**Meet the editor**

Dr. Luis Rodrigo, MD, is actually emeritus professor of Medicine at the University of Oviedo (Spain). He has been the chief of Gastroenterology Service at the University Hospital in Oviedo for 42 years. He obtained his PhD degree in 1975 and has developed a long teaching and research career during this time period. He is emeritus full professor of Medicine at the University of Ovie-

do, since 2014. He has published a total of 551 scientific papers, 270 written in English and the rest in Spanish. He has directed as main investigator in a total of 45 clinical trials. He has written around 35 chapters in books of several subjects and has been the editor of 13 books in his speciality.

## Contents

**Preface XI**

Chapter 4 **Helminthiasis: A Systematic Review of the Immune Interactions Present in Individuals Coinfected with HIV and/or Tuberculosis 65** Zilungile L. Mkhize-Kwitshana, Rebecca Tadokera and Musawenkosi H.L. Mabaso

Chapter 5 **Atherosclerosis and Helminths Infection 109** Shi-Wei Shen, Yun Lu, Feng Li, Zhen-Hai Shen, Ming Xu, Wei-Feng Yao, Hua-Jin Qi, Ling Zhou, Yin-Bo Feng, Ling Wang, Jing-Ting Yun and Da-Xin Tong

## **Section 4 Therapy and Preventive Measures 119**

## Chapter 6 **Global Control Efforts of Schistosomiasis and Soil-Transmitted Helminthiasis 121**

Takafira Mduluza, Tawanda J. Chisango, Agness F. Nhidza and Amos Marume

## Preface

**Section 4 Therapy and Preventive Measures 119**

**Helminthiasis 121**

Amos Marume

**VI** Contents

Chapter 6 **Global Control Efforts of Schistosomiasis and Soil-Transmitted**

Takafira Mduluza, Tawanda J. Chisango, Agness F. Nhidza and

Human helminthiasis is part of the neglected tropical diseases (NTDs) which are a diverse group of parasitic and bacterial diseases with distinct characteristics that thrive mainly among the poorest populations, hinder socioeconomic development, and cause substantial illness for more than one billion people globally. Seventeen NTDs have been specified by the World Health Organization (WHO). These NTDs impair physical and intellectual capacities of the affected persons, thereby perpetuating the cycle of poverty. Forty-seven countries in the African region are endemic to at least one NTD, and thirty-seven of them (79%) are coen‐ demic to at least five of these diseases.

Zoonotic parasitic diseases are increasingly impacting human populations, due to the effects of globalization, urbanization, and climate change. The authors discuss those helminth dis‐ eases which are increasing in endemic areas and consider their geographical spread into new regions within the framework of globalization, urbanization, and climate change to de‐ termine the effect these variables have on disease incidence, transmission, and the associat‐ ed challenges presented for public health initiatives, including control and elimination.

Although a considerable number of studies have been undertaken to date, it is still contro‐ versial as to whether or not coinfection with schistosomiasis increases the susceptibility to or the progression from hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. One chap‐ ter analyzes this important topic. Greater use of the HBV vaccine is particularly advisable in endemic areas for schistosomiasis.

As genomic data for parasites are increasing, novel techniques for detection incorporating nucleotide sequences are receiving widespread attention. These sensitive, specific, and rapid detection methods are particularly important in the diagnosis of low-grade and early infec‐ tions and may prove to have clinical significance. One chapter reviews the progress of nucle‐ ic acid detection in the diagnosis and prevention of schistosomiasis, including such aspects as the selection of target genes, and development and application of nucleic acid detection methods.

There is low-quality evidence that treating confirmed helminth infections in HIV-positive adults may have small, short-term favorable effects on markers of this disease progression. Further studies are required to confirm this finding. Current evidence suggests that de‐ worming with anthelmintics is not harmful, and this is reassuring for the routine treatment of confirmed or suspected helminth infections in people living with HIV in coendemic areas. Further long-term studies are required to make confident conclusions regarding the impact of presumptively deworming all HIV-positive individuals, irrespective of helminth infection status, as the only long-term trial to date did not demonstrate a clear effect.

Also, there is an increased evidence on the role of helminth infections in modifying autoim‐ mune and allergic diseases. These infections may have similar effect in other inflammatory processes, such as insulin resistance or the progression of atherosclerosis. Some studies have shown that helminth infections can be associated with improved metabolic outcomes. Un‐ derstanding the mechanisms underlying this relationship could facilitate the development of novel strategies to prevent or delay the progression of type 2 diabetes mellitus.

Interventions that lead to reductions in soil-transmitted helminths (STHs) include chemo‐ therapy with anthelmintic drugs and improvements in water, sanitation, and hygiene (WASH). In one of these chapters, the authors determine the evidence for optimal ap‐ proaches for STH control. While multicomponent integrated control may be an effective ap‐ proach to sustainably reduce STH transmission, there is a need for evidence to prove the feasibility of this approach.

I want to thank all the authors for their clear and very important contributions, as well as the speed and efficiency in sending their chapters on time. My heartfelt gratitude also to all the excellent team members from the InTech team, especially to Ms. Dajana Pemac for her continuous support through all the editorial process.

> **Prof. Luis Rodrigo, MD** Emeritus Professor of Medicine University of Oviedo Oviedo, Spain

**Section 1**

## **Epidemiology**

Also, there is an increased evidence on the role of helminth infections in modifying autoim‐ mune and allergic diseases. These infections may have similar effect in other inflammatory processes, such as insulin resistance or the progression of atherosclerosis. Some studies have shown that helminth infections can be associated with improved metabolic outcomes. Un‐ derstanding the mechanisms underlying this relationship could facilitate the development

Interventions that lead to reductions in soil-transmitted helminths (STHs) include chemo‐ therapy with anthelmintic drugs and improvements in water, sanitation, and hygiene (WASH). In one of these chapters, the authors determine the evidence for optimal ap‐ proaches for STH control. While multicomponent integrated control may be an effective ap‐

I want to thank all the authors for their clear and very important contributions, as well as the speed and efficiency in sending their chapters on time. My heartfelt gratitude also to all the excellent team members from the InTech team, especially to Ms. Dajana Pemac for her

a need for evidence to prove the

**Prof. Luis Rodrigo, MD** Emeritus Professor of Medicine

> University of Oviedo Oviedo, Spain

of novel strategies to prevent or delay the progression of type 2 diabetes mellitus.

proach to sustainably reduce STH transmission, there is

continuous support through all the editorial process.

feasibility of this approach.

VIII Preface

#### **World Wide Epidemiology of Helminths Infection World Wide Epidemiology of Helminths Infection**

Mohamed S. Abdeltawabi, Nahla El Seddik and Mohamed S. Abdeltawabi, Nahla El Seddik

Hosni K. Salem and Hosni K. Salem

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67273

## **Abstract**

Helminths (from the Greek Helmins, meaning worm) include three groups of parasitic worm, large multicellular organisms with complex tissues and organs. Helminths do not replicate within the human host except *Strongyloides stercoralis*. Prevalence is commonly combined with worm burden (intensity of infection), which is commonly measured by the number of eggs per gram (EPGs) of faeces for intestinal helminths and schisto‐ somes. Based on EPGs and their association with morbidity, individuals are classified into categories of light, moderate and heavy infection by the WHO. In the case of soil‐ transmitted helminths, the WHO recommends use of both prevalence and intensity of infection to classify communities into transmission categories—category I (high), cate‐ gory II (medium), and category III (low). The neglected status of the helminthiasis should be addressed on community levels and globally all over the world.

**Keywords:** epidemiology, helminths, infection, trematodes, cestodes, nematodes

## **1. Introduction, key determinants underlying the epidemiology of helminths infections**

Helminths are worm‐like parasites so they are commonly known as parasitic worms. They are classified mainly according to the morphology of the different stages; egg, larval and adult stages externally and internally [1].

Sexually, helminths have hermaphroditic species such as tapeworms and flukes except blood flukes. There also bisexual species like roundworms, the most common group worldwide is nematodes [1].

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2017 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.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

There are main three categories of helminths: trematodes (flukes), cestodes (tapeworms) and nematodes (roundworms) [1].

Studying epidemiology of helminths is highly important because they have a wide geograph‐ ical distribution in different regions especially in sub‐Saharan Africa, South America, Central America, Middle East, the Caribbean and Asia [2].

In 1998, the WHO Collaborating Centre for the Epidemiology of Intestinal Parasites at University of Oxford and WHO launched Global Atlas of Helminth Infections (GAHI) which is an initiative aimed at collating the available survey data as both a database and graphical tool (www.thiswormyworld.org) [2].

Based on GAHI, data collected between 1998 and 2010, on prevalence of helminths infections. Some data were collected older than these dates if no available data for a particular country.

Some countries were excluded based on their socioeconomic levels. And also three countries were excluded according to the comprehensive control such as Mauritius, Maldives and Mayotte. Seven countries in Oceania had no data, so they were assigned mean prevalence based on the regional data. Also eight countries had no data were assigned mean prevalence based on data of neighbouring countries with similar eco‐epidemiological situation such as Georgia, Iraq and Turkmenistan data from Iran; Algeria data from Morocco; Syria and Tunisia data from Turkey; PDR Korea from Jilin Sheng (A province in China); and Timor Leste from Indonesia [2, 3].

**Table 1** shows the distribution‐prevalence of different helminths all over the world.

**Table 1.** The prevalence of human helminthiasis.

## **2. Epidemiology of trematodes (flukes)**

## **2.1. Fasciola**

There are main three categories of helminths: trematodes (flukes), cestodes (tapeworms) and

Studying epidemiology of helminths is highly important because they have a wide geograph‐ ical distribution in different regions especially in sub‐Saharan Africa, South America, Central

In 1998, the WHO Collaborating Centre for the Epidemiology of Intestinal Parasites at University of Oxford and WHO launched Global Atlas of Helminth Infections (GAHI) which is an initiative aimed at collating the available survey data as both a database and graphical

Based on GAHI, data collected between 1998 and 2010, on prevalence of helminths infections. Some data were collected older than these dates if no available data for a particular country. Some countries were excluded based on their socioeconomic levels. And also three countries were excluded according to the comprehensive control such as Mauritius, Maldives and Mayotte. Seven countries in Oceania had no data, so they were assigned mean prevalence based on the regional data. Also eight countries had no data were assigned mean prevalence based on data of neighbouring countries with similar eco‐epidemiological situation such as Georgia, Iraq and Turkmenistan data from Iran; Algeria data from Morocco; Syria and Tunisia data from Turkey; PDR Korea from Jilin Sheng (A province in China); and Timor Leste from Indonesia [2, 3].

**Table 1** shows the distribution‐prevalence of different helminths all over the world.

LF *Wuchereria bancrofti*; *Brugia malayi* India, Southeast Asia and sub‐Saharan Africa

*Schistosoma mansoni* Sub‐Saharan Africa and Eastern Brazil

Cysticercosis *Taenia solium* (pork tapeworm) sub‐Saharan Africa and Sub‐Saharan Africa

*Schistosoma japonicum* (blood flukes) China and Southeast Asia *Clonorchis sinensis* (liver fluke) Developing regions of East Asia *Opisthorchis viverrini* (liver fluke) Developing regions of East Asia *Paragonimus* spp. (lung flukes) Developing regions of East Asia *Fasciolopsis buski* (intestinal fluke) Developing regions of East Asia *Fasciola hepatica* (intestinal fluke) Developing regions of East Asia

Ascariasis *Ascariasis lumbricoides* (roundworm) Asia, Africa and Latin America Trichuriasis *Trichuris trichiura* (whipworm) Asia, Africa and Latin America Hookworm *Necator americanus*; *Ancylostoma duodenale* Asia, Africa and Latin America Strongyloidiasis *Strongyloides stercoralis* (threadworm) Asia, Africa and Latin America

Onchocerciasis (river blindness) *Onchocerca volvulus* Sub‐Saharan Africa Loiasis *Loa loa* Sub‐Saharan Africa Dracunculiasis (guinea worm) *Dracunculus medinensis* Sub‐Saharan Africa Schistosomiasis *Schistosoma haematobium* Sub‐Saharan Africa

**Helminthiasis Region**

nematodes (roundworms) [1].

4 Human Helminthiasis

tool (www.thiswormyworld.org) [2].

**Table 1.** The prevalence of human helminthiasis.

America, Middle East, the Caribbean and Asia [2].

They are highly distributed worldwide, and their reservoir hosts are not the human.

There are four species of Fasciola:

## **2.2. Paragonimus westermani**

It is a lung fluke which is found in South America and Eastern Asia. Eating raw seafood is the mode of infection. It mainly affects the lung and presents there encapsulated [1, 4].

## **2.3. Schistosomes**

They are blood‐dwelling trematodes. They have separate sexes.

It is estimated that 250 million people infected with Schistosomiasis by its major types: man‐ soni, haematobium and japonicum, more than 90% of them are in sub‐Saharan Africa.

Also 500 million or more people are exposed to infection. The mode of infection is penetration of skin by cercaria. They mainly affect liver, lung, intestine and bladder.

There are three major species of Schistosomes:

There are also another two species but they have the same symptoms like *Schistosoma mansoni*:

**Figure 1.** Map of the current global distribution of schistosomiasis. Source: US Centers for Disease Control and Prevention, <http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter‐3‐infectious‐diseases‐related‐to‐travel/schistosomiasis#2696>) [5].

## **2.4. The oriental liver flukes**

They are found in the Far East and Europe. The mode of infection is eating raw fish. They affect the liver.

There are three main species: *Clonorchis sinensis*, *Opisthorchis felineus* and *Opisthorchis viverrini* [4].

## **2.5. Heterophyes heterophyes**

It is found in the Far East. The mode of infection is eating raw or pickled fish. It affects the small intestine [1].

