oligarthra* has been recorded in a wildcat (*Lynx rufus texensis*) [12], *E. granulosus* sensu stricto (s.s.) in a rural pig [13], and *E. ortleppi* (*E. granulosus* s.l., G5) in a patient [14]. However, there is no evidence that these species are being maintained in Mexico, because only isolated cases have been documented [15, 16]. Here we

review and discuss those studies conducted on *Echinococcus granulosus* s.l. in Mexico.

In the year 2000, a 38-year-old female from a community in the State of Mexico, two hours NW from Mexico City, attended the General Hospital "Dr. Manuel Gea Gonzalez". She had never lived or visited outside her community. She was treated six years before for cholecystectomy; the surgical finding was a normal bile vesicle, as commented by the patient. Her main complaint was pain in the right hypochondrium without migration that started two months before admission to the hospital, fever, malaise, fatigue, moderate pain with spontaneous exacerbation, and partial remission. Her family history had no importance for the present disease. Previous medical treatment included various antibiotics and metronidazole for the treatment of a hepatic amebic abscess. These drugs were provided simultaneously with painkillers, thus the patient partially improved. A private clinician performed an ultrasonog‐ raphy study (US) and found an undefined cyst with an internal calcification and referred her

One of the hospital clinicians undertook physical examination. No cardiopulmonary abnor‐ malities were seen and peristalsis was normal. On palpation, muscular resistance in the right hypochondria, hepatomegaly, and no signs of acute abdomen were found. Laboratory tests were normal. US showed a solitary cyst that measured around 6 cm and contained undulating hyperechogenic membranes floating inside the cyst fluid (Figure 1a). Computed tomography (CT) image showed a similar pattern (Figure 1b). Since both images resembled a hydatid cyst, an intradermal Casoni test was performed. A thickening of 4 cm at 15 minutes and at 24 hours was detected. Since this test cross-reacts with other helminthes [17], coproparasitological tests

been in immigrants or travelers but there is no information available [3].

(c) **Figure 1.** Hepatic hydatid cyst of the patient; the ultrasonographic image shows a solitary cyst that measured around 6 cm and contained undulating hyper-echogenic membranes floating inside the cyst fluid (a); the CT shows a similar hiperdense pattern (b).

A colangic-pancreatographic retrograde endoscopy (CPRE) was performed to eliminate the possibility of communicating biliary ducts. The result of this procedure was negative but the patient developed severe post-CPRE pancreatitis, thus enteral feeding was started and albendazole was stopped after one week. Laparoscopic surgery was performed two weeks later by the surgical team of the hospital, using percutaneous aspiration-instillation-reaspira‐ tion (PAIR) [18-21]. The cyst was punctured to extract the intracystic fluid (Figure 2a) and instill 100 cc of hypertonic saline solution (17.7% NaCl) as a scolicidal agent (Figure 2b) that was left for 10 minutes. The upper part of the cyst was also irrigated and opened; exploration within the cyst clearly showed the germinal membrane (Figure 2c) that was totally extracted (Figure 2d). No complications arose during the surgical procedure. Seven days later, the patient was sent home without any symptoms. A CT performed 60 days after surgery showed a fluid filled cavity with 50% size reduction. In spite of our lack of experience in the treatment of liver cystic echinococcosis, the decision to perform laparoscopy was taken since the cyst was free in the right liver lobule and the patient could have a surgical procedure of minimal invasion as a definitive treatment for the parasitic disease, which came out successful, without complica‐ tions or recurrence according to the TC performed 14 and 60 days later.

At the Center for Disease Control and Prevention in Atlanta (CDC), USA, ELISA and western blot were done with the serum samples taken before, 2, 3, and 11 weeks after surgery. All were positive to echinococcosis, and the western blot showed the 8 kDa diagnostic band [22]. At

**Figure 2.** The hydatid cyst was punctured to extract the cyst fluid (a) and instill hypertonic saline solution (b); the ger‐ minal membrane could be seen from the inner surface of the cyst capsule, in the lower part of the figure the parasite's membrane is smooth, while the host's capsule, seen in the upper side of the figure (c) is rough, probably due to the inflammatory reaction; the germinal membrane was fully extracted (d).

laparoscopy, 90 ml hydatid fluid was collected, immediately low-speed centrifuged, and the pellet was analyzed. Light microscopy showed many protoscolices, mostly damaged (Figure 3a), while the sample obtained after PAIR treatment contained only debris (Figure 3b). These images are probably due to the effect of albendazole and the hypertonic solution, respectively. We tried to confirm and pursue the infection by orally introducing around 500 protoscolices into a young dog, but coproparasitoscopic studies were negative after two months and no parasites were detected at necropsy. Worthwhile mentioning is that for several years the patient received treatments without definitive diagnosis, since bacterial infections, amebic liver abscess, and vesicular problems were presumed. CE is a well-known parasitic disease, its diagnosis, medical treatment, and epidemiology have been exhaustively published [1, 2], imaging techniques are available, and cysts are specific. Nevertheless, differential diagnosis of CE in gastrointestinal malaise is not considered in Mexico [23, 24].

DNA from the germinal layer of the cyst obtained during laparoscopic surgery was obtained and analyzed by RAPD, 18s rRNA PCR-RFLP, and *coI* PCR-sequencing techniques. Banding profiles and sequence analysis matched 100% to *Echinococcus ortleppi* (*E. granulosus* s.l. G5,

(c) **Figure 3.** Micrograph of the pellet obtained after centrifugation of the cyst fluid before instilling the hypertonic solu‐ tion (a) and after this process (b). An *Echinococcus* protoscolex can be seen in (a) but only detritus in (b).

cattle strain). Interestingly, there are few human cases documented for CE in humans by *E. ortleppi* in the world. DNA analysis of partly calcified cyst recovered from an infected Dutch boy suggested that this *Echinococcus* species can be infective to humans [25] since in regions such as the Netherlands and Switzerland where the cattle strain occurs, cattle may act as a source of human infection. In addition, a first case of cattle strain (G5) in humans in South America was also reported in a rural community of Argentina [26].

