Prevalence,risk factors,challenges,and the currently available diagnostic tools for the determination of helminths infections in human

Abstract

Soil transmitted helminthes (STH) are among the major public health issues in tropical and sub-tropical countries infecting more than 2 billion humans worldwide. STH causes considerable morbidity in children, affecting their cognitive development and physical growth. Endemic areas with poor sanitation and limited access to good quality water supply have the highest burden of STH infection. Various approaches to reduce and control the worm infections include the intermittent deworming of children with anti-helminthic drugs. Individual patient management and population based studies can only be successful upon using the diagnostic techniques with high accuracy. The lack of reliable tools for the provision of correct diagnostic results about the status and intensity of infection is a major challenge, as these factors varies considerably in areas of low infection intensities. The techniques currently available for the diagnosis of parasitic infections show limited specificity and sensitivity and as such several techniques in combination are normally used in diagnosing the variety of parasite species infection. This review article was planned to study the prevalence, risk factors, and the available diagnostic techniques for soil and water transmitted helminthes infections in humans. This article also discussed the challenges in diagnosis, treatment, and management of worm infections particularly helminthes. The articles available online on important portals like google scholar, PubMed, Digital Libraries, PakMediNet, Science direct, and the Directory of Open Access Journals (DOAJ) were searched for inclusion of the data in this review study. Duplicate studies and irrelevant reports describing the general aspects of parasite infections were excluded. This review study provides a comprehensive report on the prevalence, accurate diagnostic, and chemotherapeutic protocols to reduce the burden of worms infections. WHO suggests the chemotherapy for worm infections as feasible and cost effective strategy in schools due to the lack of proper policy for the prevention of intestinal worms.

Keywords

intestinal worms parasite soil transmitted helminthes worm infestation

Introduction

Intestinal worm infection is one among the major health problems throughout the World particularly in developing countries.¹ Intestinal worms live and feed off on another organism.² Most of the infected children may carry different types of parasites that can live throughout the body and may infect the gastro-intestinal tract.³ Adult worms lay eggs, the larvae hatched, grow, and developed into adult worm inside human body.⁴ Taenia saginata (Tap worm), Enterobius vermicularis (Pin worm) and Trichuris trichiura (Whipworm), Ascaris lumbricoides (roundworms), Necator americanus (Hookworms), and Ancyclostoma duodenale are among the most common helminths, each parasitizing hundreds of millions of people.⁵ Transmission of intestinal parasites is usually through contact with infected feces or ingestion of undercooked meat, drinking infected water, contaminated soil, and skin absorption.⁶ When unwashed, uncooked, contaminated food is ingested by mouth then parasite travels into intestine and reproduces itself causing symptoms by their toxicity like abdominal pain, diarrhea, vomiting, fatigue, dysentery, weight loss, muscle pain, skin irritation, sleep problem, and passing of worms in stool.⁷ In children, its rate is high due to soil transmission and nail biting.⁸ According to the World Health Organization (WHO) estimates, 870 million children live in the area of high prevalence. The most affected regions of the world include Africa, South Asia, and South America.^9,10 Children with long/untrimmed nail, failure to wash hands before meals, bare footed, nail biting habit, and sucking of thumb have significant association with parasitic infection.¹¹ School going children are more prone to develop intestinal worm’s infestation while playing in school lawns. Their hands become contaminated with infected soil and get infected when they put contaminated fingers in their mouth.^12,13 The adult people are also at risk due to drinking contaminated water with parasites that colonize the gastrointestinal tract.¹⁴ Children using tube well water for drinking has significantly higher prevalence of intestinal worms than using tap water.^11,15 Prevalence of intestinal worm infection is significantly lower in children using soap and water before meal and after defecation than children who just wash their hands by water.¹⁶ The prevalence of intestinal worms is 59.2% due to nail biting.¹¹ Malnutrition in school-age children is common which leads to nutritional stress, adverse health, worse school performance, and cognitive impairment.¹⁷ Diagnostics play important role in the guidance for both deploying the existing resources for STH program, and the strategies for the implementation and evaluation of STH intervention. Currently used detection and quantification methods for STH-specific eggs have practical advantages of using inexpensive test kits and can easily performed in low-resourced field settings. The most common example is the Kato Katz method. However, these methods also have significant disadvantages poor reproducibility, lower than optimal sensitivity and moderate labor costs.¹⁸ There is a wide variety of intestinal worms but two most commonly used tests for diagnostic purpose are performed from stool sample and adhesive tape may be applied in order to find eggs of worms.¹⁹ The main aim of this review is to evaluate the prevalence of water and soil transmitted helminths (STH) and to provide an insight regarding risk factors, challenges and the available diagnostic techniques for parasitic infestations.