## **2.6. Metagonimus yokogawai**

It is found in the Far East. Eating raw or pickled fish is the mode of infection. It affects the small intestine [1].

**Table 2** showing the diseases caused by flukes in the bile duct.

There are also another two species but they have the same symptoms like *Schistosoma mansoni*:

They are found in the Far East and Europe. The mode of infection is eating raw fish. They

**Figure 1.** Map of the current global distribution of schistosomiasis. Source: US Centers for Disease Control and Prevention, <http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter‐3‐infectious‐diseases‐related‐to‐travel/schistosomiasis#2696>) [5].

There are three main species: *Clonorchis sinensis*, *Opisthorchis felineus* and *Opisthorchis viverrini* [4].

It is found in the Far East. The mode of infection is eating raw or pickled fish. It affects the

It is found in the Far East. Eating raw or pickled fish is the mode of infection. It affects the

• *Schistosoma mekongi*: It is found in Mekong River basin in Southeast Asia [1, 4].

• **Figure 1** showing the current global distribution of schistosomiasis worldwide [5].

• *Schistosoma intercalatum*: It is found in Central and West Africa.

**2.4. The oriental liver flukes**

**2.5. Heterophyes heterophyes**

**2.6. Metagonimus yokogawai**

affect the liver.

6 Human Helminthiasis

small intestine [1].

small intestine [1].

**Table 2.** The diseases caused by flukes in the bile duct.

## **3. Epidemiology of Cestode (tapeworms)**

**Table 3** shows the prevalence of human Cestodiases around the world.

**Table 3.** The prevalence of human Cestodiases (tapeworms).

#### **3.1. Taenia species**

They are the most common cestodes infect humans. They affect the intestine especially the duodenum.

There are two species of Taenia:

## **3.2. Hymenolepididae**

They have a cosmopolitan distribution. The mode of infection by them is eating raw veg‐ etables. They affect the small intestine.

There are four species of Hymenolepididae:

## **3.3. Diphyllobothriidae**

There are three species of Diphyllobothriidae:

## **3.4. Dipylidium caninum**

Rarely, human is infected with *Dipylidium caninum*. Almost all reported cases are children. The mode of infection is eating raw vegetables. It affects the small intestine [7].

## **3.5. Larval cestodes**

• *Echinococcus granulosus*: It is a larval stage of dog cestode. It causes Echinococcosis which is also known as hydatid disease due to development of hydatid cysts. These cysts can be located in the liver, the lung, the brain and the kidney. This disease is found commonly in Africa, the Middle East, South America and Australia. The mode of infection is eating raw vegetables.

## **4. Epidemiology of nematodes (roundworms)**

There are two species of Taenia:

8 Human Helminthiasis

eating improperly cooked beef.

ing improperly cooked pork [6].

etables. They affect the small intestine.

There are four species of Hymenolepididae:

There are three species of Diphyllobothriidae:

**3.2. Hymenolepididae**

dren more than adults.

humans.

on Cestodes [6].

**3.3. Diphyllobothriidae**

the small intestine.

**3.4. Dipylidium caninum**

**3.5. Larval cestodes**

vegetables.

eating mammals and birds [7].

• *Taenia saginata*: It has a cosmopolitan distribution, but is more commonly found in develop‐ ing countries due to poor socioeconomic levels and bad hygiene. The mode of infection is

• *Taenia solium*: It is almost not present in the USA and Europe. The mode of infection is eat‐

They have a cosmopolitan distribution. The mode of infection by them is eating raw veg‐

• *Hymenolepis nana*: It is thought to be the most common Cestode in the world. It affects chil‐

• *Hymenolepis diminuta*: It has a cosmopolitan distribution but it is found infrequently in

• *Hymenolepis microstoma* and *Hymenolepis citelli*: They have been used extensively for studies

• *Diphyllobothrium latum*: It has a cosmopolitan distribution but it is found mostly in coun‐ tries bordering the Baltic Sea in Europe. The mode of infection is eating raw fish. It affects

• *Diphyllobothrium dendriticum* and *Diphyllobothrium ditremum* are mainly parasites of fish‐

Rarely, human is infected with *Dipylidium caninum*. Almost all reported cases are children.

• *Echinococcus granulosus*: It is a larval stage of dog cestode. It causes Echinococcosis which is also known as hydatid disease due to development of hydatid cysts. These cysts can be located in the liver, the lung, the brain and the kidney. This disease is found commonly in Africa, the Middle East, South America and Australia. The mode of infection is eating raw

The mode of infection is eating raw vegetables. It affects the small intestine [7].

**Table 4** shows the prevalence of nematodes around the world.

**Table 4.** Global prevalence of nematodes (roundworms).

## **4.1. Ascaris lumbricoides**

It is the largest intestinal nematodes found in humans. It has a cosmopolitan distribution, but it is more common in tropical and subtropical zones. The mode of infection is eating of raw vegetables. It affects the intestine and the lung [8].

## **4.2. Hookworm species**

They are considered most destructive parasitic helminths infect human. Penetration of skin by Filariform larva is the mode of infection.

**Figure 2** showing the life cycle of Ancylostoma.

**Figure 2.** The life cycle of Ancylostoma.

## **4.3. Trichuris trichiura**

It is known as the whipworm. Children are most commonly affected. It is found in tropi‐ cal zones with poor sanitation. The mode of infection is eating raw vegetables. It affects the caecum and the upper part of the colon [1].

## **4.4. Strongyloides stercoralis**

**4.2. Hookworm species**

10 Human Helminthiasis

small intestine.

**4.3. Trichuris trichiura**

**Figure 2.** The life cycle of Ancylostoma.

caecum and the upper part of the colon [1].

by Filariform larva is the mode of infection.

**Figure 2** showing the life cycle of Ancylostoma.

America, and Oceania. It affects the small intestine.

They are considered most destructive parasitic helminths infect human. Penetration of skin

• *Necator americanus*: It is a New World hookworm. It is found in Far East, Asia, Africa, South

• *Ancylostoma duodenale*: It is an Old World hookworm. It is found in the Middle East, North China, Europe, the Mediterranean countries, Africa, Asia and South America. It affects the

• Cutaneous Larva migrans (*Ancylostoma braziliense* or *Ancylostoma caninum*): The mode of infection is contacting with hookworms larva of the dog or cat. They affect the skin [8].

It is known as the whipworm. Children are most commonly affected. It is found in tropi‐ cal zones with poor sanitation. The mode of infection is eating raw vegetables. It affects the It is found in sub‐tropical zones. Most of the 52 species of *Strongyloides* do not infect humans. *S stercoralis* is the most common human pathogen. Other species include *S myopotami* and *S procyonis*. These species have animal hosts and are thus responsible for zoonotic infections. Infections are initiated when exposed skin contacts contaminated soil. Autoinfection com‐ monly occurs allowing infection to persist decades. Hyperinfection is typically triggered by drug‐induced or disease‐associated defects in cellular immunity, which allows a massive increase in parasite burden and dissemination to nearly all organ systems [9].

## **4.5.** *Angiostrongylus* **(Parastrongylus)** *cantonensis*

It is also known as the rat nematodes. It is found in the Pacific areas, Central America and Cuba. The mode of infection is eating raw vegetables. It causes eosinophilic meningitis [1].

## **4.6.** *Angiostrongylus* **(Parastrongylus)** *costaricensis*

It is found mainly in Costa Rica, but also some cases have been reported in Mexico, Central and South America. The mode of infection is eating raw vegetables. It affects the bowel wall [1].

## **4.7. Anisakiasis**

It is found in Netherlands, North America, Canada, Chile, and the UK with the increasing spread of popularity of 'sushi'. There are two species causing human anisakiasis: *Anisakis simplex* and *Pseudoterranova osculatum*. The mode of infection is eating raw fish. It affects the intestine [1].

## **4.8. Gnathostoma spinigerum**

It is found in Japan, China, the Far East and the Philippines; the mode of infection is eating raw freshwater fish. It affects the intestine and the cutaneous tissues [1].

## **4.9. Trichinella spiralis**

It has a cosmopolitan distribution, and it is most commonly found in China and Europe. The mode of infection is eating raw pork. It affects the small intestine, the blood and the central nervous system.

According to the zones, temperature and infectivity, there are four types of it: *Trichinella spiralis* spiralis, *Trichinella spiralis* nelson, *Trichinella spiralis* nativa and *Trichinella spiralis* pseu‐ dospiralis [1].

## **4.10. Wuchereria bancrofti**

It causes lymphatic filariasis. It is found in tropics and subtropics zones.

The mode of transmission is puncturing the skin by the vector, a mosquito.

Mosquitoes responsible for its transmission are *Culex quinquefasciatus*, *Anopheles gambiae* and *Anopheles funestus*.

It causes Lymphangitis commonly in the lower extremities and obstruction of the genital organs.

There are two strains of *Wuchereria bancrofti*:

## **4.11. Brugia malayi**

It is found in South East Asia. There are two strains of *Brugia malayi*:

The mode of transmission is puncturing the skin by the vector, Mansonia.

It affects the lymph vessels causing the same clinical picture of *Wuchereria bancrofti* [1].

## **4.12. Brugia timori**

It is found in Indonesia. The mode of transmission is puncturing the skin by the vector, Mansonia. It affects the lymph vessels [1].

## **4.13. Loa loa**

It is known as the African eye worm as it is found in West and Central Africa. The mode of transmission is puncturing the skin by the vector, Chrysops species.

It affects the skin causing Calabar swellings, which are inflammatory swellings resulting in a localized subcutaneous oedema [1].

## **4.14. Mansonella species**

It is found in tropics and subtropics zones. The mode of transmission is puncturing the skin by the vector, Culicoides species. It affects the skin.

There are three species of Mansonella: *Mansonella perstans*, *Mansonella ozzardi* and *Mansonella streptocerca* [1, 8].

## **4.15. Onchocerca volvulus**

It is found in central and West Africa. It is found also in South America.

The mode of transmission is puncturing the skin by the vector, Simulium (black flies), espe‐ cially *Simulium damnosum*. It affects the skin and the eye [1, 8].

## **4.16. Dracunculus medinensis**

The mode of transmission is puncturing the skin by the vector, a mosquito.

• The nocturnal periodic strain which is found in Africa, India and the Far East.

• The sub‐periodic strain which is found in Malaysia, Indonesia and the Philippines.

It affects the lymph vessels causing the same clinical picture of *Wuchereria bancrofti* [1].

It is found in Indonesia. The mode of transmission is puncturing the skin by the vector,

It is known as the African eye worm as it is found in West and Central Africa. The mode of

It affects the skin causing Calabar swellings, which are inflammatory swellings resulting in a

It is found in tropics and subtropics zones. The mode of transmission is puncturing the skin

There are three species of Mansonella: *Mansonella perstans*, *Mansonella ozzardi* and *Mansonella*

The mode of transmission is puncturing the skin by the vector, Simulium (black flies), espe‐

• The sub‐periodic strain which is found in the Pacific region [1].

It is found in South East Asia. There are two strains of *Brugia malayi*:

transmission is puncturing the skin by the vector, Chrysops species.

It is found in central and West Africa. It is found also in South America.

cially *Simulium damnosum*. It affects the skin and the eye [1, 8].

The mode of transmission is puncturing the skin by the vector, Mansonia.

• The nocturnal periodic strain which is spread widely in Asia.

*Anopheles funestus*.

12 Human Helminthiasis

**4.11. Brugia malayi**

**4.12. Brugia timori**

**4.13. Loa loa**

There are two strains of *Wuchereria bancrofti*:

Mansonia. It affects the lymph vessels [1].

localized subcutaneous oedema [1].

by the vector, Culicoides species. It affects the skin.

**4.14. Mansonella species**

**4.15. Onchocerca volvulus**

*streptocerca* [1, 8].

organs.

Mosquitoes responsible for its transmission are *Culex quinquefasciatus*, *Anopheles gambiae* and

It causes Lymphangitis commonly in the lower extremities and obstruction of the genital

It is found in Iran, India and Ghana in zones of little clean drinking water. The mode of trans‐ mission is ingestion of unclean water containing Cyclops.

It affects intestine and the lung [8].

## **4.17. Capillaria philippinensis**

It is found in mainly Philippines and Thailand, but there were also some few unusual reports from Japan, Taiwan, Egypt and Iran. The source of infection is eating raw or improperly cooked fish. It affects the intestine [9–11].

## **4.18. Enterobius vermicularis**

It has a cosmopolitan distribution. The modes of transmission are finger suckling, nail biting and eat raw vegetables.

It affects the small intestine [9–11].

According to a study performed in April 2008, the epidemiology of nematodes in develop‐ ing regions of Asia, Africa, Latin and America as follows: *Ascaris lumbricoides* (807 millions), *Trichuris trichiura* (604 millions), hookworm (576 millions) and *Strongyloides stercoralis* (30–100 millions) [11].

Here are some examples of Nematodes' prevalence in different parts of the world:

## **a. Mazandaran province, northern Iran**

Data analysis performed after examining unwashed raw vegetables for soil transmitted hel‐ minths showed below:

## **b. Kampala, Uganda**

Cross sectional survey in September and October 2013 applied on five groups of people:

## **c. Bungoma county, Western Kenya**

Cross sectional study held in 2016 by examining cryopreserved stool samples of 796 persons with age ranges from 2 to 81 years revealed; prevalence of Ascaris lumbricoides (17%) and *Trichuris trichiura*, Ancylostoma and Strongyloides (less than 1.0%) [14].

## **d. Tezi Town, Puge county of Liangshan prefecture, Southwestern China**

Cross sectional study from October 23 to November 3, 2014 showed prevalence of *Ascaris lumbricoides* (13.5%), *Trichuris trichiura* (30.6%) and both(7.1%) [15].