#### **3. Epidemiologic study**

laparoscopy, 90 ml hydatid fluid was collected, immediately low-speed centrifuged, and the pellet was analyzed. Light microscopy showed many protoscolices, mostly damaged (Figure 3a), while the sample obtained after PAIR treatment contained only debris (Figure 3b). These images are probably due to the effect of albendazole and the hypertonic solution, respectively. We tried to confirm and pursue the infection by orally introducing around 500 protoscolices into a young dog, but coproparasitoscopic studies were negative after two months and no parasites were detected at necropsy. Worthwhile mentioning is that for several years the patient received treatments without definitive diagnosis, since bacterial infections, amebic liver abscess, and vesicular problems were presumed. CE is a well-known parasitic disease, its diagnosis, medical treatment, and epidemiology have been exhaustively published [1, 2], imaging techniques are available, and cysts are specific. Nevertheless, differential diagnosis

**Figure 2.** The hydatid cyst was punctured to extract the cyst fluid (a) and instill hypertonic saline solution (b); the ger‐ minal membrane could be seen from the inner surface of the cyst capsule, in the lower part of the figure the parasite's membrane is smooth, while the host's capsule, seen in the upper side of the figure (c) is rough, probably due to the

DNA from the germinal layer of the cyst obtained during laparoscopic surgery was obtained and analyzed by RAPD, 18s rRNA PCR-RFLP, and *coI* PCR-sequencing techniques. Banding profiles and sequence analysis matched 100% to *Echinococcus ortleppi* (*E. granulosus* s.l. G5,

of CE in gastrointestinal malaise is not considered in Mexico [23, 24].

inflammatory reaction; the germinal membrane was fully extracted (d).

34 Current Topics in Echinococcosis

Between September and November 2002, a cross-sectional study was undertaken in the area of the index case, Santa Cruz, Tepozotlan, State of Mexico [3]. The objective of this work was to determine the prevalence and distribution of human echinococcosis and its probable risk factors. This US-based study was a pilot to search for autochthonous human echinococcosis in Mexico. Initially, authorization to perform the study was obtained from the jurisdiction, local authorities, and the clinician in charge of the Health Center. Ethics and Research Commissions of the Hospital General "Dr. Manuel Gea González" approved the research protocol. House‐ hold census was used to randomly select houses, visits were done in order to obtain cartog‐ raphy from the community and to identify street names and house numbers. Demographic, household characteristics, and animals were registered. The community, Santa Cruz, is located in the municipality of Tepozotlan, State of Mexico at 19o 42´50" N and 99o 13´24" W at 2300 masl. The municipality belongs to a warm zone bordered by 4 mountain ranges: Sierra Madre Occidental, Eje Neovolcanico, Sierra Madre Oriental y Sierra Madre del Sur (Figure 4a).

An initial field exercise was performed in the community Cañadas de Cisneros, close to Santa Cruz, in order to train the field workers, to evaluate if the content of the family questionnaire was clearly understood and to register the average time that each worker needed to fill one family questionnaire. One hundred people were interviewed during the exercise and the

(c) **Figure 4.** Scenery of the community of Santa Cruz (a), an ultrasound participant of the community (b).

average time for answering one questionnaire was 10 minutes. Most questions were clearly understood, except when asked if inhabitants had animals, 60% answered no but many had dogs, so another question specific on having dogs was added. This questionnaire was used for the investigation of risk factors in a larger population.

Santa Cruz had then 405 houses (11 were uninhabited), 98% of the streets were paved, electric power, telephone, and drinking water were available, the latter by a chlorine facility. There was no drainage thus latrines were used. Of the 375 houses that were submitted to a census 1,911 inhabitants were found, with an average of 5 (from 1 to 17) per house. Women constituted 63%, average age was 27; 20% of men and women were between 3 and 24 years and 30% were between 25 and 44 years; 48% were single, 41% married, the remaining were widows, couples separated or living together; 39% studied primary school, 24% middle school, 17% high school, 5% kindergarten, 3% bachelor, and 7% illiterate. Male workers made almost half of the population; students (30%) and house workers (23%) constituted the other half. Most houses were built of brick, sand, and had latrines, although 3% used the field. Drinking water was available in 86% of the houses, half of which boil the water before drinking, those lacking drink water from a well. Dogs were present in 83% of houses, 79% were vaccinated for rabies, most slept outside, although half of the dogs were kept tied. Of the 426 dogs in the community, feces were collected from 414 to examine for cestode eggs. After treatment and purge, 3 dogs had *Taenia pisiformis* but none had *Echinococcus*. Also, 83% of people had livestock, 77% were kept close to the house.

Sample size was defined for prevalence, each house was considered a unit; 94 houses were randomly selected and the index case and her family were also included. Families were visited to inform them about the study and invite them to participate; a written informed consent was requested. All members of the selected families were also invited to attend the Health Center for a free US; those units that did not accept, the house to the right replaced them. Cysts were searched in liver, spleen, and kidneys and cyst characteristics were recorded. Blood was drawn from people with images suggestive of CE, serum samples were sent to CDC for diagnosis confirmation, and an in-depth questionnaire was applied to them.

Only 331 people out of the 543 that were invited to have a US attended (Figure 4b). To avoid bias, families that attended and those that did not attend were compared for differences in demographic data. A high proportion of students and house workers were observed; the opposite was found with male workers who were less in the group that undertook a US; this was to be expected because workers could not lose a working day. Regarding knowledge of the disease, 8% of participants older than 18 years said they heard of hydatid cysts or "sand (arenilla)" and 15% referred having seen cysts in livestock viscera. Concerning interaction with dogs, 37% said dogs were guardians, 34% said they were friends or pets, 20% were companions or toys, and 9% said that dogs transmit diseases. How do people show kindness to dogs? 32% pet them, 25% talk to them, and 9% hug and kiss their dogs. While 26% of dogs lick their owners, 22% play or jump, 15% touch their legs, 12% bark, and 25% did not answer. Out of 331 US performed, 2 people had images suggestive of hydatid cysts; added to the index case give a prevalence of 0.9%. Cases were distributed in the same sector of the community (Figure 5). The close distribution of cases suggests the same exposure source.

The index case has been described at the beginning of this chapter in all clinical aspects. Epidemiologic data showed that the husband was a veterinarian. They lived with 12 other family members; all were submitted to diagnostic assays; Casoni test was positive in 3 people at 15 minutes but negative at 48 hours, all 12 were negative in ELISA, and in coproparasito‐ scopic studies. The house had a cement floor and walls, they had a small farm with 4 sheep, 3 cows, 2 horses, and 1 pig; they also grew vegetables and had 4 dogs that are fed with fresh viscera from livestock slaughtered in the backyard. Dogs were held in the same place for 3 days in order to collect feces to look for the adult parasite, and the results were negative.

average time for answering one questionnaire was 10 minutes. Most questions were clearly understood, except when asked if inhabitants had animals, 60% answered no but many had dogs, so another question specific on having dogs was added. This questionnaire was used for

**Figure 4.** Scenery of the community of Santa Cruz (a), an ultrasound participant of the community (b).

(a) (b) (c)

Santa Cruz had then 405 houses (11 were uninhabited), 98% of the streets were paved, electric power, telephone, and drinking water were available, the latter by a chlorine facility. There was no drainage thus latrines were used. Of the 375 houses that were submitted to a census 1,911 inhabitants were found, with an average of 5 (from 1 to 17) per house. Women constituted 63%, average age was 27; 20% of men and women were between 3 and 24 years and 30% were between 25 and 44 years; 48% were single, 41% married, the remaining were widows, couples separated or living together; 39% studied primary school, 24% middle school, 17% high school, 5% kindergarten, 3% bachelor, and 7% illiterate. Male workers made almost half of the population; students (30%) and house workers (23%) constituted the other half. Most houses were built of brick, sand, and had latrines, although 3% used the field. Drinking water was available in 86% of the houses, half of which boil the water before drinking, those lacking drink water from a well. Dogs were present in 83% of houses, 79% were vaccinated for rabies, most slept outside, although half of the dogs were kept tied. Of the 426 dogs in the community, feces were collected from 414 to examine for cestode eggs. After treatment and purge, 3 dogs had *Taenia pisiformis* but none had *Echinococcus*. Also, 83% of people had livestock, 77% were kept

the investigation of risk factors in a larger population.

close to the house.