Literature search and inclusion criteria

For this general review, the articles available online on important portals like google scholar, PubMed, Digital Libraries, PakMediNet, Science direct, and Directory of Open Access Journals (DOAJ) were searched using the key words: diagnostics, intestinal helminths, water and soil-transmitted helminths, risk factors, challenges, and the diagnostic techniques used for determining the parasitic infection were included. The examples of diagnostic methods for parasitic infections are Kato-Katz method, formol ether floatation method, McMaster method; merthiolate-iodine-formaldehyde, sodium nitrate flotation, and the polymerase chain reaction (PCR). Textbooks information about parasitic infections are also included in this review.

Prevalence of STHs infections

World Health Organization (WHO) estimates that the major soil transmitted helminths contribute 5.18 million disability adjusted life years all around the world in 2010 and these main STHs include the hookworms (Necator americanus and Ancyclostoma duodenale), the whipworm (Trichuris trichiura), and the roundworm (Ascaris lumbricoides). Globally, an estimated 440 million individuals are infected with hookworms, 460 million with whipworms and 820 million with round worms.²⁰

Intestinal parasitic infections remain a major public health problem with vast socioeconomic devastation among vulnerable rural communities and occurring predominantly in developing countries.^21,22 According to 2014 WHO report, over 3.5 billion people were infected with intestinal helminths mainly by hookworms, T. trichiura, A. lumbricoides, H. nana, S. stercoralis, and T. saginata.²³ Helminth infections grouped under geohelminthic; hook worm, T. trichiura and A. lumbricoides are extensively distributed in Eat Asia, China, the Americas, and sub-Saharan Africa.^24,25 Geo-helminthic infections affect over 2 billion people worldwide and 2/3rd of African countries having high risk areas has the prevalence of over 50%.²⁶ A. lumbricoides is still one of the most prevalent IPIs with worldwide cases of 807 to 1221 million particularly in East Asia and sub-Saharan Africa. Additionally, the whipworm (T. trichiura) and hookworm are liable for 604 to 795 million and 576 to 740 million infections, respectively.²⁷ WHO stated that worldwide 2.4 billion people do not have access to improved sanitation facility and 1.1 billion people do not have access to improved water supply sources, whereas approx. 2 billion people are infected with STHs. Consequently, about 2 million people, mostly children less than 5 years age die annually due to diarrheal diseases, and the populations of developing countries are the most affected ones, living under poverty, in poor health conditions and rural populations or periurban dwellers.²⁸

Hookworm infections, in addition to Malaysia (21.0%), Bangladesh (22.3%), Nepal (30.7%), and Papua New Guinea (60.6%), remain common throughout sub-Saharan Africa. Contrary to that, hookworm infections are not found in North Africa and most of the Central Asia, Except Egypt with 6.0% prevalence. The prevalence of T. trichiura infections is 49.9% in Malaysia and 45.5% in Philippines as well as in Central America (28.4% in Venezuela to 5.1% in El Salvador) and Central Africa (38.8% in Equatorial Guinea to 11.8% in Central African republic). Figure 1 below shows the global distribution of the prevalence of each STH infection.²⁹

Figure 1.