*Mansonella ozzardi* is a rare nematode but mainly present in The Americas from southern Mexico to northwestern Argentina with prevalence rate (0–46%) [16].

## **5. Future perspectives**

There is being some changes in several countries due to the efforts to treat actively these para‐ sites. Nevertheless, some negative circumstances such as wars, climate changes and poverty may influence in this growing problem.

The neglected status of the helminthiasis should be addressed on community levels and glob‐ ally all over the world.

## **Author details**

Mohamed S. Abdeltawabi, Nahla El Seddik and Hosni K. Salem\*

\*Address all correspondence to<EMAIL_ADDRESS>
Faculty of Medicine, Cairo University, Cairo, Egypt

## **References**

• Slum dwellers at risk of flooding.

• Slum dwellers without work of flooding.

• Highest prevalence in urban farmers = 75.9%

• Hookworm is the predominant by 27.8%

**c. Bungoma county, Western Kenya**

and above [13].

14 Human Helminthiasis

**5. Future perspectives**

ally all over the world.

**Author details**

may influence in this growing problem.

• They were tested using Kato‐Katz stool sampling which showed:

• Lowest prevalence in workers managing faecal sludge = 35.8%

*Trichuris trichiura*, Ancylostoma and Strongyloides (less than 1.0%) [14].

*lumbricoides* (13.5%), *Trichuris trichiura* (30.6%) and both(7.1%) [15].

Mexico to northwestern Argentina with prevalence rate (0–46%) [16].

Mohamed S. Abdeltawabi, Nahla El Seddik and Hosni K. Salem\*

\*Address all correspondence to<EMAIL_ADDRESS>
Faculty of Medicine, Cairo University, Cairo, Egypt

**d. Tezi Town, Puge county of Liangshan prefecture, Southwestern China**

• In urban farmers, the prevalence of Trichuris, Schistosoma mansoni and Ascaris was 15%

Cross sectional study held in 2016 by examining cryopreserved stool samples of 796 persons with age ranges from 2 to 81 years revealed; prevalence of Ascaris lumbricoides (17%) and

Cross sectional study from October 23 to November 3, 2014 showed prevalence of *Ascaris*

*Mansonella ozzardi* is a rare nematode but mainly present in The Americas from southern

There is being some changes in several countries due to the efforts to treat actively these para‐ sites. Nevertheless, some negative circumstances such as wars, climate changes and poverty

The neglected status of the helminthiasis should be addressed on community levels and glob‐

parasites of humans: field‐based inferences on the impact of mass deworming. Parasites & Vectors [Internet]. 2016; 9(1): 1–12. doi:10.1186/s13071‐016‐1314‐y

**Section 2**

## **Diagnosis**

parasites of humans: field‐based inferences on the impact of mass deworming. Parasites

[15] Xiao P‐L, Zhou Y‐B, Chen Y, Yang Y, Shi Y, Gao J‐C, et al. Prevalence and risk factors of *Ascaris lumbricoides* (Linnaeus, 1758), *Trichuris trichiura* (Linnaeus, 1771) and HBV infections in Southwestern China: a community‐based cross sectional study. Parasites &

[16] Lima NF, Veggiani Aybar CA, Dantur Juri MJ, Ferreira MU. *Mansonella ozzardi* : a neglected New World filarial nematode. Pathog Glob Health [Internet]. 2016; 110(3): 97–107. Available from: http://www.tandfonline.com/doi/full/10.1080/20477724.2016.1190544

& Vectors [Internet]. 2016; 9(1): 1–12. doi:10.1186/s13071‐016‐1314‐y

16 Human Helminthiasis

Vectors [Internet]. 2015; 8(1): 1–11. doi:10.1186/s13071‐015‐1279‐2

## **Mass Spectrometry and Metabolomics—New Approaches for Helminth Biochemical Studies Mass Spectrometry and Metabolomics—New Approaches for Helminth Biochemical Studies**

Estela O. Lima, Cibele Z. Esteves, Diogo N. Oliveira, Tatiane M. Guerreiro, Carlos F. O. R. Melo and Rodrigo R. Catharino Estela O. Lima, Cibele Z. Esteves, Diogo N. Oliveira, Tatiane M. Guerreiro, Carlos F. O. R. Melo and Rodrigo R. Catharino

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65040

#### **Abstract**

Metabolomics, the study of the endogenously synthesized small molecules repertoire (nonproteinaceous), is of great relevance for establishing a wide view of cell physiology at specific moments, linking metabolic profiles to phenotypes and genotypes. To better understand biological systems, such as helminths life cycle, helminthic infection, and host-parasite interaction, metabolomics studies are crucial. For that, mass spectrometrybased metabolomics is the most popular strategy. Nontargeted metabolomics allows researchers to profile entire metabolomes present in cells, tissues, biofluids, or even samples as complex as stools. Through different mass spectrometric techniques, it is possible to unveil chemical markers for helminths, such as *Schistosoma mansoni* (a trematode) and *Ascaris lumbricoides* (a nematode), in addition to study mechanisms of action for different drugs, which targets parasites. Therefore, mass spectrometry allows designing biochemical pathways that may clarify the processes of parasite life cycle, helminthic infection, and host-parasite interaction, providing targets to further interference for parasite control or even infection treatment.

**Keywords:** metabolomics, mass spectrometry, helminths, *Ascaris lumbricoides* , *Schisto‐ soma mansoni*

© 2017 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. © 2017 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**

## **1.1. Metabolomics and mass spectrometry in biomedical research**

The composition of the intracellular environment depends on harmonic and orchestrated synthesis and interaction of molecules that are essential for cellular survival and healthy life maintenance [1], and it is true for both eukaryotic and prokaryotic organisms. Eukaryotic cells present a higher degree of intricacy compared with prokaryotic cells because of the huge biochemical complexity they present [2]. Helminths represent a pluricellular eukaryotic group of worms that establish parasitic relationships with humans, which has been registered since ancient Greek writings [3]. Structurally, helminths are completely different from *Homo sapiens*, and it is assured that peculiar metabolic differences exist between these organisms. These differences enable understanding and studying helminth-human interaction, transmission routes, and life cycles through molecular studies, the cornerstone in all fields of biomedical research.

The search for faster, more accurate methodologies that describe the biochemical/pathophysiological status of an organism of interest has driven major efforts in biomedical research in the late years in order to meet the clinical needs for a health community [4]. Bioanalytical methodologies have then emerged as suitable choices for expeditious approaches that exhibit great resolution and sensitivity, thus generating the development of more accurate and precise methodologies. In instrumental bioanalysis, several compounds of interest can be measured and/or monitored depending on their molecular nature. As there is no "one-size-fits-all" approach, different molecular classes have demanded the creation of different analytical strategies in which both comprehensive and specific analyses can be performed. This, therefore, has elicited the development of the "omics" strategies, which were subdivided according to the needs of the researchers. For example, for large and bulky molecules such as proteins, a wide array of analytical methods compose what is called the proteomics approach, which is dedicated to study each and every aspect of proteins and their levels, interactions, and structure in biological systems [5]. The same rationale was then applied for other biological components, such as genes (genomics) [6], lipids (lipidomics) [7], and small molecules/ metabolites (metabolomics) [8].

Metabolomics can be understood in more detail as the systematic study of small, nonprotein, both endogenous and exogenous molecules, which represent the end of the entire biological chain started by gene expression (i.e., phenotype) [9]. Metabolomics studies usually measure compound classes such as lipids, nucleic acids, amino acids, and glycans, composing a useful biochemical platform for studying mechanisms of interactions, signalling synthetic pathways of systems and molecules under different metabolic conditions (e.g., diseases), providing specific molecules or molecular groups that characterize such conditions: the biomarkers. This assists in understanding, diagnosing, preventing, and predicting conditions, as well as helps assessing risks and benefits of pharmacological interventions [10].

Cell metabolism is one of the challenges that have been the driving force of metabolomics over the years [11]. The efforts on development were to obtain increasingly accurate and wellresolved methods, introducing more sophisticated and specific methods such as nuclear magnetic resonance (NMR) and mass spectrometry (MS) [12]. This made room for a number of improvements also in data processing and bioinformatics, where multivariate analyses and big data analytics found a whole new application in resolving and integrating complex biological matrices and systems [13], giving rise to the four main metabolomics strategies: metabolic profiling, the identification and quantification of known metabolites that are related to a specific number of metabolic pathways [14]; metabolic fingerprinting, which comprises fast-paced analysis to classify a determined sample during high-throughput screenings (positive/negative or disease/health) [15]; metabolic footprint, which analyses the extracellular metabolites (i.e., excretion/secretion products) of a given organism [16]; and targeted analysis, a series of quali-quantitative analyses of any given known metabolite in an orga-nism or a metabolic pathway [17].

**1. Introduction**

20 Human Helminthiasis

research.

metabolites (metabolomics) [8].

**1.1. Metabolomics and mass spectrometry in biomedical research**

The composition of the intracellular environment depends on harmonic and orchestrated synthesis and interaction of molecules that are essential for cellular survival and healthy life maintenance [1], and it is true for both eukaryotic and prokaryotic organisms. Eukaryotic cells present a higher degree of intricacy compared with prokaryotic cells because of the huge biochemical complexity they present [2]. Helminths represent a pluricellular eukaryotic group of worms that establish parasitic relationships with humans, which has been registered since ancient Greek writings [3]. Structurally, helminths are completely different from *Homo sapiens*, and it is assured that peculiar metabolic differences exist between these organisms. These differences enable understanding and studying helminth-human interaction, transmission routes, and life cycles through molecular studies, the cornerstone in all fields of biomedical

The search for faster, more accurate methodologies that describe the biochemical/pathophysiological status of an organism of interest has driven major efforts in biomedical research in the late years in order to meet the clinical needs for a health community [4]. Bioanalytical methodologies have then emerged as suitable choices for expeditious approaches that exhibit great resolution and sensitivity, thus generating the development of more accurate and precise methodologies. In instrumental bioanalysis, several compounds of interest can be measured and/or monitored depending on their molecular nature. As there is no "one-size-fits-all" approach, different molecular classes have demanded the creation of different analytical strategies in which both comprehensive and specific analyses can be performed. This, therefore, has elicited the development of the "omics" strategies, which were subdivided according to the needs of the researchers. For example, for large and bulky molecules such as proteins, a wide array of analytical methods compose what is called the proteomics approach, which is dedicated to study each and every aspect of proteins and their levels, interactions, and structure in biological systems [5]. The same rationale was then applied for other biological components, such as genes (genomics) [6], lipids (lipidomics) [7], and small molecules/

Metabolomics can be understood in more detail as the systematic study of small, nonprotein, both endogenous and exogenous molecules, which represent the end of the entire biological chain started by gene expression (i.e., phenotype) [9]. Metabolomics studies usually measure compound classes such as lipids, nucleic acids, amino acids, and glycans, composing a useful biochemical platform for studying mechanisms of interactions, signalling synthetic pathways of systems and molecules under different metabolic conditions (e.g., diseases), providing specific molecules or molecular groups that characterize such conditions: the biomarkers. This assists in understanding, diagnosing, preventing, and predicting conditions, as well as helps

Cell metabolism is one of the challenges that have been the driving force of metabolomics over the years [11]. The efforts on development were to obtain increasingly accurate and well-

assessing risks and benefits of pharmacological interventions [10].

The technical aspects of both NMR and MS place them both as the main tools for metabolomics studies, especially for their complementary nature. NMR is a spectroscopy-based technique that relies on the incidence of a magnetic field over the nuclei of atoms with nonzero spin counts, such as 1 H, 13C, and 15N, thus providing accurate structural and quantitative information of the metabolic profile under study [18]. There are several advantages associated with the use of NMR as an analytical tool for metabolomics; as it is not a destructive analysis, i.e., it is possible to recover the sample after the analysis, which is useful especially for target analyses. Moreover, NMR requires little sample preparation and shows great reproducibility, two desirable features for bioanalyses [19]. In contrast, some disadvantages hamper the wide distribution of NMR as a routine approach; the high costs associated with instrument maintenance and the need for relatively high amounts of the analyte usually turn down NMR as a high-throughput choice, which finds its best use as a research tool.

Mass spectrometry, on the other hand, despite being sample-destructive, shows great potential for routine analyses. MS-based bioanalytical systems show superior performance in detection and quantification limits, easily reaching a working range of nano to picomoles [20]. Relying on a basic structure of ionization source-mass analyser, MS brings a wide array of combinations of both, which comprehends virtually any type of sample for metabolomics studies [21], in addition to the possibility of coupling with chromatographic approaches such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) [22]. Moreover, structural elucidation strategies with MS are diverse and can be suited according to the needs and to the instrument available, either with mass accuracy on high-resolution instruments or with fragmentation profile (MS/MS) in standard devices—or even a combination of the two [23]. These factors contributed to the popularization of MS among researchers and clinical experts and have, therefore, provided the basis for great development in this area.

Hence, this chapter will provide a comprehensive overview on the application of metabolomics analysis and strategies developed for the analyses of helminths, with a particular focus on MS, reinforcing the great importance of these organisms for general public health. The main aim is to address recent analytical chemistry methodologies, challenges to overcome and promising results, expanding possibilities at helminthiasis research, and thus contributing with advances in the area.

## **2. Biochemical characterization of helminthic parasites**

## **2.1.** *Schistosoma mansoni* **biochemical characterization**

*S. mansoni* metabolomics studies are conducted to improve the knowledge of disease pathology, to evaluate the behaviour and infectivity of different strains, to elucidate mechanisms of action, and to evaluate the efficacy of available drugs in addiction to discover biomarkers that can be applied as diagnostic and drug targets [24].