36 Current Topics in Echinococcosis

The second case detected during the US survey was an 89-year-old married male, without digestive or abdominal symptomatology. The US showed one irregular 76 x 72 mm lesion with a defined wall in the right hepatic lobule, having two densities, one anechoic and internal echoes like snowflakes (Figure 6a). The serum sample was negative. He lived with 6 people in a brick house with a cement floor; he grew vegetables, fresh water was kept in a cistern, drinking water in flasks and had a latrine. He had 2 dogs that were held at the entrance of the house, slept outside, and were fed with leftovers, never with fresh viscera.

The third case, also detected during the US screening survey, was a 46-year-old married female. The US showed one 55 x 48 mm lesion in the left hepatic lobule with a wall slightly limited by a hyperechoic round ring (Figure 6b). The serum sample was negative. There were 4 house‐ holds, she finished primary school, lived in a brick house with a cement floor, had a latrine,

**Figure 5.** Location of the community of Santa Cruz in Mexico and of the cases in the community. Kindly reproduced with permission of the American Society of Tropical Medicine.

fresh water was kept in a small cistern, they boiled drinking water and kept it in flasks. They did not grow vegetables and did not have dogs.

These 2 cases were not confirmed by molecular techniques since, being asymptomatic, they were not offered any treatment at that time. Nevertheless, interestingly, in addition to the fact that the cases lived more or less close by, is the finding that the hydatid cysts of both cases detected in the community had similar sizes (between 5 cm and 7 cm). It is known that hydatid cysts grow approximately 1 cm per year [1, 2], suggesting that exposure occurred around 6 years earlier, and also that possibly one dog harboring *E. granulosus* s.l. adult parasites, living at that time in close contact with their houses, could have infected both patients.

(c) **Figure 6.** US of the second (a) and third probable (b) cases. Kindly reproduced with permission of the American Soci‐ ety of Tropical Medicine.

#### **4. Population genetics study for** *Echinococcus*

Due to scarce source of genetic information and as an attempt to clarify the status of *Echino‐ coccus granulosus* s.l. and genetic variability in Mexico, the presence of the intestinal tapeworms in dogs and of CE in livestock of north and central areas from Mexico with informal reports of this parasite was conducted [16].

### **5. CE in slaughterhouses**

fresh water was kept in a small cistern, they boiled drinking water and kept it in flasks. They

**Figure 5.** Location of the community of Santa Cruz in Mexico and of the cases in the community. Kindly reproduced

These 2 cases were not confirmed by molecular techniques since, being asymptomatic, they were not offered any treatment at that time. Nevertheless, interestingly, in addition to the fact that the cases lived more or less close by, is the finding that the hydatid cysts of both cases detected in the community had similar sizes (between 5 cm and 7 cm). It is known that hydatid cysts grow approximately 1 cm per year [1, 2], suggesting that exposure occurred around 6 years earlier, and also that possibly one dog harboring *E. granulosus* s.l. adult parasites, living

at that time in close contact with their houses, could have infected both patients.

did not grow vegetables and did not have dogs.

with permission of the American Society of Tropical Medicine.

38 Current Topics in Echinococcosis

Several sanitary inspections to the municipal slaughterhouse of Calera, Zacatecas took place between December 2005 and June 2006 to identify CE in visceral organs, as well as to obtain data regarding the infected animals, place of origin, owner information, and number and size of cysts. In this slaughterhouse, formal farm and backyard livestock animals are accepted, coming from Zacatecas and nearby states (Aguascalientes, Morelos, and San Luis Potosi), thus offal from 387 pigs, 243 bovines, and 32 sheep were inspected for the larval stage of *Echino‐ coccus*. Of the more than 660 animal viscera inspected in the Calera slaughterhouse, 18 pigs showed CE (7 males and 11 females, P<0.05), with a mean of 8 hepatic cysts per animal with average size of 3.7±1.7 cm. Interestingly, a similar tendency about gender in infected animals was observed in the experimental model of CE in mice, where female have a higher number of cysts in liver than male [27]. The global frequency of 5% of CE in pigs seen in the municipal slaughterhouse of Calera and the community studied are similar to previous data of Mexico [9, 13], but lower than those from other Latin American countries that can be as high as 12% for livestock [28]. Samples of fluid cysts from 9 pigs (6 from Zacatecas, 2 from Aguascalientes, and 1 from Morelos) were recovered and stored in 70% ethanol and processed for molecular analysis. PCR for amplifying a partial sequence of mitochondrial cytochrome oxidase I (*coI)* of size ≈450 bp [29], were performed to fluid cysts and amplicons were purified, sequenced, and compared with several *coI* sequences from the GenBank.

#### **5.1. Epidemiological study for CE in livestock**

Based on the presence of the parasite in the municipal slaughterhouse of Calera, Zacatecas, a rural community, was selected to identify the adult and larval stages. The community is located in the northern state of Zacatecas, in the central area at 22°55´N, 102°48´W; it has a semi-ariddry weather, mild temperature (mean 15.4o C), with average rainfall of 448 mm H2O.

In 2006, an initial census showed that the community was organized in 39 blocks with 155 houses, 140 were inhabited with 526 peoples, showing a mean of 4±2 people/house, a range from 1 to 11 and a mode of 2. Also, 266 were women and 260 were men, 174 (33%) were younger than 18 years old. On the other hand, 26% were economically active population (136/526) and 74% of households derive their income from a single person; the main activity was agriculture (83%) followed by crafts (10%) and trade (7%). The community had electricity, chlorinated drinking water, and telephone services; 45% of the streets are paved. Of the 140 housing, 49 (35%) had 60 dogs, while in 79 (56%) several livestock were recorded, such as sheep (586 animals/18 houses), cattle (441 animals/58 houses), pigs (124 animals/44 houses), and horses (31 animals/ 24 houses).

#### **5.2. Identification of Echinococcus and treatment of dogs**

With the owners' verbal consent, feces obtained by rectal spoon of all 60 dogs in the community were recovered and analyzed by Faust technique [3, 30] and by Coproantigen ELISA (CpAg ELISA) [31, 32]. Coprological study showed that 5 dogs were infected with *Toxocara canis* (8.3%), 2 with teniids (3.4%) and 1 with *Ancylostoma caninum* (1.6%), while 11 samples positive for *E. granulosus* antigens (18.3%) were detected. Fecal samples were obtained after treatment and used for egg and antigen detection; none was positive.