The global distribution of STH infection prevalence by STH species.²⁹ (a) Hookworm, (b) Ascaris lumbricoides, and (c) Trichuris trichiura; based on geostatistical models for sub-Saharan Africa and available emperical information for all other regions.

It was found that of the three STH infections, A. lumbricoides have the widest distribution with increased transmission rates seen in Nigeria (25.4%), Cameroon (30.8%), and northwestern countries of Central sub-Saharan Africa (38.8% in Equatorial Guinea to 32.2% in Congo). This infection is found dispersed geographically among the southern countries of Central Latin America (Ecuador 35.8%, Colombia 26.0%, and Venezuela 28.4%) and Asian countries (the Philippines 33.6%, Afghanistan 36.0%, Malaysia 41.7%, and Bangladesh 38.4%).²⁹

Global estimates suggest that over 568 million school aged children and 270 million pre-school children are included among the nearly 1.5 billion people globally infected with STHs. All these infected people are in need of treatment and prevention interventions. People carrying light intensity infections are usually asymptomatic whereas high morbidity are observed in people harboring heavy infections. Thus, variety of non specific and specific adverse effects such as cognitive impairment in children and reduced physical growth are the debilitating outcomes from the heavily infected people,³⁰ as well as intestinal occlusion and anemia. Recent estimates suggest that four STHs namely the hookworms (Necator americanus and Ancyclos-toma duodenale), Trichuris trichiura and Ascaris lumbricoides infect over 480, 508, and 700 million people worldwide, respectively.²⁹ In tropical countries, the highest prevalence is recorded with a total number of annually estimated deaths higher than 135,000. Crowded households are the more common source of clusters of infections.³¹ The transmission of STHs depend on the three principal conditions such as fecal contamination of soil, favorable soil conditions for the survival of eggs/larvae, direct oral infestation or skin contact with contaminated soil and through the consumption of contaminated water and/or food.³² School going children (5–15 years) and the pregnant women are the most vulnerable groups for STHs infections. Endemic countries with limited healthcare facilities, absence of portable water and inadequate sanitary conditions are reported to have higher STHs infection.³³ Prevalence data of intestinal worm infection in different countries is given in Table 1.

Table 1.

Prevalence data of intestinal parasites.