The main analytical metabolomics approaches are MS and NMR [25], and both of them allow performing target or untargeted analysis, which are mostly focused on two classes of metabo-lites: lipids and oligosaccharides. In case of living organisms, such as parasites, lipids are necessary for cell-cell signalling, for protection from host immunity, for maintenance of integrity and parasite structure during the life cycle and are also involved in the mechanisms of sexual development, egg production, and cercaria penetration [26–28]. Concerning oligosaccharides, these molecules are precursors of glycoconjugates such as glycoproteins and glycolipids that are mainly involved in development of innate and adaptive immune response in the host, parasite survival, and the chronic infection establishment [29, 30]. Lipids as well glycans may change as long as the parasite develops, thus it is crucial to understand metabolomics patterns according to the different lifecycle stages for each parasite studied.

## *2.1.1. Metabolomics approach for S. mansoni lifecycle stages*

## *<IP_ADDRESS>. Mature and immature eggs*

The main class of lipids present at *S. mansoni* eggs is glycosphingolipids [31], which are involved in interactions with the different affected organs, such as gut, lungs, or liver, facilitating its passage through the tissues [32]. Glycosphingolipids are also reported to be associated with egg's high immunogenicity and pathogenicity in different strains [33]. For example, the granuloma formation occurs by the reaction of host immune response mediated by parasite glycoconjugates; therefore, the greater abundance of glucosylceramides (GlcCer) in one strain compared to another, the higher pathogenicity [32, 34]. The same study using high-resolution mass spectrometry (HRMS) showed that phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), triacylglycerol (TG), and diacylglycerol (DG) are involved in the differentiation of *S. mansoni* strains [34]. In respect to maturation, immature and mature eggs present also been differentiated using Matrix-Assisted Laser Desorption/ Ionization—Time of Flight Mass Spectrometry (MALDI-TOF-MS). This research showed that immature eggs have a common N-glycan profile and are formed predominantly of nonspecific oligomanoses, while mature eggs have highly complex and fucosylated N-glycans [30]. Therefore, lipid molecules in eggs show differences according to maturation phases and diverge between distinct strains.

## *<IP_ADDRESS>. Miracidium*

**2. Biochemical characterization of helminthic parasites**

*S. mansoni* metabolomics studies are conducted to improve the knowledge of disease pathology, to evaluate the behaviour and infectivity of different strains, to elucidate mechanisms of action, and to evaluate the efficacy of available drugs in addiction to discover biomarkers that

The main analytical metabolomics approaches are MS and NMR [25], and both of them allow performing target or untargeted analysis, which are mostly focused on two classes of metabo-lites: lipids and oligosaccharides. In case of living organisms, such as parasites, lipids are necessary for cell-cell signalling, for protection from host immunity, for maintenance of integrity and parasite structure during the life cycle and are also involved in the mechanisms of sexual development, egg production, and cercaria penetration [26–28]. Concerning oligosaccharides, these molecules are precursors of glycoconjugates such as glycoproteins and glycolipids that are mainly involved in development of innate and adaptive immune response in the host, parasite survival, and the chronic infection establishment [29, 30]. Lipids as well glycans may change as long as the parasite develops, thus it is crucial to understand metabolomics patterns according to the different lifecycle stages for each parasite

The main class of lipids present at *S. mansoni* eggs is glycosphingolipids [31], which are involved in interactions with the different affected organs, such as gut, lungs, or liver, facilitating its passage through the tissues [32]. Glycosphingolipids are also reported to be associated with egg's high immunogenicity and pathogenicity in different strains [33]. For example, the granuloma formation occurs by the reaction of host immune response mediated by parasite glycoconjugates; therefore, the greater abundance of glucosylceramides (GlcCer) in one strain compared to another, the higher pathogenicity [32, 34]. The same study using high-resolution mass spectrometry (HRMS) showed that phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), triacylglycerol (TG), and diacylglycerol (DG) are involved in the differentiation of *S. mansoni* strains [34]. In respect to maturation, immature and mature eggs present also been differentiated using Matrix-Assisted Laser Desorption/ Ionization—Time of Flight Mass Spectrometry (MALDI-TOF-MS). This research showed that immature eggs have a common N-glycan profile and are formed predominantly of nonspecific oligomanoses, while mature eggs have highly complex and fucosylated N-glycans [30]. Therefore, lipid molecules in eggs show differences according to maturation phases and

**2.1.** *Schistosoma mansoni* **biochemical characterization**

can be applied as diagnostic and drug targets [24].

*2.1.1. Metabolomics approach for S. mansoni lifecycle stages*

*<IP_ADDRESS>. Mature and immature eggs*

diverge between distinct strains.

studied.

22 Human Helminthiasis

This is about a free-living and motile form that penetrates in *Biomphalaria sp.* snails, the intermediate host of the *S. masoni* lifecycle. Different strains were analyzed by HRMS, and the results showed the possibility to differentiate two strains of miracidium by identifying different lipids: triacylglycerol (TG), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol-ceramide (PI-Cer), and dodecanoid acid (DA). The most interesting lipid among them is the last compound, reported to be responsible for facilitating the penetration of miracidium in snails [27]. Considering glycolipid studies through MALDI-TOF-MS analysis, miracidium glycolipids have been almost devoid of LeX- or LN-motifs, what is coherent with previous data that showed snail infection is apparently independent of LeX-motifs, but LDNmotifs are fairly present in miracidium and snail. Observing that the parasite presented the same spectra compared to the intermediate host, miracidium appear to develop a snailcompatible glycosylation pattern [30]. These molecular studies show lipid variability and/or similarity among different lifecycle stages, opening opportunities to better understand interactions between parasite and host/intermediate host or to interfere at metabolic pathways, as long as we know where and how [30, 35].

## *<IP_ADDRESS>. Cercaria*

Cercaria, the final larval stage of *S. mansoni* lifecycle, produces secretions that facilitate skin penetration. These fluids are reported to be weak immunogenic and composed of glucoproteins and glycolipids. Mass spectrometry approaches demonstrate the predominance of ceramides-linked glucose and/or galactose with fucose and xylose cores at cercaria secretions [30, 33, 36]. Another study also determined the cercaria differentiation between two *S. mansoni* strains by HRMS. The lipids TG, DG, PE, PA, and phosphatidylglycerol (PG) were reported to be responsible for this discrimination [27].

## *<IP_ADDRESS>. Adults*

The tegument surface structure of *S. mansoni* adult worms is crucial for parasite survival and modulation of host immune response. This structure is syncytial and is covered by two closely apposed lipid bilayers, one of them is an inner plasma membrane and the other an outer membranocalyx complex [28, 37]. Mass spectrometry studies conducted with tegument and whole worm demonstrate an enrichment of the phosphatidylcholine (PC) and PS of tegumental compositions, but this phenomena does not occur with PE and PI, which presents the same composition for the two worm fractions. The tegumental membranes are also enriched with lysophospholipids, mainly lyso-PS and lyso-PE containing eicosanoic acid (20:1) [28].

As regards glycans, their composition dramatically changes during the development stages. Comparing with early life-stages, adult worms present smaller ratio between complex typeglycans and oligomannosidic glycans and the antigenic core composed by xylose is undetectable [30].

Mass spectrometry imaging (MSI) enables the study of spatial distribution of biomarkers. Ferreira and co-workers (2014) used MALDI-MSI to image two different *S. mansoni* strains. **Figure 1** shows the sum of all ions obtained by the imaging analysis where it is possible to detect both male and female worms' bodies. The spectra obtained from the single worms were compared with the pair and enabled the election of specific ions for male and female worms [34].

**Figure 1.** Representative fingerprinting of *Schistosoma mansoni* adult couple worms. The imaging represents a sum of all ions within mass range 600–2000 m/z. (A) Picture of worms analyzed. Gynaecophoric channel in male's body (clear) holding female's body (dark). (B) Worms' image generated by MALDI-MSI instrument. Illustrative picture of fingerprinting analysis. (C) Representative fingerprinting (total ion current) spectra. Positive ion mode.

The same study determined the ions responsible for the differentiation of male and female worms of two different strains and their localization in a worm body. The fingerprint of each worm was submitted to Principal Component Analysis (PCA), and the groups of strains are discriminated, enabling the selection of chemical markers for each group. New MS/MS imaging was performed and the distribution of the parental ions selected by PCA can be seen in **Figures 2** and **3**.

**Figure 2** shows the male and female image of BH strain obtained by MALDI-MSI analysis. Female worm presents the predominance of PC (823, 665 and 761 m/z) and PG (764 m/z) distributed to reproductive and digestive systems. Male worm presents diacylcglycerol (DAG) (635 and 651 m/z) in ventral sucker and gynaecophoric channel, triacylglycerol (TAG) (846 and 886 m/z) in ventral sucker and reproductive system, phosphatidic acid (PA) (675 m/z) in ventral sucker and gynaecophoric channel, PI (862 m/z) in reproductive and digestive systems and ventral sucker, and finally, PC (887 m/z) in reproductive system and ventral sucker.

**Figure 1** shows the sum of all ions obtained by the imaging analysis where it is possible to detect both male and female worms' bodies. The spectra obtained from the single worms were compared with the pair and enabled the election of specific ions for male and female worms

**Figure 1.** Representative fingerprinting of *Schistosoma mansoni* adult couple worms. The imaging represents a sum of all ions within mass range 600–2000 m/z. (A) Picture of worms analyzed. Gynaecophoric channel in male's body (clear) holding female's body (dark). (B) Worms' image generated by MALDI-MSI instrument. Illustrative picture of finger-

The same study determined the ions responsible for the differentiation of male and female worms of two different strains and their localization in a worm body. The fingerprint of each worm was submitted to Principal Component Analysis (PCA), and the groups of strains are discriminated, enabling the selection of chemical markers for each group. New MS/MS imaging was performed and the distribution of the parental ions selected by PCA can be seen

printing analysis. (C) Representative fingerprinting (total ion current) spectra. Positive ion mode.

[34].

24 Human Helminthiasis

in **Figures 2** and **3**.

**Figure 2.** Adult worms generated by MS, selected using each of the parent ions in MS/MS mode. Images were acquired as one run for each m/z. Female and male BH strain (FBH and MBH, respectively). Pos, posterior portion; Ant, anterior portion; R, reproductive system; D, digestive system; VS, ventral sucker; OS, oral sucker; and GC, gynaecophoric channel.

**Figure 3.** Adult worm images generated by MS, selected using each of the parent ions in MS/MS mode. Images were acquired as one run for each m/z. Female and male SE strain (FSE and MSE, respectively). Pos, posterior portion; Ant, anterior portion; R, reproductive system; VS, ventral sucker; and OS, oral sucker.

**Figure 3** shows male and female image of SE strain obtained by MALDI-MSI analysis. Female worm presents the predominance of PI (623 m/z) and DAG (606 m/z) distributed mainly in tegument while male worm presents TAG (786 and 850 m/z) and PC (825 m/z) in ventral and oral suckers and reproductive system.

Ferreira and coworkers (2014) demonstrate that it is possible to determine spatial distribution of biomarkers for parasites and also show that more studies are necessary to understand the function of each chemical markers according to the organs they are located, searching for its influence through pathogenicity of different strains. Furthermore, a new platform of study putting together metabolomic and mass spectrometry techniques was proposed and was named as Parasitomics.

## **2.2.** *Ascaris lumbricoides* **and chemical markers at** *moulting* **process**

According to Pullan and coworkers [38] and CDC [39], it is estimated that 800–1200 million people are infected with *Ascaris lumbricoides* worldwide and more than 60,000 deaths occur annually because of *Ascariasis* (spectrum of disease caused by *Ascaris lumbricoides* infection). World Health Organization has applied some strategies for disease burden control, such as deworming programmes [40]. In case of soil-transmitted helminthiasis, the main public health strategy is the mass drug administration (MDA), which consists of anthelmintic drug treatment regularly provided to risk population, mainly school-aged children [40, 41]. Despite the great efforts, approximately 30% of children are really receiving the anthelmintic treatment in the whole world [42]. Besides the absence of total coverage of MDA worldwide, there is still another issue to pay attention, the development of anthelmintic drug resistance [43–45].

Taking into account all of these issues cited above, beyond the disability-adjusted life years (DALYs) in consequence of morbidity and mortality indexes, it is necessary to better comprehend and to deal with helminthic parasites, such as *Ascaris lumbricoides*, in a different point of view. It is urgent to evaluate and discover different and better ways to control and eliminate it. Therefore, to outline strategies that are suitable for its life cycle could bring light to management of helminthiasis. Since part of the development of *A. lumbricoides* happens in the soil [46, 47], it is therefore reasonable to consider that breaking the life cycle during the phases in the environment would be a valuable attempt in containing the infection in endemic areas [48].

Molecular detailed analysis of the *diverse A. lumbricoides* forms may elucidate new targets for parasite control. Metabolic fingerprinting using mass spectrometry has been widely employed, targeting parasite-related metabolites [34, 49, 50]. The trend of establishing target molecules and linking them with important metabolic pathways in the parasite life cycle broadens the knowledge in the field, generating a platform that will assist in better knowledge on their biology, elucidation of survival mechanisms, pharmacodynamics, diagnostic methodologies, development of pharmaceutical products to eradicate these diseases, whether in soil or living hosts, etc. [50, 51].

Melo and coworkers aimed to characterize, in a simple way, the larval development stages of *A. lumbricoides* (the traditional human ascariasis agent) by metabolic fingerprinting using HRMS from stool samples [52]. The molecular elucidation in different life stages presents a huge potential for application in parasite infection control, breaking the life cycle and reducing infection and re-infection of exposed human beings, mainly at endemic zones.