For Copro-PCR, only in those 11 positive samples by CpAg ELISA were analyzed; JB3 and JB4 primers [29] only 3 samples yielded one amplicon of ≈450 bp and those were purified and sequenced, showing a high identity with *E. canadensis* G7 genotype.

Positive dogs for taeniid eggs or coproantigens were purged and treated; for this they were isolated in cages with fecal collection facilities, which were maintained in the backyards of their homes, and were treated orally with praziquantel and with arecoline bromide. Dogs were kept in the cage and after collection of feces the dogs were released. However, no tapeworms were recovered after treatment, probably because it was administered 3 months after diagno‐ sis, since arecoline bromide is rarely available in Mexico. Fecal samples were obtained after treatment and used for egg and antigen detection; none was positive.

In addition, a questionnaire was applied to search for possible risk factors in the population, and showed that having fed pig and/or sheep raw offal to dogs was significantly associated with *Echinococcus* antigen positivity (odds ratio=5.9, 95% confidence interval=1.11-31.3, P=0.023).

#### **5.3. Identification of CE in pigs and sheep**

**5.1. Epidemiological study for CE in livestock**

dry weather, mild temperature (mean 15.4o

**5.2. Identification of Echinococcus and treatment of dogs**

and used for egg and antigen detection; none was positive.

sequenced, showing a high identity with *E. canadensis* G7 genotype.

treatment and used for egg and antigen detection; none was positive.

(31 animals/ 24 houses).

40 Current Topics in Echinococcosis

P=0.023).

Based on the presence of the parasite in the municipal slaughterhouse of Calera, Zacatecas, a rural community, was selected to identify the adult and larval stages. The community is located in the northern state of Zacatecas, in the central area at 22°55´N, 102°48´W; it has a semi-arid-

In 2006, an initial census showed that the community was organized in 39 blocks with 155 houses, 140 were inhabited with 526 peoples, showing a mean of 4±2 people/house, a range from 1 to 11 and a mode of 2. Also, 266 were women and 260 were men, 174 (33%) were younger than 18 years old. On the other hand, 26% were economically active population (136/526) and 74% of households derive their income from a single person; the main activity was agriculture (83%) followed by crafts (10%) and trade (7%). The community had electricity, chlorinated drinking water, and telephone services; 45% of the streets are paved. Of the 140 housing, 49 (35%) had 60 dogs, while in 79 (56%) several livestock were recorded, such as sheep (586 animals/18 houses), cattle (441 animals/58 houses), pigs (124 animals/44 houses), and horses

With the owners' verbal consent, feces obtained by rectal spoon of all 60 dogs in the community were recovered and analyzed by Faust technique [3, 30] and by Coproantigen ELISA (CpAg ELISA) [31, 32]. Coprological study showed that 5 dogs were infected with *Toxocara canis* (8.3%), 2 with teniids (3.4%) and 1 with *Ancylostoma caninum* (1.6%), while 11 samples positive for *E. granulosus* antigens (18.3%) were detected. Fecal samples were obtained after treatment

For Copro-PCR, only in those 11 positive samples by CpAg ELISA were analyzed; JB3 and JB4 primers [29] only 3 samples yielded one amplicon of ≈450 bp and those were purified and

Positive dogs for taeniid eggs or coproantigens were purged and treated; for this they were isolated in cages with fecal collection facilities, which were maintained in the backyards of their homes, and were treated orally with praziquantel and with arecoline bromide. Dogs were kept in the cage and after collection of feces the dogs were released. However, no tapeworms were recovered after treatment, probably because it was administered 3 months after diagno‐ sis, since arecoline bromide is rarely available in Mexico. Fecal samples were obtained after

In addition, a questionnaire was applied to search for possible risk factors in the population, and showed that having fed pig and/or sheep raw offal to dogs was significantly associated with *Echinococcus* antigen positivity (odds ratio=5.9, 95% confidence interval=1.11-31.3,

C), with average rainfall of 448 mm H2O.

From July 2006 to January 2007, 586 sheep and 55 adult pigs were examined using US for hydatid cysts in the community. For this, standing sheep were immobilized and US was performed with a transductor between the 4th and the 7th intercostal ribs of the shaved right side, no sedation was required for the sheep. Pigs were intramuscularly sedated and positioned for the transducing process. All sheep in the community were submitted to US, except: piglets, pregnant or nursing females, as well as stallions or pigs destined for early human consumption. In the community, sheep and pigs are confined in small flocks inside semi-closed backyard areas where they had access to open feeding, and that were accessible to dogs, cats, and other small wilds animals.

Three local abattoirs in the community were identified; regular visits were undertaken, mainly during pig and sheep sacrifice in order to recover potential infected viscera. Before sacrifice, animals were submitted to US and afterwards, during viscera inspection, spleen, liver, and lungs were meticulously checked. Only 3 female pigs had CE during necropsy, but only in 1 case was the diagnosis previously established using US. However, it was not possible to recover any fluid cysts for DNA analysis.

#### **5.4. Phylogenetic and population genetics analyses**

All sequences obtained of 9 hydatid cysts and 3 by Copro-PCR, were subjected to BLAST search in the GenBank database; multiple alignments were performed with the CLUSTAL W [33] and MUSCLE [34] programs, with manual adjusted in MEGA program v5 [35]. The multiple alignments were used to determine the appropriate model of molecular evolution in the Modeltest 3.7 program [36]. The *coI* sequences for *E. granulosus* s.l. were analyzed with the General Time Reverse model, using gamma distribution (GTR+G). The phylogenetic recon‐ struction using Bayesian inference was performed with Mr Bayes 3.2.1 program [37-39]. Analysis was executed for 10 million generations; trees were sampled every 100 generations. Trees with scores lower than those at the stationary phase were discarded from the analysis, while those that reached the stationary phase were collected and used to build consensus trees. In addition, unrooted haplotype networks were created using NETWORK 4.611 software. These networks were nested according to the rules in Median-Joining networks [40], in which interior/tip status was considered a clade/haplotype and the ancestral haplotype had priority over missing haplotypes. An analysis of genetic diversity within and between populations was performed using DnaSPv4 [41] and included nucleotide diversity (π), haplotype polymor‐ phism (θ), expected heterozygosity (HS), genetic differentiation index (FST), and Tajima's D test.

Up to 335 sequences for *coI* of *E. granulosus*, *E. canadensis*, *E. equinus*, *E. ortleppi, E. shiquicus* and *E. multilocularis* were analyzed, 68 haplotypes were found. GenBank has many more sequences of *E. granulosus* (232) than of other species, HS and π values were lower than *E. canadensis* (Table 1). Also, θ values were very small (~0.3 for *E. granulosus* and *E. canadensis* and <0.01 for other species), whilst Tajima's D test exhibited negative values. When we matched different *Echinococcus* species, the FST value was close to 1, suggesting a very high differentiation between species and corroborating the taxonomic status among these species (Table 2).