Country	Study location	Type of infection	Percentage	Reference
Africa	Tanzania	Hookworms	25.2%	Mazigo et al.³⁴
		Ascaris lumbricoides	1.6%
		Enterobius vermicularis	0.67%
		Trichuris trichiura	0.79%
		Schistosoma mansoni	5.6%
		Taenia species	0.57%
	Sub Saharan Africa	Hymenolepis nana	13.8%	Gelaw et al.³⁵
		Entamoeba histolytica	9.2%
		Ascaris lumbricoides	5.9%
Cuba	Matanzas	Ascaris lumbricoides	7%	Cañete et al.³⁶
		Trichuris trichiura	3%
		Enterobius vermicularis	2%
		Taenia species	1%
Egypt	Damanhur	Ascaris lumbricoides	14%	Hegazy et al.³⁷
		Enterobius vermicularis	3.4%
		Ancyclostoma duodenale	5%
		Hymenolepis nana	0.2%
Ethiopia	Gambo area	Ascaris lumbricoides	7.8%	Wegayehu et al.³⁸
		Hymenolepis nana	0.6%
		Hookworms	4.9%
		Schistosoma mansoni	0.12%
		Taenia species	2.9%
		Trichuris trichiura	2.8%
		Ascaris lumbricoides	48%	Asrat et al.³⁹
		Giardia lamblia	41.9%
		Entamoeba histolytica	27.3%
		Schistosoma mansoni	15.9%
		Hookworm	11.5%
	North Gondar	Ascaris lumbricoides	48%	Ayalew et al.⁴⁰
		Schistosoma mansoni	15.9%
		Hookworm	11.5%
		Trichuris trichiura	7%
		Hymenolepis nana	6.8%
		Taenia species	5.3%
		Enterobius vermicularis	2.6%
	Jawe Woreda	Hookworm	21.1%	Hailu⁴¹
		Schistosoma mansoni	3.5%
		Ascaris lumbricoides	3.9%
India	Pulwama	Ascaris Lumbricoides	69.84%	Wani and Ahmad⁴²
	Chennaei, Tamil Nadu	Hymenolepis nana	1.1%	Rayan et al.⁴³
		Ascaris lumbricoides	21%
		Trichuris trichiura	8%
	Srinagar, Indian Occupied Kashmir	Ascaris lumbricoides	28.4%	Wani et al.⁴⁴
		Giardia lamblia	7.2%
		Trichuris trichiura	4.9%
		Taenia saginata	3.7%
	Mangalore	Taenia saginata	4.37%	Hart and Hart⁴
		Hymenolepis nana	0.38%
		Trichuris trichiura	0.08%
Iraq	Baghdad	Hymenolepis nana	1.17%	Al-Taie⁴⁵
		Entrerobius vermicularis	0.38%
		Ascaris lumbricoides	8.21%
Kenya	Nairobi	Trichuris trichiura	4.6%	Mwanthi et al.⁴⁶
		Hookworm	0.1%
		Schistosoma mansoni	0.4%
Nepal	Kaski	Ascaris lumbricoides	2.1%	Chandrashekhar et al.⁴⁷
		Trichuris trichiura	1.3%
		Hymenolepis nana	1.6%
		Necator americanus	0.4%
		Enterobius vermicularis	1%
Nigeria	Ilaje	Ascaris lumbricoides	41.27%	Awolaju and Morenikeji⁴⁸
	Ikoyl	Ascaris lumbricoides	36.67%
	Aragan (Araromi)	Ascaris lumbricoides	33.33%
	Oke –Omiru	Ascaris lumbricoides	39.58%
	Cappa	Ascaris lumbricoides	38.46%
Pakistan	Muzaffarabad	Ascaris lumbricoides	3.8%	Chaudhry et al.⁴⁹
		Hookworm	2.4
		Enterobius vermicularis	1.3%
		Trichuris trichiura	1.0%
		Hymenolepis nana	1.7%
		Taenia saginata	1.0%
	Bannu	Ascaris lumbricoides	15%	Khan et al.⁵⁰
		Enterobius vermicularis	12%
		Hymenolepis nana	10%
		Taenia saginata	7%
		Ancyclostoma duodenale	1%
	Rural Peshawar	Round worm	45.5%	Ullah et al.⁵¹
		Pin worm	4%
		Hook worm	3.5%
		Whip worm	3.5%
		Hymenolepis nana	8.0%
		Taenia saginata	1.5%
	Muzaffarabad	Giardia lamblia	11.8%	Chaudhry et al.⁴⁹
		Ascaris lumbricoides	3.8%
		Hookworm	2.4%
	Quetta	Hymenolepis nana	34%	Ahsan-ul-Wadood et al.⁵²
		Giardia lamblia	32%
		Entamoeba histolytica	29%
		Ascaris lumbricoides	4%
		Ancyclostoma duodenale	1%
	Sukkar	Giardia lamblia	36.19%	Shaikh et al.⁵³
		Entamoeba histolytica	18.57%
		Hymenolepis nana	16.19
		Ascaris lumbricoides	14.76
		Ancyclostoma duodenale	7.24
		Enterobius vermicularis	4.19
		Trichuris trichiura	1.81
		Taenia saginata	1.05
Sudan	Elengaz area, Khartoum	Hymenolepis nana	26.4%	Gabbad and Elawad⁵⁴
		Taenia saginata	8.6%
		Enterobius vermicularis	6.2%
		Schistosoma mansoni	4.4%
United Arab Emirates	Sharjah	Entamoeba histolytica	71.8%	Dash et al.⁵⁵
		Giardia lamblia	17.5%
Vietnam	Southern Vietnam	Hookworm	52%	Verle et al.⁵⁶
		Trichuris trichiura	50%
		Ascaris lumbricoides	45%
		Giardia lamblia	3.0%