From *Ascaris lumbricoides* specimens, it is possible to separate different development larval stages using optical microscopy. Melo's researchers group analyzed stool samples collected from school-aged children at Brazil. Nonembryonated and embryonated eggs, containing L1 and L3 larvae, could be separated from stool pool. L2 stage larvae were excluded from samples because it is an overlapping period between the end of L1 and the beginning of L3, what makes it difficult to identify and isolate. Considering the high sensitivity of mass spectrometer analyser, it was necessary to clean as much as possible this complex sample, what favors the real identification of significant molecules [52].

For molecular screening like this, starting from a complex sample, it is necessary to do a multivariate statistical analysis for correct identification of targets. For the present study, Principal Component Analysis (PCA) was chosen to statistically determine relevant groups of ions, what led to identification of the correspondent molecular biomarkers for each development stage under analysis. From the elected ions by PCA, it was possible to achieve the most suitable chemical markers through metabolome databases analysis, along with a good literature review. ESI-HRMS analysis of *Ascaris lumbricoides* specimens showed different spectral signals among the egg, L1 and L3, and PCA statistical analysis elected distinct chemical markers for each structure/larval stage.

## *2.2.1. Metabolomics approach for A. lumbricoides lifecycle stages*

## *<IP_ADDRESS>. Eggs*

putting together metabolomic and mass spectrometry techniques was proposed and was

According to Pullan and coworkers [38] and CDC [39], it is estimated that 800–1200 million people are infected with *Ascaris lumbricoides* worldwide and more than 60,000 deaths occur annually because of *Ascariasis* (spectrum of disease caused by *Ascaris lumbricoides* infection). World Health Organization has applied some strategies for disease burden control, such as deworming programmes [40]. In case of soil-transmitted helminthiasis, the main public health strategy is the mass drug administration (MDA), which consists of anthelmintic drug treatment regularly provided to risk population, mainly school-aged children [40, 41]. Despite the great efforts, approximately 30% of children are really receiving the anthelmintic treatment in the whole world [42]. Besides the absence of total coverage of MDA worldwide, there is still another issue to pay attention, the development of anthelmintic drug resistance [43–45].

Taking into account all of these issues cited above, beyond the disability-adjusted life years (DALYs) in consequence of morbidity and mortality indexes, it is necessary to better comprehend and to deal with helminthic parasites, such as *Ascaris lumbricoides*, in a different point of view. It is urgent to evaluate and discover different and better ways to control and eliminate it. Therefore, to outline strategies that are suitable for its life cycle could bring light to management of helminthiasis. Since part of the development of *A. lumbricoides* happens in the soil [46, 47], it is therefore reasonable to consider that breaking the life cycle during the phases in the environment would be a valuable attempt in containing the infection in endemic areas [48].

Molecular detailed analysis of the *diverse A. lumbricoides* forms may elucidate new targets for parasite control. Metabolic fingerprinting using mass spectrometry has been widely employed, targeting parasite-related metabolites [34, 49, 50]. The trend of establishing target molecules and linking them with important metabolic pathways in the parasite life cycle broadens the knowledge in the field, generating a platform that will assist in better knowledge on their biology, elucidation of survival mechanisms, pharmacodynamics, diagnostic methodologies, development of pharmaceutical products to eradicate these diseases, whether in soil or living

Melo and coworkers aimed to characterize, in a simple way, the larval development stages of *A. lumbricoides* (the traditional human ascariasis agent) by metabolic fingerprinting using HRMS from stool samples [52]. The molecular elucidation in different life stages presents a huge potential for application in parasite infection control, breaking the life cycle and reducing

From *Ascaris lumbricoides* specimens, it is possible to separate different development larval stages using optical microscopy. Melo's researchers group analyzed stool samples collected from school-aged children at Brazil. Nonembryonated and embryonated eggs, containing L1 and L3 larvae, could be separated from stool pool. L2 stage larvae were excluded from samples because it is an overlapping period between the end of L1 and the beginning of L3, what makes it difficult to identify and isolate. Considering the high sensitivity of mass spectrometer

infection and re-infection of exposed human beings, mainly at endemic zones.

**2.2.** *Ascaris lumbricoides* **and chemical markers at** *moulting* **process**

named as Parasitomics.

26 Human Helminthiasis

hosts, etc. [50, 51].

Considering eggs analysis, it was possible to identify five biomarkers: hexadecenal (515 m/z), 21-Methyl-8Z-pentatriacontene (522 m/z), 3,7,11,15-Tetramethyl tetratriacontane (565 m/z), and two unsaturated fatty acids, corresponding to 537 m/z. The phospholipid hexadecenal may probably be a by-product of sphingosine-1-phosphate (S1P) degradation. Hexadecenal is associated with cytoskeleton reorganization, JNK-dependent apoptosis, and proliferation signalling pathways. Since the analyzed eggs were under embryogenesis, JNK pathway may favor cell proliferation and embryo development [53–55]. Taking into account that hexadecenal appears only in the mass spectra of eggs, it may be acting in signalling during embryonic development process according to weather conditions, food availability and population, among others [56].

Two other signals elected by PCA, 21-Methyl-8Z-pentatriacontene (522 m/z) and 3,7,11,15- Tetramethyl Tetratriacontane (565 m/z), correspond to signalling molecules in insects, although their function is not clear yet. There were still two unsaturated fatty acids elected for eggs (537 m/z) that, based at their carbon chain, it is supposed they have a role at preventing water loss, considering that fertilized eggs are constantly at high risk of dehydration in the soil [57, 58].

## *<IP_ADDRESS>. L1 moulting stage*

Considering biomarkers statistically elected for L1, a total of four molecules were selected. One of them, 2-deoxyecdysone-22-phosphate (563 m/z), is a steroid hormone source found during insects embryogenesis. These steroid hormones, commonly called ecdysones, are associated with control of development in insects and nematodes, processes called moulting [59]. Taking into account that ecdysone synthesis depends on cholesterol [60], it is necessary for the nematode to absorb it. Therefore, it could explain the election of cholesteryl ester (CE) as a second biomarker at L1 stage [61, 62].

The lipid composition may vary according to environmental conditions and parasite life stage. Elected as L1 biomarker, cardiolipins are examples of molecules present at the inner mitochondrial membranes [63], whose constitution may vary to adapt to different aeration conditions. Considering that the development of *A. lumbricoides* occurs through different life stages with distinct air availability [64], it is coherent that oxidative stress conditions interfere with mitochondrial lipid composition. In addition to cardiolipins, sphingolipids (SPL) were one of the chemical markers elected at L1 stage. SPL is an important class of lipids and may be modified according to the cycle life stage, just similarly as it happens with insects' development [65].

## *<IP_ADDRESS>. L3 moulting stage*

The last larval stage studied at Melo's work was L3, and one of the possible markers was Dimethylheptatriacontane. It is a signalling molecule involved with sexual communication between arthropods, better known as pheromone [66]. Considering nematodes and arthropods are closely related, some hypothesis were raised about its function at *A. lumbricoides* L3: (i) it could play the same role as in arthropods; (ii) to stimulate sexual differentiation during parasite development [67]; (iii) to act as a "messenger" during development according to environmental conditions [56]. Depending on the life stage, this *signalling* molecule may present more than one function, but the exact role of dimethylheptatriacontane at nematodes is still to be unveiled.

Considering all biomarkers elected for different lifecycle stages for *Ascaris lumbricoides*, it is possible to observe that there is a whole metabolic parasite world to discover and establish connections. Therefore, it is too important to keep looking for new biochemical pathways that explain parasite similarities, survival, development, host invasiveness, and immunogenicity. Only the entire knowledge will answer the unsolved questions and allow human interference to improve health conditions and social development.

## **3. Searching for drug targets at helminthiasis—Praziquantel and** *Schistosoma mansoni* **chemical targets**

It is estimated that about 2 billion people in the world, especially the inhabitants of developing regions such as sub-Saharan Africa, East Africa, Asia, and the Americas are infected with one or more helminths, making parasitic diseases very expensive for public health [68–70]. The drugs used until now to treat these diseases are far from ideal, and many of them were introduced decades ago [71, 72]. Most of these drugs are related to serious adverse effects, which supports the low adherence to treatment by patients. Moreover, these drugs have limited efficacy and can have their activity paralyzed due to the phenomenon of drug resistance [72, 73]. The treatment of such parasitic diseases is mainly related to two factors: sanitation and health education, which when combined with the use of anthelmintic drugs promote the success of treatment and avoids occurrence of new cases [73, 74].

Ascariasis is a parasitic disease caused by infection with the nematode *Ascaris lumbricoides*, which is one of the soil-transmitted helminthiasis that head the list of the so-called "Neglected Diseases" [69, 75]. This parasitic disease has been associated with intestinal pathology, respiratory symptoms, and malnutrition in children from endemic areas [76, 77]. The indicated treatment for Ascariasis is based at anti-helminthic drugs against *A. lumbricoides*, which have been found to be safe and effective. Single oral doses of Albendazole, Mebendazole, and Pyrantel pamoate have demonstrated high cure rates against this parasite [69].

The lipid composition may vary according to environmental conditions and parasite life stage. Elected as L1 biomarker, cardiolipins are examples of molecules present at the inner mitochondrial membranes [63], whose constitution may vary to adapt to different aeration conditions. Considering that the development of *A. lumbricoides* occurs through different life stages with distinct air availability [64], it is coherent that oxidative stress conditions interfere with mitochondrial lipid composition. In addition to cardiolipins, sphingolipids (SPL) were one of the chemical markers elected at L1 stage. SPL is an important class of lipids and may be modified according to the cycle life stage, just similarly as it happens with insects' devel-

The last larval stage studied at Melo's work was L3, and one of the possible markers was Dimethylheptatriacontane. It is a signalling molecule involved with sexual communication between arthropods, better known as pheromone [66]. Considering nematodes and arthropods are closely related, some hypothesis were raised about its function at *A. lumbricoides* L3: (i) it could play the same role as in arthropods; (ii) to stimulate sexual differentiation during parasite development [67]; (iii) to act as a "messenger" during development according to environmental conditions [56]. Depending on the life stage, this *signalling* molecule may present more than one function, but the exact role of dimethylheptatriacontane at nematodes is still to be unveiled. Considering all biomarkers elected for different lifecycle stages for *Ascaris lumbricoides*, it is possible to observe that there is a whole metabolic parasite world to discover and establish connections. Therefore, it is too important to keep looking for new biochemical pathways that explain parasite similarities, survival, development, host invasiveness, and immunogenicity. Only the entire knowledge will answer the unsolved questions and allow human interference

opment [65].

28 Human Helminthiasis

*<IP_ADDRESS>. L3 moulting stage*

to improve health conditions and social development.

success of treatment and avoids occurrence of new cases [73, 74].

*Schistosoma mansoni* **chemical targets**

**3. Searching for drug targets at helminthiasis—Praziquantel and**

It is estimated that about 2 billion people in the world, especially the inhabitants of developing regions such as sub-Saharan Africa, East Africa, Asia, and the Americas are infected with one or more helminths, making parasitic diseases very expensive for public health [68–70]. The drugs used until now to treat these diseases are far from ideal, and many of them were introduced decades ago [71, 72]. Most of these drugs are related to serious adverse effects, which supports the low adherence to treatment by patients. Moreover, these drugs have limited efficacy and can have their activity paralyzed due to the phenomenon of drug resistance [72, 73]. The treatment of such parasitic diseases is mainly related to two factors: sanitation and health education, which when combined with the use of anthelmintic drugs promote the

Ascariasis is a parasitic disease caused by infection with the nematode *Ascaris lumbricoides*, which is one of the soil-transmitted helminthiasis that head the list of the so-called "Neglected

Albendazole and Mebendazole are Benzimidazole-based compounds, which are the most widely used anthelmintics until now [75]. These medicines are known as antimicrotubule agents due to their therapeutic effect by relating with the capacity of binding to free β-tubulin. This attachment inhibits tubulin polymerization and disrupts microtubules, which loses structure and function, leading the organism to death [78, 79]. The selective toxicity of these agents results because Benzimidazoles bind parasite β-tubulin with much higher affinity than the mammalian protein [80]. However, there are evidences of drug resistance due to existence of cases where loss of efficacy has been reported after administration of a single dose [44, 81].

Schistosomiasis is also part of the list of "Neglected Diseases" and is responsible for infecting more than 200 million people in tropical and subtropical regions of the world, especially in sub-Saharan Africa [82, 83]. Introduced in the early 1980, Praziquantel (PZQ) is the drug of choice in the treatment of schistosomiasis. The detailed molecular mechanism of action of PZQ has not yet been fully explained, but it is related with tegumental damage and paralytic muscular contraction of parasites via influx of Ca2+ across the tegument [74, 84].

PZQ is a safe anthelmintic, but cases of drug resistance are already known, such as reported for ascariasis. The first field report came from a new and epidemic focus in northern Senegal [85]. Many infections that persisted after treatment and were not detected by the usual diagnostic methods (with eggs per gram below the detection limit of the coprological techniques) required repeated or very sensitive examinations [86, 87]. Thus, the recommended doses of medicine for schistosomiasis should be considered subcurative [88], and it is safe to assume that in schistosome populations, some individual parasites are tolerant to the drug, at least at the usual dosages [73].

The phenomenon of anthelmintic resistance is a genetic modification that can be defined as a state of insensitivity or decreased sensitivity to the effect of a determined drug concentration, conferring a number of parasites of a given population the capacity to survive the pharmacological effect of recommended therapeutic doses of an anthelmintic [75]. In veterinary practice, frequent treatment of closed populations has led to a serious problem of anthelmintic drug resistance, which is now largely irreversible [73]. Thus, it is urgently needed to search and develop sensitive tools for early detection and monitoring of drug resistance, as well as new anthelmintic evaluation of drug combinations for parasite control.