1 N, number of sequences analyzed

2 π, nucleotide diversity

3 θ, haplotype polymorphism

4 ND, not determined

**Table 1.** Genetic polymorphism indexes among *Echinococcus* species obtained with the use of *coI* GenBank sequences.


**Table 2.** Genetic differentiation index values between different paired sequences of *Echinococcus* species.

Sequences for *coI* of *E. canadensis* from Africa, Asia, Europe, and Latin and North America deposited in the GenBank databases (58), as well as our 12 sequences were analyzed (from KF734649 to KF734660, GenBank accession numbers). High identity (>98%) between Mexican, Peruvian, and Polish pig sequences was found. Globally, G6 was compared to G7 and to G8 and showed lower value of HS (~0.26) and the pair between G6 and G7 exhibited the lowest value of FST (~0.10); the other genotypes had FST ≥0.6. When G6 and G7 were separated by geographic area, the main pairs had FST ≤0.1, except Europe vs. Africa. We did not find available sequences of G7 in humans, however, there are some sequences for G6 in humans from Africa (Mauritania) and Asia (China, Iran, Mongolia) that presented similar HS and FST values as those sequences for G6 and G7 obtained from livestock (Table 3).


1 HS, expected heterozygosity within each population

2 FST, genetic differentiation index

**Species N1 Haplotypes π<sup>2</sup> θ<sup>3</sup> Tajima's D** *E. granulosus* s.s. 232 31 0.0085 0.0423 -2.3523 *E. canadensis* 70 18 0.0118 0.0336 -2.1235 *E. shiquicus* 16 9 0.0054 0.0076 -1.0886 *E. equines* 10 6 0.0061 0.0075 -0.7831 *E. ortleppi* 3 2 0.0023 0.0022 ND4 *E. multilocularis* 4 2 0.0019 0.0015 ND

**Table 1.** Genetic polymorphism indexes among *Echinococcus* species obtained with the use of *coI* GenBank sequences.

**Table 2.** Genetic differentiation index values between different paired sequences of *Echinococcus* species.

**Population A (Species A) Population B (Species B) FST** *E. canadensis E. equinus* 0.8498 *E. canadensis E. granulosus* s.s. 0.8243 *E. canadensis E. ortleppi* 0.7023 *E. canadensis E. shiquicus* 0.8578 *E. canadensis E. multilocularis* 0.8840 *E. equinus E. granulosus* s.s*.* 0.9156 *E. equinus E. ortleppi* 0.9390 *E. equinus E. shiquicus* 0.9198 *E. equinus E. multilocularis* 0.9517 *E. granulosus* s.s*. E. ortleppi* 0.9068 *E. granulosus* s.s*. E. shiquicus* 0.9269 *E. granulosus* s.s. *E. multilocularis* 0.9429 *E. ortleppi E. shiquicus* 0.9455 *E. ortleppi E. multilocularis* 0.9540 *E. shiquicus E. multilocularis* 0.9557

1

2

3

4

N, number of sequences analyzed

π, nucleotide diversity

42 Current Topics in Echinococcosis

ND, not determined

θ, haplotype polymorphism

FST, genetic differentiation index

3 include sequences from Italy, Poland, and Rumania

4 include sequences from Mexico and Peru

5 include sequences from Algeria, Ethiopia, Mauritania, and Sudan

6 include sequences from Iran and Kazakhstan

7 include sequences of patients from China, Iran, Mauritania, Mongolia, and Russia

**Table 3.** Expected heterozygosity and genetic differentiation indexes between different paired sequences of *Echinococcus canadensis* genotypes obtained from animals.

Regarding the phylogenetic analyses, a Bayesian tree showed that all Mexican sequences obtained were grouped in the *E. canadensis* cluster with a value of 0.80 of posterior probability (Figure 7a), while a second Bayesian inference performed with only *E. canadensis* grouped to Mexican sequences with a G6 and G7 cluster from different reservoirs and different countries (Figure 7b).

**Figure 7.** Bayesian phylogenetic trees of *Echinococcus* (c) species (a) and *Echinococcus canadiensis* (b) using *coI* sequences; Bayesian analysis with the *coI* data; Values at nodes indicate the posterior percentage probabilities using 10 million generations. The Mexican sequences are identified by arrows.

The haplotype network inferences showed consistent results with the values of genetic differentiation previously obtained (Figure 8); some sequences of *E. granulosus* were placed in the *E. canadensis* clade, probably because at the time they were submitted to GenBank, their taxonomic status had not been fully established. However, clearly 6 clades can be identified that correspond to *E. granulosus*s.s., *E. multilocularis, E. canadensis, E. equinus*, *E. oligarthra*, and *E. shiquicus*. Other network trees, which were built only with sequences of *E. canadensis*, exhibited a similar distribution profile according to their host (Figure 9a) and origin country (Figure 9b). Thus, 3 clusters were identified: one for Canada, USA, Finland, Estonia, Mongolia, Kazakhstan with elk, wolf, and moose; the 2nd, for African, Asian, Latin-American, and European countries with camel, cattle, sheep, dog, pig, goat, and human and interestingly, the Mexican G7 haplotypes were placed around core dispersion center; finally, the 3rd cluster grouped other haplotypes G7 from Romania, Mauritania, and Peru and identified in pigs, humans, camels, and cattle.

Regarding the phylogenetic analyses, a Bayesian tree showed that all Mexican sequences obtained were grouped in the *E. canadensis* cluster with a value of 0.80 of posterior probability (Figure 7a), while a second Bayesian inference performed with only *E. canadensis* grouped to Mexican sequences with a G6 and G7 cluster from different reservoirs and different countries

(a) (b)

**Figure 7.** Bayesian phylogenetic trees of *Echinococcus* (c) species (a) and *Echinococcus canadiensis* (b) using *coI* sequences; Bayesian analysis with the *coI* data; Values at nodes indicate the posterior percentage probabilities using 10 million

The haplotype network inferences showed consistent results with the values of genetic differentiation previously obtained (Figure 8); some sequences of *E. granulosus* were placed in the *E. canadensis* clade, probably because at the time they were submitted to GenBank, their taxonomic status had not been fully established. However, clearly 6 clades can be identified that correspond to *E. granulosus*s.s., *E. multilocularis, E. canadensis, E. equinus*, *E. oligarthra*, and *E. shiquicus*. Other network trees, which were built only with sequences of *E. canadensis*, exhibited a similar distribution profile according to their host (Figure 9a) and origin country (Figure 9b). Thus, 3 clusters were identified: one for Canada, USA, Finland, Estonia, Mongolia, Kazakhstan with elk, wolf, and moose; the 2nd, for African, Asian, Latin-American, and European countries with camel, cattle, sheep, dog, pig, goat, and human and interestingly, the Mexican G7 haplotypes were placed around core dispersion center; finally, the 3rd cluster grouped other haplotypes G7 from Romania, Mauritania, and Peru and identified in pigs,

generations. The Mexican sequences are identified by arrows.

humans, camels, and cattle.

(Figure 7b).

44 Current Topics in Echinococcosis

**Figure 8.** Median joining network of *Echinococcus* species using *coI* sequences. Bigger sizes of circles represent higher frequency of each haplotype in each species. Numbers in branches refer to mutational changes.

#### **5.5. What can we interpret about the population genetics study about Echinococcus in Mexico?**

Current revisions accept 9 *Echinococcus* species based on clinical, molecular, epidemiological, and biological data, as well as characteristics of their host-parasite relationship: *E. granulosus* s.s. *, E. felidis*, *E. multilocularis*, *E. shiquicus*, *E. equinus*, *E. oligarthra*, *E. vogeli*, *E. ortleppi* and *E. canadensis* [5-7, 10]. Field and laboratory findings have revealed a significant phenotypic and genetic variability among isolates of *Echinococcus*, mainly for *E. granulosus* s.l. from different intermediate hosts. Recent phylogenetic studies based on both mitochondrial and nuclear DNA genes have showed that *E. granulosus* s.l. consists of 10 strains or genotypes (G1 to G10)

(c) **Figure 9.** Median joining network trees of *Echinococcus canadensis* from different reservoirs (a) and countries (b), built using *coI* sequences. Numbers in branches refer to mutational changes; sizes of circles are proportional to haplotype frequencies, thus, bigger circles represent ancestral haplotypes, small red circles represent missing haplotypes.

with at least 4 valid species: *E. granulosus* s.s. (G1-G3; sheep strains [G1 and G2] and buffalo strain [G3]), *Echinococcus equinus* (G4; horse strain), *Echinococcus ortleppi* (G5; cattle strain), and *Echinococcus canadensis* (G6-G10: camel strain [G6], pig strain [G7], cervid strains [G8 and G10] and human strain [G9, under a controversial status]) [4, 5, 42, 43]. Nonetheless, the species status of *E. canadensis* is still controversial [4-8, 11, 44], because few biological differences have been identified among all strains (from G6 to G10) proposing them as a single species. However, ecological and geographical differences between camel and pig strains to cervid strains show clear differences, suggesting a diversification process. In this way, some authors have suggested that G6 and G7 should be treated as a single species designated as *E. interme‐ dius* [4, 8, 11], and even G8 as *E. borealis* and G10 as *E. canadensis* have also been proposed [8]. In contrast, Nakao et al. [6, 7, 45], advise that G6 to G10 should be retained by the specific name *E. canadensis*, pending further population genetic studies, using particularly nuclear DNA markers to clarify the status of the taxon.

A contrasting phenomenon is observed for CE by *E. canadensis* (G7) in humans, because the pig strain is the main genotype identified in patients from Eastern and Southeastern European countries [46, 47]. There are few human cases of CE reported in Mexican patients, although none by G7 has been identified by molecular techniques. There are only 2 genetic studies performed in samples of CE from Mexico; Cruz-Reyes et al. [11] documented that G7 parasites of Mexican and Polish pig isolates showed similar patterns by RFLP of rDNA ITS1 and RAPD techniques, although PCR-sequencing analysis of mitochondrial *coI* gen fragment was performed, no polymorphism data were reported. Saarma et al. [48] identified 2 variants (A, B) inside of G6/G7 group consisting of samples from Mexico and Argentina, using 5 nuclear markers (elongation factor 1 alpha, transforming growth factor beta receptor kinase, thiore‐ doxin peroxidase, calreticulin, and ezrin-radixin-moesin-like protein).

In general, the genetic population parameters reported by Rodriguez-Prado et al. [16] were similar to those reported by Haag et al.[49], who using mitochondrial (*nad*) and nuclear (*ActII, Hbx2, AgB*) sequences of *Echinococcus*, found π=0.0005 for *E. multilocularis*, and π=0.0090 for *E. granulosus*. In addition, a study focused in the genetic diversity of *Echinococcus granulosus* s.s. hydatid cysts from 4 European countries (Bulgaria, Hungary, Romania, and Italy) evaluated by DNA sequence analysis of the *coI,* showed FST values that ranged from 0.018 to 0.187 with higher negative values for Tajima's D that ranged from -0.515 to -1.066 [50]. Analyzing Mexican and other sequences, it was observed that among *E. canadensis* populations, they have a moderate differentiation (FST ~0.1), while the other genotypes of *E. canadensis* exhibited a high differentiation among them (FST >0.6). When G6 and G7 were divided in geographic areas, similar genetic differentiation was observed with FST <0.1, except when Europe (G7) was matched with Africa or Asia (FST=0.2). However, in both cases, this phenomenon could be interpreted as a moderate genetic differentiation within these populations. Also, we found negative values for Tajima's D, similarly to Casulli et al. [50] data, indicating that a size expansion due to a bottleneck event might have occurred in the past.

Based in the network analyses and according to Rodriguez-Prado et al. [16], the following inferences can be done: 1) *E. canadensis* in Mexico is very diverse and has probably been introduced from abroad several times from different sources because 6 Mexican isolates have from 4 to 14 mutational changes between the isolate and the main haplotype; 2) haplotypes grouped in the North American wildlife cluster (G10) are closer within them, with 1 or 2 mutational changes and they are placed far of the Mexican isolates, thus they might be ruled out as sources of introduction to Mexico; 3) differentiation between G6 and G7 would not make any sense based on the differentiation of genetic indexes found for both genotypes (FST close to 0.1), besides one of the main ancestral dispersion centers in the network analysis, clustered identical haplotypes of G6 and G7 from China, Mexico, Peru, Sudan, and Russia.

#### **6. Final remarks**

with at least 4 valid species: *E. granulosus* s.s. (G1-G3; sheep strains [G1 and G2] and buffalo strain [G3]), *Echinococcus equinus* (G4; horse strain), *Echinococcus ortleppi* (G5; cattle strain), and *Echinococcus canadensis* (G6-G10: camel strain [G6], pig strain [G7], cervid strains [G8 and G10] and human strain [G9, under a controversial status]) [4, 5, 42, 43]. Nonetheless, the species status of *E. canadensis* is still controversial [4-8, 11, 44], because few biological differences have been identified among all strains (from G6 to G10) proposing them as a single species. However, ecological and geographical differences between camel and pig strains to cervid strains show clear differences, suggesting a diversification process. In this way, some authors have suggested that G6 and G7 should be treated as a single species designated as *E. interme‐ dius* [4, 8, 11], and even G8 as *E. borealis* and G10 as *E. canadensis* have also been proposed [8]. In contrast, Nakao et al. [6, 7, 45], advise that G6 to G10 should be retained by the specific name *E. canadensis*, pending further population genetic studies, using particularly nuclear DNA

(a) (b)

(c) **Figure 9.** Median joining network trees of *Echinococcus canadensis* from different reservoirs (a) and countries (b), built using *coI* sequences. Numbers in branches refer to mutational changes; sizes of circles are proportional to haplotype frequencies, thus, bigger circles represent ancestral haplotypes, small red circles represent missing haplotypes.