Drug discovery is an iterative process where different strategies can be applied. Usually, it can be divided into several distinct stages, starting with target identification and validation, proceeding to assay development and screening (whole cell or target molecular basis) and ending with hits identification. Thenceforward, it is necessary to synthesize and evaluate analogues to verify the structure-activity relationships and identify the elected hit; to optimize its structure, interactive medicinal chemistry is used, and finally, it is done on all preclinical development before the clinical evaluation [71].

Target-based drug discovery starts with the identification and elucidation of the function of a potential therapeutic drug target and understanding its role in the disease process. Drug targets are, basically, molecular structures that will undergo a specific interaction with chemicals (drugs), which are administered to treat or diagnose a disease. Most of the times, drug targets are enzymes or proteins involved in biological activity, which are produced by expression of active genes in a cell [70]. Furthermore, it is interesting to develop drugs whose targets include biochemical pathways, such as cellular physiological process or cell *signalling* molecules. For example, it has been reported as an inhibitor of programmed cell death pathway (apoptosis) to be a potential small molecule acting on a caspase to control schistosomiasis caused by *S. japonicum* [89]. Therefore, metabolic pathways can also be explored as therapeutic targets, where there should be a clear understanding of anthelmintics mechanisms of action operating in helminth parasites [70].

For exploratory studies as cited above, metabolomics is a very powerful tool to connect parasite metabolism and its interaction with the drug of choice. To better understand some of these mechanisms, chromatographic techniques combined with MS have been employed for chemical characterization of adult schistosomes and PZQ metabolites in the host [90, 91]. In a study from 2015, researchers used MS Imaging (MALDI-MSI) to better understand some of the mechanisms of interaction between PZQ and schistosomes. The researchers were able to characterize both sexes of *S. mansoni* adult worms (BH strain) treated with PZQ and identified the spatial distribution of chemical markers. In addition to tegumental damage, PZQ significantly changed the metabolic profile of both sexes of *S. mansoni* [49]. These data are supported by a recent work where tegumental damage of schistosomes under PZQ exposure was demonstrated through scanning electronic microscopy and optical microscopy [92]. These findings could be helpful for understanding possible targets and pathways of this antischistosomal drug and demonstrates the versatility and potential use of mass spectrometrybased metabolomics for identification of drug targets.

## **4. Conclusions**

New tools are now available for biomedical research, and mass spectrometry techniques are among them. In "omics" research, huge opportunities are rising and enable to understand mechanistic basis for biological differences. For example, the absence of an enzyme in determined disease might be compared with healthy samples, unveiling significant molecular differences that clarify the mechanisms of action associated with a genetic failure. This new approach for biological systems allows the correlation between genotype and phenotype aspects, which is a complex process nowadays. Metabolomics studies are now giving the molecular profile of health or disease just like a "snapshot" of cell physiology. It is up to us scientists to establish the right and coherent correlations between metabolic patterns and biological alterations.

Although helminthiasis still consists of a neglected disease, concerning all the discussed topics, it is possible to keep looking for new strategies that help to control and treat these infections. The groups of molecules identified by metabolomics approaches, exemplified as MS, might be tested and correlated with the proposed hypothesis, and then it may be possible to finally understand the parasites metabolism. In this way, it will be possible to comprehend the mechanisms of all different lifecycle stages, expanding the field of action for parasite control. In addition, metabolomics can bring light to the designing of new therapeutic drugs, highly effective and specific, reducing collateral effects and drug resistance.

Therefore, mass spectrometry and metabolomics show huge potential as new tools to unveil metabolism of different biological systems, including helminths. The more scientists unveil about metabolism of parasites and parasite-host interactions, the greater the chances of improving public health aiming a better future.

## **Author details**

its structure, interactive medicinal chemistry is used, and finally, it is done on all preclinical

Target-based drug discovery starts with the identification and elucidation of the function of a potential therapeutic drug target and understanding its role in the disease process. Drug targets are, basically, molecular structures that will undergo a specific interaction with chemicals (drugs), which are administered to treat or diagnose a disease. Most of the times, drug targets are enzymes or proteins involved in biological activity, which are produced by expression of active genes in a cell [70]. Furthermore, it is interesting to develop drugs whose targets include biochemical pathways, such as cellular physiological process or cell *signalling* molecules. For example, it has been reported as an inhibitor of programmed cell death pathway (apoptosis) to be a potential small molecule acting on a caspase to control schistosomiasis caused by *S. japonicum* [89]. Therefore, metabolic pathways can also be explored as therapeutic targets, where there should be a clear understanding of anthelmintics mechanisms of action

For exploratory studies as cited above, metabolomics is a very powerful tool to connect parasite metabolism and its interaction with the drug of choice. To better understand some of these mechanisms, chromatographic techniques combined with MS have been employed for chemical characterization of adult schistosomes and PZQ metabolites in the host [90, 91]. In a study from 2015, researchers used MS Imaging (MALDI-MSI) to better understand some of the mechanisms of interaction between PZQ and schistosomes. The researchers were able to characterize both sexes of *S. mansoni* adult worms (BH strain) treated with PZQ and identified the spatial distribution of chemical markers. In addition to tegumental damage, PZQ significantly changed the metabolic profile of both sexes of *S. mansoni* [49]. These data are supported by a recent work where tegumental damage of schistosomes under PZQ exposure was demonstrated through scanning electronic microscopy and optical microscopy [92]. These findings could be helpful for understanding possible targets and pathways of this antischistosomal drug and demonstrates the versatility and potential use of mass spectrometry-

New tools are now available for biomedical research, and mass spectrometry techniques are among them. In "omics" research, huge opportunities are rising and enable to understand mechanistic basis for biological differences. For example, the absence of an enzyme in determined disease might be compared with healthy samples, unveiling significant molecular differences that clarify the mechanisms of action associated with a genetic failure. This new approach for biological systems allows the correlation between genotype and phenotype aspects, which is a complex process nowadays. Metabolomics studies are now giving the molecular profile of health or disease just like a "snapshot" of cell physiology. It is up to us scientists to establish the right and coherent correlations between metabolic patterns and

development before the clinical evaluation [71].

30 Human Helminthiasis

operating in helminth parasites [70].

based metabolomics for identification of drug targets.

**4. Conclusions**

biological alterations.

Estela O. Lima, Cibele Z. Esteves, Diogo N. Oliveira, Tatiane M. Guerreiro, Carlos F. O. R. Melo and Rodrigo R. Catharino\*

\*Address all correspondence to<EMAIL_ADDRESS>
Innovare Laboratory of Biomarkers, University of Campinas, Brazil

## **References**

[24] Canuto GA, da Cruz PL, Faccio AT, Klassen A, Tavares MF. Neglected diseases prioritized in Brazil under the perspective of metabolomics: a review. Electrophoresis. 2015;36(18):2336–47.

[8] Kafsack BF, Llinás M. Eating at the table of another: metabolomics of host-parasite

[9] Roessner U, Bowne J. What is metabolomics all about? Biotechniques. 2009;46(5):363. [10] Weckwerth W. Metabolomics: an integral technique in systems biology. Bioanalysis.

[11] Patti GJ, Yanes O, Siuzdak G. Innovation: metabolomics: the apogee of the omics trilogy.

[12] Pan Z, Raftery D. Comparing and combining NMR spectroscopy and mass spectrometry in metabolomics. Analytical and Bioanalytical Chemistry. 2007;387(2):525–7. [13] Katajamaa M, Orešič M. Data processing for mass spectrometry-based metabolomics.

[14] Harrigan GG, Goodacre R. Metabolic Profiling: Its Role in Biomarker Discovery and Gene Function Analysis. Springer Science & Business Media; Dordrecht, Netherlands.

[15] Ellis DI, Goodacre R. Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst. 2006;131(8):875–85.

[16] Kell DB, Brown M, Davey HM, Dunn WB, Spasic I, Oliver SG. Metabolic footprinting and systems biology: the medium is the message. Nature Reviews Microbiology.

[17] Weljie AM, Newton J, Mercier P, Carlson E, Slupsky CM. Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Analytical Chemistry.

[18] Barding Jr GA, Salditos R, Larive CK. Quantitative NMR for bioanalysis and metabo-

[19] Pettegrew JW. NMR: Principles and Applications to Biomedical Research. Springer

[20] Han X, Gross RW. Structural determination of picomole amounts of phospholipids via electrospray ionization tandem mass spectrometry. Journal of the American Society for

[21] Venter A, Nefliu M, Cooks RG. Ambient desorption ionization mass spectrometry.

[22] DeHaven CD, Evans AM, Dai H, Lawton KA. Organization of GC/MS and LC/MS metabolomics data into chemical libraries. Journal of Cheminformatics. 2010;2(1):9.

[23] Eppe G, Focant J-F, Pirard C, De Pauw E. PTV-LV-GC/MS/MS as screening and complementary method to HRMS for the monitoring of dioxin levels in food and feed.

lomics. Analytical and Bioanalytical Chemistry. 2012;404(4):1165–79.

Science & Business Media; New York - USA. 2012.

Trends in Analytical Chemistry. 2008;27(4):284–90.

Mass Spectrometry. 1995;6(12):1202–10.

Talanta. 2004;63(5):1135–46.

interactions. Cell Host & Microbe. 2010;7(2):90–9.

Nature Reviews Molecular Cell Biology. 2012;13(4):263–9.

Journal of Chromatography A. 2007;1158(1):318–28.

2010;2(4):829–36.

32 Human Helminthiasis

2012.

2005;3(7):557–65.

2006;78(13):4430–42.

[50] Keiser J, Duthaler U, Utzinger J. Update on the diagnosis and treatment of food-borne trematode infections. Current Opinion in Infectious Diseases. 2010;23(5):513–20.

[37] Van Hellemond JJ, Retra K, Brouwers JF, van Balkom BW, Yazdanbakhsh M, Shoemaker CB, et al. Functions of the tegument of schistosomes: clues from the proteome and

[38] Pullan RL, Smith JL, Jasrasaria R, Brooker SJ. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasites & Vectors. 2014;7(1):1.

[39] Malaria GH-DoPDa. The Burden of Ascariasis 2011 Available from: http://

[40] Committee WE. Prevention and Control of Schistosomiasis and Soil-Transmitted Helminthiasis. World Health Organization, Technical Report Series, No. 912; Geneva,

[41] Lo NC, Andrews JR, Bogoch II. Improving helminth treatment access: costs and

[42] Organization WH. Investing to Overcome the Global Impact of Neglected Tropical Diseases: Third WHO Report on Neglected Diseases 2015. World Health Organization;

[43] Shalaby HA. Anthelmintics resistance; how to overcome it? Iranian Journal of Parasi-

[44] Albonico M, Engels D, Savioli L. Monitoring drug efficacy and early detection of drug resistance in human soil-transmitted nematodes: a pressing public health agenda for

[45] Osei-Atweneboana MY, Eng JK, Boakye DA, Gyapong JO, Prichard RK. Prevalence and intensity of *Onchocerca volvulus* infection and efficacy of ivermectin in endemic communities in Ghana: a two-phase epidemiological study. The Lancet. 2007;369(9578):

[46] Araujo P. Observations on the early ecdyses of larvae of *Ascaris lumbricoides*, *A. suum* and *Toxocara canis*. Revista do Instituto de Medicina Tropical de São Paulo. 1972;14(2):

[47] Artigas PdT, Ueta MT. About the evolution of *Ascaris lumbricoides* Linnaeus, 1758, in the larval stage inside the egg. Memories of Butantan Institute. 1989;51(1):15–24.

[48] Ligocka A, Paluszak Z. Effectiveness of different sanitization technologies on the inactivation of *Ascaris suum* eggs in organic waste. Bulletin of the Veterinary Institute

[49] Ferreira MS, de Oliveira RN, de Oliveira DN, Esteves CZ, Allegretti SM, Catharino RR. Revealing praziquantel molecular targets using mass spectrometry imaging: an expeditious approach applied to *Schistosoma mansoni*. International Journal for Parasi-

helminth control. International Journal for Parasitology. 2004;34(11):1205–10.

lipidome. International Journal for Parasitology. 2006;36(6):691–9.

www.cdc.gov/globalhealth/ntd/diseases/ascaris\_burden.html

opportunities. The Lancet Infectious Diseases. 2016;16(7):762–4.

Switzerland. 2002; vol. i–vi:1–57.

Geneva, Switzerland. 2015.

tology. 2013;8(1):18.

2021–9.

34 Human Helminthiasis

83–90.

in Pulawy. 2009;53:641–4.

tology. 2015;45(6):385–91.

[79] Lacey E. Mode of action of benzimidazoles. Parasitology Today. 1990;6(4):112–5.

[65] Guan XL, Cestra G, Shui G, Kuhrs A, Schittenhelm RB, Hafen E, et al. Biochemical membrane lipidomics during Drosophila development. Developmental Cell.

[66] Carlson D, Langley P, Huyton P. Sex Pheromone of the Tsetse Fly: Isolation, Identification, and Synthesis of Contact Aphrodisiacs. American Association for the Advance-

[67] Steiger S, Schmitt T, Schaefer HM. The origin and dynamic evolution of chemical information transfer. Proceedings of the Royal Society of London B: Biological Sciences.

[68] Brooker S, Kabatereine NB, Smith JL, Mupfasoni D, Mwanje MT, Ndayishimiye O, et al. An updated atlas of human helminth infections: the example of East Africa.

[69] Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J. Helminth infections: the great neglected tropical diseases. The Journal of Clinical Investigation. 2008;118(4):

[70] Rana AK, Misra-Bhattacharya S. Current drug targets for helminthic diseases. Parasi-

[71] Pink R, Hudson A, Mouriès M-A, Bendig M. Opportunities and challenges in antipar-

[72] Renslo AR, McKerrow JH. Drug discovery and development for neglected parasitic

[73] Geerts S, Gryseels B. Drug resistance in human helminths: current situation and lessons

[74] van den Enden E. Pharmacotherapy of helminth infection. Expert Opinion on Phar-

[75] Hagel I, Giusti T. *Ascaris lumbricoides*: an overview of therapeutic targets. Infectious Disorders-Drug Targets (Formerly Current Drug Targets-Infectious Disorders).

[76] Hagel I, Cabrera M, Hurtado M, Sanchez P, Puccio F, Di Prisco M, et al. Infection by *Ascaris lumbricoides* and bronchial hyper reactivity: an outstanding association in Venezuelan school children from endemic areas. Acta Tropica. 2007;103(3):231–41.

[77] Stephenson LS, Latham MC, Ottesen E. Malnutrition and parasitic helminth infections.

[78] Lacey E. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism ofdrug resistance to benzimidazoles.InternationalJournalforParasitology.

asitic drug discovery. Nature Reviews Drug Discovery. 2005;4(9):727–40.

from livestock. Clinical Microbiology Reviews. 2000;13(2):207–22.

2013;24(1):98–111.

36 Human Helminthiasis

2010:rspb20102285.

1311–21.

ment of Science. Washington DC - USA. 1978.

tology Research. 2013;112(5):1819–31.

macotherapy. 2009;10(3):435–51.

Parasitology. 2000;121(S1):S23–S38.

2010;10(5):349–67.

1988;18(7):885–936.

International Journal of Health Geographics. 2009;8(1):1.

diseases. Nature Chemical Biology. 2006;2(12):701–10.

#### **Helminths and their Role in Environmental Engineering Helminths and their Role in Environmental Engineering**

Blanca Jiménez, Catalina Maya, José A. Barrios and Blanca Jiménez, Catalina Maya, José A. Barrios and

Inés Navarro Inés Navarro

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64878

#### **Abstract**

Helminth eggs represent an important challenge to environmental engineers as they are among the most difficult biological parasites to inactivate in wastewater and sludge. Even though no official data on helminthiasis exist, it is estimated that more than 2.6  billion people are affected. These parasites are of concern in developing countries, particularly in those areas where the reuse of wastewater and sludge for agriculture is common. With regard to this, the unrestricted use of wastewater for irrigation presents a serious health risk due to the dissemination of pathogens, particularly helminth eggs. Helminth eggs survive in water, soil, and crops for several months and over much longer periods than other microorganisms. Therefore, and in order to minimize risk, several guidelines and regulations exist which limit their content in wastewater and sludge. Risk assessment estimates that such regulations may be less strict in developing countries where higher concentrations of helminth eggs occur in wastewater and sludge. These eggs need to be removed from wastewater and inactivated in sludge using certain treatment processes, some of which are not feasible in developing countries. Adequate methods are needed to precisely identify and quantify helminth eggs in environmental samples. Therefore, a multidisciplinary approach is needed to address helminthiasis in environmental engineering issues.

**Keywords:** Biosolids, guidelines and regulations, helminth eggs, helminthiasis, inacti‐ vation processes, microbial risk assessment, wastewater

## **1. Introduction**

For centuries, wastewater has been used for agricultural irrigation throughout the world, and this is still a common practice in several countries. There are many examples that show wastewater reuse is key to increasing food production and improving local economies. This

© 2017 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. © 2017 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.

is simply due to the high water demand for irrigation. In developing countries, this amounts to 81% of the total water extracted and to around 45% in developed countries [1]. As an example, in Mexico, where 75% of the territory is semiarid, wastewater reuse for agriculture is often practiced. Mexican farmers have long appreciated wastewater use because it contains, in addition to water, organic matter, nitrogen, and phosphorus, which act as fertilizers, increasing crop yield. Jiménez [2] summarized the reasons why wastewater is reused for irrigation: (a) it significantly saves fresh water; (b) it provides nutrients to soil; (c) it reduces or eliminates the need for chemical fertilizers; (d) it contributes to the expansion of agricultural land in arid and semi‐arid areas; (e) it increases farmers' income; and (f) it is a relatively cheap disposal method for wastewater, reducing surface water pollution. It is estimated that there are 20 million hectares in at least 50 developing countries where wastewater is used to irrigate [3], representing around 10% of the total irrigated land. Even though the reuse of wastewater for agricultural irrigation has several benefits, it poses a public health risk due its pathogenic content. Among these pathogens, helminth eggs are of particular concern.

Helminths are parasitic worms transmitted to humans via their eggs (infective life stage, **Figure 1**). They are considered to be the biological structures most resistant to inactivation in the environmental engineering field [4]. Most helminths are transmitted by direct contact with contaminated soil, crops, or wastewater (e.g., *Ascaris lumbricoides*, *Trichuris trichiura*, and hookworms), but some require the presence of intermediate hosts (e.g., freshwater snails in the case of schistosomiasis) [5].

**Figure 1.** Fertilized eggs with larval growth and development of the worm for (a) *Ascaris lumbricoides* and (b) *Toxocara canis* (Photographic archives, Treatment and Reuse Group, Instituto de Ingeniería, UNAM).

Concerns about their presence are related to their very low infectious dose, their high rate of survival in the environment for extended periods of time (up to several years, compared to weeks for other pathogens), and their high resistance to conventional disinfection processes [6]. Helminth eggs are a particular threat to health where sanitation conditions are poor, polluted water is used for irrigation, or where excreta or non‐treated sludge is disposed of in an uncontrolled manner. They cause a set of diseases call "helminthiasis" that are given specific names based on the genera involved (for instance, ascariasis for genus *Ascaris*). Symptoms include deterioration of the intestines, toxic effects, blood loss, diarrhea, undernourishment, and anemia. Some helminths gnaw away at the intestinal wall causing hemorrhages while simultaneously secreting an anti‐coagulant. The damage caused by inflammation, and the wounds they open generate tumors and excrescences. In addition, helminths can block conduits (for example, biliary) or cause intestinal obstruction and perforation of the digestive tract causing peritonitis [6, 7].

is simply due to the high water demand for irrigation. In developing countries, this amounts to 81% of the total water extracted and to around 45% in developed countries [1]. As an example, in Mexico, where 75% of the territory is semiarid, wastewater reuse for agriculture is often practiced. Mexican farmers have long appreciated wastewater use because it contains, in addition to water, organic matter, nitrogen, and phosphorus, which act as fertilizers, increasing crop yield. Jiménez [2] summarized the reasons why wastewater is reused for irrigation: (a) it significantly saves fresh water; (b) it provides nutrients to soil; (c) it reduces or eliminates the need for chemical fertilizers; (d) it contributes to the expansion of agricultural land in arid and semi‐arid areas; (e) it increases farmers' income; and (f) it is a relatively cheap disposal method for wastewater, reducing surface water pollution. It is estimated that there are 20 million hectares in at least 50 developing countries where wastewater is used to irrigate [3], representing around 10% of the total irrigated land. Even though the reuse of wastewater for agricultural irrigation has several benefits, it poses a public health risk due its pathogenic

Helminths are parasitic worms transmitted to humans via their eggs (infective life stage, **Figure 1**). They are considered to be the biological structures most resistant to inactivation in the environmental engineering field [4]. Most helminths are transmitted by direct contact with contaminated soil, crops, or wastewater (e.g., *Ascaris lumbricoides*, *Trichuris trichiura*, and hookworms), but some require the presence of intermediate hosts (e.g., freshwater snails in

**Figure 1.** Fertilized eggs with larval growth and development of the worm for (a) *Ascaris lumbricoides* and (b) *Toxocara*

*canis* (Photographic archives, Treatment and Reuse Group, Instituto de Ingeniería, UNAM).

content. Among these pathogens, helminth eggs are of particular concern.

the case of schistosomiasis) [5].

40 Human Helminthiasis

Helminthiases may lead to serious collateral effects limiting the physical and mental devel‐ opment of children, notably when repeated infection occurs between 5 and 15 years of age. It is estimated that 182 million children of preschool age are infected, that is, around 33% of those living in developing countries [8–10]. These children display lower height and weight due to undernourishment compared to well‐nourished children living in healthy environments. At the same time, malnutrition results in a low school performance and a decrease in intelligence quotient (IQ) [11, 12]. Other effects, such as epileptic seizures, violent headaches, vertigo, local paralysis, vomiting, and optical and physical perturbations have been reported [7].

In spite of that, the control of helminth eggs was neglected until the late 1980s when the World Health Organization (WHO) developed guidelines for water and sludge reuse in agriculture. In 2006 the World Health Organization [6] confirmed the need to control the associated health hazards through an updated version of the guidelines for safe use of wastewater, excreta, and sludge in agriculture and aquaculture. These guidelines recommend limiting the helminth egg content in wastewater, excreta, and sludge; however, to achieve this, it is necessary to remove and inactivate the eggs. The following sections discuss the data collected for helminth eggs in environmental matrices, public health risks, regulations in different countries, treatment methods for wastewater and sludge, and analytical methods to determine their concentrations in environmental matrices.

## **2. Helminth egg content in wastewater and sludge**

Helminths are multi‐cellular organisms (worms) with a wide variety of shapes and sizes. Some of them are free‐living (like earthworms), while others are parasites. Helminths can be classified in different ways, but the classification most frequently used considers their body shape. Roundworms are called Nematodes while flatworms are called Plathelminthes; if the latter have segments then are referred to as Cestodes while non‐segmented ones are termed Trematodes [5]. As mentioned previously, helminths reproduce via the production of eggs, which are microscopic structures whose shape, size (20–180 m), and density (1.06–1.23) vary. A fertilized *Ascaris lumbricoides* female roundworm can lay around 200,000 eggs per day and up to 27,000,000 eggs over a lifespan of 10–24 months, while the *Taenia saginata*, a beef tape‐ worm, can produce as many as 800,000 eggs per segment per day (a typically sized worm may have around 1000 segments) [13–15]. Helminth eggs contained in wastewater, sludge, or excreta may be viable (alive) but not infective. To be infective, the eggs need to develop into larvae, which require a temperature of around 25°C combined with a moisture content of at least 5% [16]. These conditions are found in soils or crops where eggs develop into larvae within a few days, remaining viable for several months or years [6]. In addition, infective doses are very low, within the range of 1–10 eggs. Most helminthiases are, therefore, transmitted via the eggs through a human‐water‐soil‐crop‐human pathway.

The content of helminth eggs in developing countries' wastewater and sludge differs consid‐ erably from that of developed ones (**Table 1**). Helminth egg concentrations may be 7–80 times greater in developing countries. For instance, the helminth egg content in developing countries ranges from 70 to 3000 helminth eggs per liter (HE/L) in wastewater and 70–735 helminth eggs per gram of total dry solids (HE/g TS) in sludge. In comparison, for developed countries, the content in wastewater is only 1–9 HE/L and that for sludge is 2–13 HE/g TS [17]. Not all eggs are viable, but, in general, mean viability generally reaches 88%, with an even higher percent‐ age for *Ascaris* spp. (90%) as a result of their high environmental resistance [4, 18, 19].

**Table 1.** Helminth ova content in wastewater and sludge for selected countries.

## **3. Prevalence of helminthiasis**

A fertilized *Ascaris lumbricoides* female roundworm can lay around 200,000 eggs per day and up to 27,000,000 eggs over a lifespan of 10–24 months, while the *Taenia saginata*, a beef tape‐ worm, can produce as many as 800,000 eggs per segment per day (a typically sized worm may have around 1000 segments) [13–15]. Helminth eggs contained in wastewater, sludge, or excreta may be viable (alive) but not infective. To be infective, the eggs need to develop into larvae, which require a temperature of around 25°C combined with a moisture content of at least 5% [16]. These conditions are found in soils or crops where eggs develop into larvae within a few days, remaining viable for several months or years [6]. In addition, infective doses are very low, within the range of 1–10 eggs. Most helminthiases are, therefore, transmitted via the

The content of helminth eggs in developing countries' wastewater and sludge differs consid‐ erably from that of developed ones (**Table 1**). Helminth egg concentrations may be 7–80 times greater in developing countries. For instance, the helminth egg content in developing countries ranges from 70 to 3000 helminth eggs per liter (HE/L) in wastewater and 70–735 helminth eggs per gram of total dry solids (HE/g TS) in sludge. In comparison, for developed countries, the content in wastewater is only 1–9 HE/L and that for sludge is 2–13 HE/g TS [17]. Not all eggs are viable, but, in general, mean viability generally reaches 88%, with an even higher percent‐

age for *Ascaris* spp. (90%) as a result of their high environmental resistance [4, 18, 19].

Japan 80 1–51 [25] Jordan 300 [14] Mexico 6–98 (up to 330 in poor areas) 73–177 (viable eggs) [17] Morocco 840 [26] South Africa 772 2–40 [27, 28] Syria 800 [29] Tunisia 30 [15] Ukraine 60 [13] United States of America 1–8 2–13 [20]

**Country/region Helminth eggs in wastewater (HE/L) Helminth eggs in sludge (HE/g TS) References** Developing countries 70–3000 67–735 [13, 18, 20] Tropical countries 300–3000 [18] Brazil 166–202 1–76 [21] Cayman islands 100–1230 [14] China 840 2300 [22] France 9 5–7 [20] Egypt (liquid primary) 6–42 Mean: 67, Max: 735 [23–24] Egypt (dewatered primary) Mean: 8, Max: 124 [24] Ghana 76 [20, 24]

eggs through a human‐water‐soil‐crop‐human pathway.