A contrasting phenomenon is observed for CE by *E. canadensis* (G7) in humans, because the pig strain is the main genotype identified in patients from Eastern and Southeastern European countries [46, 47]. There are few human cases of CE reported in Mexican patients, although none by G7 has been identified by molecular techniques. There are only 2 genetic studies performed in samples of CE from Mexico; Cruz-Reyes et al. [11] documented that G7 parasites of Mexican and Polish pig isolates showed similar patterns by RFLP of rDNA ITS1 and RAPD techniques, although PCR-sequencing analysis of mitochondrial *coI* gen fragment was

markers to clarify the status of the taxon.

46 Current Topics in Echinococcosis

The first human CE case identified by molecular techniques was reported in 2006 in the United Kingdom (UK) in a male with clinical and CT scan images consistent with CE that was surgically treated for the removal of a hydatid cyst, which was subsequently confirmed as *E. granulosus* G1 [51]. This case was similar to the Mexican one: it was unexpected and wrongly diagnosed for a long time. Also in the UK, recent *Echinococcus* isolates from intermediate and definitive animal hosts, as well as from human CE cases were analyzed to determine species and genotypes within these hosts. *E. equinus* was identified from horse hydatid isolates, while *E. granulosus* s.s. was identified from hydatid cysts of sheep and cattle, as well as in DNA extracted from a farm dog, foxhound fecal samples, and from human cystic echinococcosis isolates. Low genetic variability for *E. equinus* using *coI* sequences (π=0.000666±0.000620) indicates the presence of a dominant haplotype; in contrast, greater haplotypic variation was observed for *E. granulosus* s.s. (π=0.000955±0.000790). Furthermore, a haplotype phylogenetic inference showed a star-shaped network with a centrally placed main haplotype that was reported from other world regions [52].

Four new cases of CE in Mexican patients were reported since 2009, a 38-year-old female from the State of Mexico, who was submitted to surgery to remove a liver cyst [53]; 2 cases referred to a tertiary-care oncology hospital with an initial diagnosis of liver and lung cancer, respec‐ tively. The liver cyst case was resolved with percutaneous drainage and administration of albendazole for 2 months, while the lung cyst was resolved solely with 2 months of albendazole [54]. The last case was a 16-year-old female from the state of Puebla, Mexico with a liver cyst that was surgically removed [55]. Unfortunately, in all cases, molecular assays were not performed so identification of *Echinococcus* strains was not possible.

#### **7. Conclusion**

We wish to finalize this chapter by pointing out that the correct diagnosis and adequate management of the initial case, the subsequent epidemiological follow-up of human echino‐ coccosis at a community level in Mexico and the in-depth genetic study of the parasite, integrate a comprehensive and unique study of *Echinococcus granulosus,* s.l. and support the very low frequency of this disease in Mexico.

Although no data on cystic echinococcosis caused by *E. canadensis* G7 have previously been documented in Mexican patients, the high number of infected patients in central Europe by the G7 genotype, points to the potential public health importance of this strain or species, as well as the necessity to perform molecular and epidemiological studies focused to clarify this contrasting situation and the controversial taxonomic status of *E. canadensis* species.

#### **Author details**

Ana Flisser1\*, Pablo Maravilla2 , Pilar Mata-Miranda3 and Fernando Martinez-Hernandez2

\*Address all correspondence to: flisser@unam.mx

1 Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, DF, Mexico

2 Departamento de Ecologia de Agentes Patogenos, Hospital General "Dr. Manuel Gea Gonzalez", Secretaria de Salud, DF, Mexico

3 Coordinación Auxiliar de Investigación en Salud, Delegación Veracruz Norte, Xalapa, Veracruz, Mexico

#### **References**

observed for *E. granulosus* s.s. (π=0.000955±0.000790). Furthermore, a haplotype phylogenetic inference showed a star-shaped network with a centrally placed main haplotype that was

Four new cases of CE in Mexican patients were reported since 2009, a 38-year-old female from the State of Mexico, who was submitted to surgery to remove a liver cyst [53]; 2 cases referred to a tertiary-care oncology hospital with an initial diagnosis of liver and lung cancer, respec‐ tively. The liver cyst case was resolved with percutaneous drainage and administration of albendazole for 2 months, while the lung cyst was resolved solely with 2 months of albendazole [54]. The last case was a 16-year-old female from the state of Puebla, Mexico with a liver cyst that was surgically removed [55]. Unfortunately, in all cases, molecular assays were not

We wish to finalize this chapter by pointing out that the correct diagnosis and adequate management of the initial case, the subsequent epidemiological follow-up of human echino‐ coccosis at a community level in Mexico and the in-depth genetic study of the parasite, integrate a comprehensive and unique study of *Echinococcus granulosus,* s.l. and support the

Although no data on cystic echinococcosis caused by *E. canadensis* G7 have previously been documented in Mexican patients, the high number of infected patients in central Europe by the G7 genotype, points to the potential public health importance of this strain or species, as well as the necessity to perform molecular and epidemiological studies focused to clarify this

contrasting situation and the controversial taxonomic status of *E. canadensis* species.

, Pilar Mata-Miranda3

1 Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad

2 Departamento de Ecologia de Agentes Patogenos, Hospital General "Dr. Manuel Gea

3 Coordinación Auxiliar de Investigación en Salud, Delegación Veracruz Norte, Xalapa,

and Fernando Martinez-Hernandez2

performed so identification of *Echinococcus* strains was not possible.

reported from other world regions [52].

48 Current Topics in Echinococcosis

very low frequency of this disease in Mexico.

\*Address all correspondence to: flisser@unam.mx

Nacional Autonoma de Mexico, DF, Mexico

Gonzalez", Secretaria de Salud, DF, Mexico

**7. Conclusion**

**Author details**

Veracruz, Mexico

Ana Flisser1\*, Pablo Maravilla2


[24] Palacios-Ruíz JA, Ramírez-Solís E, Moreno-Moller M, Cárdenas-Mejía A, Mata-Mir‐ anda P, Maravilla P, Flisser A. Identificación y manejo integral de un caso autóctono de hidatidosis en México. Rev Mex Gastroenterol. 2003;68:41-45.