42 Human Helminthiasis

Germany <1 Great Britain <6

**Table 1.** Helminth ova content in wastewater and sludge for selected countries.

Stoll N. [30] calculated that there were 2200 million nematode infections worldwide and predicted that by 1991, there would be 5000 million infected people. Similarly, he estimated that the Chinese population produced a total of 18,000 tons of *Ascaris* eggs per year. Currently, it is estimated that more than 2.6 billion people are infected with helminths (**Table 2**), and although helminthiases are globally distributed, the prevalence of disease is mostly limited to developing countries [5, 7, 31]. They are endemic in regions of Africa, Central America, South America, and the Far East, with incidence rates as high as 90% for specific regions and sectors of the population [6, 7]. The dominant species depends on local conditions. However, most of the information related to procedures to control helminth eggs in sludge was gathered in developed countries where the low concentrations found render it difficult to utilize the data collected [20].

Around 96% of the helminthiases reported are induced by infection by Nematoda and Trematoda classes, with 4% due to Cestoda [5, 32]. The most common genus is *Ascaris lumbricoides* [33] with more than 819 million people infected. The eggs are highly infective (commonly, one egg suffices), very persistent in the environment, and very resistant to conventional disinfection/inactivation processes [10–12, 34]. For example, embryonated eggs of *Ascaris* spp*.* can survive for 20 days at temperatures of ‐20.9 to ‐27°C and survive in frozen Siberian soils for 10 years [35]. This resistance comes from the fact that eggs are surrounded by a series of layers functioning as a barrier that protects the larva against harsh environmental conditions. Indeed, the different helminth egg species possess 3–4 layers with different physical and chemical characteristics: (a) The 1–2 outer layers are formed of mucopolysac‐ charides and proteins; (b) the middle layers have chitin which provides structure and me‐ chanical resistance; and (c) the inner layer, composed of lipids and proteins, protect the egg from desiccation, strong acid and bases, oxidants, reductive agents, detergents and proteolytic compounds [36–39].

The interaction of the layers' components with external agents may cause a change in their chemical composition, as well as in the specific function of each layer, which in turn modifies their permeability, their mechanical and chemical resistance and, finally, the egg viability. These mechanisms may be similar to those that take place during the hatching process, during which the larva secretes certain enzymes that induce the loss of permeability of the ascaroside layer and attack the outer eggshell layers. However, in this case, the layer remains intact until the hatching of the larva with no detectable chemical change [40–41].

The impact on the quality of life of infected populations may be illustrated using an estimation made in 2013 for years lived with disabiliy (YLDs) [42]. This amounted worldwide to 1,004,000 years for anemia caused by hookworm disease, and 671,000 years for schistosomiasis. Based on the analysis of YLDs estimates, ascariasis was among the eight most prevalent causes of disease, affecting more than 10% of world's population in 2013, such that if the mean prevalence of chronic sequelae is selected, for periods longer than 3 months, the impact of ascariasis was estimated in 819 million cases.

**Table 2.** Common helminth species and helminthiasis by region.

## **4. Guidelines and regulations**

As mentioned previously, the use of domestic wastewater for irrigation, even without treat‐ ment, is an established practice in many countries. However, it leads to the proliferation of helminthiasis in the exposed population [6, 43]. Following a series of epidemiological studies, since 1989, the World Health Organization has recommended a limit for the content of nematode eggs in wastewater reused for agricultural irrigation [44]. Similarly, sludge pro‐ duced during wastewater treatment may be reused in agriculture or soil remediation after adequate treatment to reduce its microbial content. Once treated, wastewater and sludge need to meet national regulations or international criteria in order to be safely reused. For instance, in 2006, the World Health Organization suggested that a limit of less than 1 viable helminth egg per liter (HE/L) in wastewater makes it acceptable for agricultural reuse, while less than 1 viable helminth egg per gram of total dry solids (1 HE/g TS) is required in sludge [6]. However, these limits are difficult to meet in countries or regions with endemic diseases, requiring effective inactivation processes to control the high concentrations of helminth eggs.

**Helminth species Common name Prevalence**

S*trongyloides stercoralis* 370

*Taenia saginata* Beef tapeworm 50 *Taenia solium* Pork tapeworm

*Fasciolopsis buski* Intestinal fluke

tapeworm

Food‐borne Trematodes

Other groups 100 Worldwide

**Table 2.** Common helminth species and helminthiasis by region.

**4. Guidelines and regulations**

Total Over 2.6 billion infections worldwide

**Nematoda**

44 Human Helminthiasis

*Trichostrongylus orientalis*

*Hymenolepis nana* Dwarf

**Cestoda**

**Trematoda**

*Clonorchis sinensis Echinostoma* spp. *Fasciola gigantica Fasciola hepatica Fasciolopsis buski Heterophyes* spp. *Metagonimus* spp. *Opisthorchis felineus Opisthorchis viverrini Paragonimus* spp.

Source: Ref. [5].

**(million inhabitants)**

Pacific islands *Necator americanus*

**Regional presence**

South America

Central and South America, the Caribbean, Asia and in the

may be found around the world in South America, Southeast Asia, Western Africa and East Africa; and in areas of the tropics and subtropics and some areas of Southern and Eastern Europe, the United States of America and Mexico.

throughout Africa and South America, especially in Brazil, Venezuela, Surinam, Guyana and several Caribbean islands, including Puerto Rico, St. Lucia, Martinique, and Guadalupe

56 Largely in Southern and Eastern Asia but also in Central and

South America, and the Caribbean islands

*Ascaris lumbricoides* Roundworm 819 Many regions of South‐East Asia, Africa, and Central and

*Ancylostoma duodenale* Hookworm 439 Tropical and subtropical countries (Sub‐Saharan Africa),

*Trichuris trichiura* Whipworm 465 Moist, warm, tropical regions of Asia, Africa, Central and

Roundworm Several Mainly rural communities in Asia

*Hymenolepis diminuta* Rat tapeworm 50 Most occurrences in areas which lack adequate sanitation but

*Schistosoma mansoni* Blood fluke 207 Tropical and subtropical regions. This species occurs widely

As mentioned previously, the use of domestic wastewater for irrigation, even without treat‐ ment, is an established practice in many countries. However, it leads to the proliferation of helminthiasis in the exposed population [6, 43]. Following a series of epidemiological studies, since 1989, the World Health Organization has recommended a limit for the content of nematode eggs in wastewater reused for agricultural irrigation [44]. Similarly, sludge pro‐ duced during wastewater treatment may be reused in agriculture or soil remediation after adequate treatment to reduce its microbial content. Once treated, wastewater and sludge need

Eastern Europe.

Following the WHO guidelines [6], many countries have set standards for helminth eggs in treated water, including Brazil, Colombia, Costa Rica, Chile, Israel, Jordan, Mexico, Saudi Arabia, and Tunisia. These standards also set recommendations for the types of crops to be irrigated, irrigation methods, and other intervention measures to manage risks. In addition, sludge and biosolids (treated sludge) regulations have been developed by several countries, led principally by the United States (**Tables 3** and **4**).

U: Unrestricted irrigation: For agricultural irrigation of all kinds of crops, including those that are eaten uncooked (lettuce, onion); R: Restricted irrigation: For agricultural irrigation of all kinds of crops, except that are eaten uncooked (highly mechanized, labor intensive).

a When children under 15 years of age are exposed, additional health protection measures should be used (e.g., treatment to ≤0.1 egg per liter, protective equipment such as gloves or shoes /boots or chemical treatment);

b An arithmetic mean should be determined throughout the irrigation season. The mean value of ≤1 egg per liter should be obtained for at least 90% of samples in order to allow for the occasional high‐value sample (i.e., with >10 eggs per liter). In some wastewater treatment processes (e.g., waste stabilization ponds), the hydraulic retention time can be used as a surrogate to assure compliance with a limit of ≤ 1 egg per liter;

c Including fruit trees and olives, not crops harvested directly from the soil. Source: Refs. [6, 45–47].

**Table 3.** Regulations for helminth egg content in treated wastewater use in agriculture.

**Table 4.** Regulations for helminth egg content in sludge.

In general, the standard values have been set to protect human health, allowing sludge application at sites where human contact may occur [48]. Under those conditions, a value of <1 HE/g is to be achieved (Class A). Only Brazil and Mexico allow higher concentrations of helminth eggs. However, in the case of Brazil, the regulation states that Class B sludge may be land applied only after risk assessment determines that this is a secure practice. In contrast, the limits for Class B and C sludge in the Mexican regulations refer to total eggs, assuming not all of them are viable, and is intended for land application without human contact (e.g., agriculture, soil restoration, forestry). However, to enforce these limits, it is necessary to correctly measure the helminth egg content in wastewater and sludge (treated or untreated).

## **5. Quantitative microbial risk assessment (QMRA)**

WHO guidelines and US EPA limits for helminth eggs were based on limited epidemiological evidences, and on the performance of different sludge treatment methods, rather than the results of a risk assessment. Neither of the organizations based their considerations on results of a dose response‐curve "because methodologies had not been developed sufficiently enough to make such calculations" [6, 49]. As a result, no human risk assessment was conducted, due to the lack of a dose‐response model appropriate to describe helminth eggs infection. At the time, of the three different methods used to evaluate microbial risks: microbiology laboratory analysis, epidemiological studies, and quantitative microbial risk assessment (QMRA), only the first two have been applied to helminth data [50–52]. In contrast, a number of human dose‐ response relationships have been developed for bacteria, viruses, and protozoa [53–59]; consequently, a dose‐response function for helminth infections was required for applying a QMRA.

**Country Regulation standard guidelines Class/type Helminth egg limit,**

CFR 40 Part 503 /1993 A 0.25

In general, the standard values have been set to protect human health, allowing sludge application at sites where human contact may occur [48]. Under those conditions, a value of <1 HE/g is to be achieved (Class A). Only Brazil and Mexico allow higher concentrations of helminth eggs. However, in the case of Brazil, the regulation states that Class B sludge may be land applied only after risk assessment determines that this is a secure practice. In contrast, the limits for Class B and C sludge in the Mexican regulations refer to total eggs, assuming not all of them are viable, and is intended for land application without human contact (e.g., agriculture, soil restoration, forestry). However, to enforce these limits, it is necessary to correctly measure the helminth egg content in wastewater and sludge (treated or untreated).

WHO guidelines and US EPA limits for helminth eggs were based on limited epidemiological evidences, and on the performance of different sludge treatment methods, rather than the results of a risk assessment. Neither of the organizations based their considerations on results of a dose response‐curve "because methodologies had not been developed sufficiently enough to make such calculations" [6, 49]. As a result, no human risk assessment was conducted, due to the lack of a dose‐response model appropriate to describe helminth eggs infection. At the time, of the three different methods used to evaluate microbial risks: microbiology laboratory analysis, epidemiological studies, and quantitative microbial risk assessment (QMRA), only

Brazil CONAMA 375/2006 A 0.25

Chile No. 123 (30/08/2006) A 0.25

France Directive 86/278 ‐ 0.3 Mexico NOM‐004‐SEMARNAT‐2002 A 1

The WHO WHO, 2006 1

New Zealand Guidelines for the safe application of biosolids to land in

Norway Regulations for the treatment, use and disposal of sewage

South Africa Guidelines for the utilisation and disposal of wastewater

**5. Quantitative microbial risk assessment (QMRA)**

New Zealand

sludge (Vol. 2)

**Table 4.** Regulations for helminth egg content in sludge.

sludge

The United States of

46 Human Helminthiasis

America

VViable eggs.

**eggs/g TS**

B 10

B ‐

B 10 C 35

A 0.25

A 0.25 B 1

– Absent

QMRA is a modeling approach used to predict the human health risks from exposure to pathogens and has been shown to be effective in assessing the transmission of water and food‐ borne infections. It estimates the risk of infection or illness based on the concentration of infectious pathogens in wastewater, on surfaces or in drinking water, or from other environ‐ mental sources (e.g., crops, biosolids, air), the estimated ingestion rate, and the established dose‐response models for a given population.

Since 2009 a dose‐response curve is only available for *Ascaris* [60], although other helminths genus are highly relevant in wastewater and sludge reuse, especially in developing countries [61]. This curve was derived from *A. lumbricoides* prevalence data obtained from stools of a large sample of children in the Mezquital Valley in Mexico, rather than conducting human *Ascaris* dose‐challenge studies. Navarro et al. [60] found that their *Ascaris* infection data best fitted the β‐Poisson dose‐response equation [Eq. (1)].

$$P(d) = 1 - \left[1 + \left(d \wedge N\_{50}\right)\left(2^{1/\alpha} - 1\right)\right]^{-\alpha} \tag{1}$$

where P(d) is the risk of infection in an individual who has ingested *d Ascaris* eggs on one occasion; *N*<sup>50</sup> is the mean *Ascaris* infective dose; and α is an *Ascaris* 'infectivity constant'. They found the values of *N*50 and α to be 859 and 0.104, respectively.

Use oftheQMRAtool allowedan initialtarget estimation of *Ascaris* concentration in sludge and wastewater.These values were greaterthanWHOandUSEPAlimits [62], but withanestimated probability of infection smaller than the prevalence rate observed on site. Such scenarios may allow a gradual improvement in population health conditions, as well as working towards an *Ascaris* eggs content in sludge and/or wastewater which gradually should approach regulation limits.The authors concludedfromthese riskanalyses thatregulations targetingbiosolids reuse in developing countries should address the challenge of firstly deciding an acceptable infection risk and, secondly, putting in place an integrated framework for risk management, involving additional health protection measures. They suggested factors to be considered, which include, amongst others: (a) an affordable treatment process, (b) crop restriction policy, (c) different sludge application rates, and (d) efficient produce washing methods.