[12] Salinas-Lopez N, Jimenez-Guzman F, Cruz-Reyes A: Presence of *Echinococcus oli‐ garthrus* (Diesing, 1863) Lühe, 1910 in *Lynx rufus texensis* Allen, 1895 from San Fer‐ nando, Tamaulipas state, in North-east Mexico. Int J Parasitol. 1996;26:793-796. [13] Villalobos N, González LM, Morales J, de Aluja AS, Jiménez MI, et al. Molecular identification of *Echinococcus granulosus* genotypes (G1 and G7) isolated from pigs in

[14] Maravilla P, Thompson RCA, Palacios-Ruiz JA, Estcourt A, Ramirez-Solis E, et al. *Echinococcus granulosus* cattle strain identification in an autochthonous case of cystic

[15] Rodriguez-Prado U, Gutiérrez-Marín A, Martínez-Ocaña J, Romero-Valdovinos M, Olivo-Díaz A, Hernández-Castro R, Flisser A, Maravilla P, Martínez-Maya JJ. Estudio coprológico y ultrasonogáfico en perros y borregos de una comunidad suburbana en el Estado de México para la búsqueda de *Echinococcus spp.* Vet Mex. 2014;Supp:53-58.

[16] Rodriguez-Prado U, Jimenez-Gonzalez DE, Avila G, Gonzalez AE, Martinez-Flores WA, Mondragon de la Peña C, Hernandez-Castro R, Romero-Valdovinos M, Flisser A, Martinez-Hernandez F, Maravilla P, Martinez-Maya JJ: Genetic variation of *Echi‐ nococcus canadensis* (G7) in Mexico. Am J Trop Med Hyg. 2014;91:1149-1153. doi:

[17] Ceruzzi O, Osimani JJ, Botto C, Rodríguez N, Cabrera R. Algunos factores a consid‐ erar en la evaluación de la intradermorreacción de Casoni para hidatidosis. Bol Chile

[18] Alper A, Emre A, Hazar H, Özden I, Bilge O, Acarli K, Ariogul O. Laparoscopic sur‐ gery of hepatic hydatid disease: Initial results and early follow-up of 16 patients.

[19] Khoury G, Jabbour-Khoury S, Bikhazi K. Results of laparoscopic treatment of hyda‐

[20] Saglam A. Laparoscopic treatment of liver hydatid cysts. Surg Laparosc Endosc.

[21] Sayek I, Onat D. Diagnosis and treatment of uncomplicated hydatid cyst of the liver.

[22] Maddison SE, Slemenda SB, Schantz PM, Fried JA, Wilson M, Tsang VCW. A specific diagnostic antigen of *Echinococcus granulosus* with an apparent molecular weight of 8

[23] Palacios JA, Ramírez-Solis ME, Cárdenas-Mejia A, Maravilla P, Flisser A. Hypertonic

echinococcosis in central Mexico. Acta Trop. 2004;92:231-236.

Mexico. Vet Parasitol. 2007;147:185-189.

10.4269/ajtmh.14-0317.

50 Current Topics in Echinococcosis

Parasitol. 1976;31:50.

1996;6:16.

World J Surg 1995;19:725.

World J Surg. 2001;25:21.

tid cyst of the liver. Surg Endosc. 1996;10:57.

KDa. Am J Trp Med Hyg. 1989;40:377-383.

saline in hydatid disease. World J Surg. 2002;26:1398.


*coccus granulosus* sensu stricto from the United Kingdom: Genetic diversity and hap‐ lotypic variation. Int J Parasitol. 2015;45:161-6. doi: 10.1016/j.ijpara.2014.10.005.

[53] Steta J, Torre A. Mexican-native human echinococcosis: Case report of an underesti‐ mated disease. Ann Hepatol. 2009;8:251-254.

[39] Ronquis F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed

[40] Bandelt HJ, Forster P, Röhl A. Median-joining networks for inferring intraspecific

[41] Rozas J, Sanchez-Del Barrio JC, Messeguer X, Rozas R. DNA sequence Polymor‐ phism (DNAsp) ver. 4.10.9. Departamento de Genetica, Universidad de Barcelona.

[42] Thompson RC, McManus DP. Towards a taxonomic revision of the genus *Echinococ‐*

[43] Alvarez Rojas CA, Romig T, Lightowlers MW. *Echinococcus granulosus* sensu lato gen‐ otypes infecting humans—Review of current knowledge. Int J Parasitol. 2014;44:9-18.

[44] Yanagida T, Mohammadzadeh T, Kamhawi S, Nakao M, Sadjjadi SM, et al. Genetic polymorphisms of *Echinococcus granulosus* sensu stricto in the Middle East. Parasitol

[45] Nakao M, McManus DP, Schantz PM, Craig PS, Ito A. A molecular phylogeny of the genus *Echinococcus* inferred from complete mitochondrial genomes. Parasitology.

[46] Schneider R, Gollackner B, Schindl M, Tucek G, Auer H. *Echinococcus canadensis* G7 (pig strain): An underestimated cause of cystic echinococcosis in Austria. Am J Trop

[47] Dybicz M, Gierczak A, Dąbrowska J, Rdzanek Ł, Michałowicz B. Molecular diagnosis of cystic echinococcosis in humans from central Poland. Parasitol Int.

[48] Saarma U, Jõgisalu I, Moks E, Varcasia A, Lavikainen A, Oksanen A, Simsek S, An‐ dresiuk V, Denegri G, González LM, Ferrer E, Gárate T, Rinaldi L, Maravilla P. A novel phylogeny for the genus *Echinococcus*, based on nuclear data, challenges rela‐

[49] Haag KL, Zaha A, Araújo AM, Gottstein B. Reduced genetic variability within cod‐ ing and non-coding regions of the *Echinococcus multilocularis* genome. Parasitology.

[50] Casulli A, Interisano M, Sreter T, Chitimia L, Kirkova Z, La Rosa G, Pozio E. Genetic variability of *Echinococcus granulosus* sensu stricto in Europe inferred by mitochondri‐

[51] Craig PS, Woods ML, Boufana B, O´Loughlin B, Gimpel J, San Lett W, Mcmanus DP. Cystic echinococcosis in a fox-hound hunt worker, UK. Pathogens and Global

[52] Boufana B, Lett WS, Lahmar S, Buishi I, Bodell AJ, Varcasia A, Casulli A, Beeching NJ, Campbell F, Terlizzo M, McManus DP, Craig PS. *Echinococcus equinus* and *Echino‐*

tionships based on mitochondrial evidence. Parasitology. 2009;136:317-28.

al DNA sequences. Infect Genet Evol. 2012;12:377-383.

models. Bioinformatics. 2003;19:1572-1574.

phylogenies. Mol Biol Evol. 1999;16:37-48.

*cus.* Trends Parasitol. 2002;18:452-457.

Int. 2012;61:599-603.

2007;134(Pt 5):713-722.

2013;62:364-367.

1997;115:521-529.

Health. 2012;106:373-375.

Med Hyg. 2010;82:871-874.

2006.

52 Current Topics in Echinococcosis


#### **Chapter 3**