The overall prevalence rate of intestinal worms in different cities of Pakistan is increasing. Prevalence and intensity of intestinal worm infections are very high in rainy season while low in winter whereas, the infection rate is high in males compared to females that happen due to the suppression of immune system and the stimulatory effect of estrogen on immune response against GIT. Khan et al. investigated the prevalence of intestinal worm infection in primary school children (5–10 years age) living in district Bannu, Pakistan. They found that 54% of the children in the study population have infection with seven types of intestinal worms including Ancyclostoma duodenale, Giardia lamblia, Entamoeba histolytica, Taenia saginata, Hymenolepis nana, Pinworm, and Ascaris lumbricoides.⁵⁰ The prevalence data of each worm infection is given in Table 1. Most common infection was Ascaris lumbricoides (15%) and least infection was Ancyclostoma duodenale (1%). Abdominal discomfort and pain was the most common clinical presentation in children suffering from intestinal worm infection. Other complaints were cough, anemia, itching, urticaria, vomiting, bloody diarrhea, and diarrhea in percentage of 7.41%, 18.52%, 9.26%, 3.7%, 7.4%, 3.7%, and 12.9%, respectively.⁸ Mehraj et al. reported that helminthiasis is endemic Worldwide. Humid and hot tropical climate, lack of access to potable water, poor hygiene, illiteracy, and poverty are the major risk factor for intestinal worm infections.⁵⁷

Risk factors

The risk of acquiring STH infection cannot be attributed by only one factor but because of coexistence and amalgamation of multiple factors including environmental, behavioral, social, and biological factors both at individual and community level. These factors include the lack of personal hygiene, substandard living conditions and poverty. Studies in other tropical countries postulated that the rate of infection is influenced by the environment and the behavior of local residents.⁵⁸

Potential risk factors explored were markers of social advantage (home ownership, occupation, education, and TV/radio ownership), demographic factors (ethnicity, place of residence either urban/rural, mother’s age, and gender), and the factors associated with lifestyle and housing (use of soap, place of cooking, domestic animals, method of waste disposal, water source, type of toilet, and roof type). Poor personal hygiene, inadequate availability of clean water, poor sanitation, low socioeconomic status, and the rural areas are the major risk factors for STH infection.⁵⁹ Densely populated home environment, dirty playground, and the poor hygiene of child and the mother or caregiver are also the risk factors for STH infection in preschool children.⁶⁰

Challenges

The control of STHs involves several challenges, among which few are highlighted. First challenge for the control of STH is the proper mobilization of funds required for successful action. In contrast to some of the neglected tropical diseases (NTDs) with visible morbidity like lymphatic filariasis (LF) and onchocerciasis which have raised funding successfully for implementing the control activities and globally little funds are available for implementing the control of STH alone. Therefore, it is advantageous for the endemic countries to avail the opportunities generated by the impetus and new partnership for NTD control resulting from the growing financial resources all over the world.²⁵

Second challenge is the expansion of the coverage of regular deworming as a public health intervention to approach the individuals at risk of morbidity caused by STH infections. In context of multidisease integrated control, the integration of helminth control within community directed interventions in sub-Saharan Africa is taking advantage of the long experience, LF control programs and gains of onchocerciasis through the establishment of community directed treatment with ivermectin (CDTI) in all countries supported by the African Programme for Onchocerciasis Control—APOC.⁶¹ Yet, STHs are more widely distributed in most of the countries than both the LF and onchocerciasis. Hence, the challenge will be to expand the MDA coverage in areas with no CDTI.

The third challenge is the failure of the cost-effective anti-helminthic drugs in achieving the desired results because of inadequate waste drainage system, inaccessibility of safe water, poor sanitation, and favorable climate for intestinal parasitic infection transmission in developing countries.⁶²

The fourth challenge is the proper monitoring and evaluation of diagnostic tools. Regular monitoring and evaluation is crucial for ensuring the efficient implementation of the programs providing maximum benefits to the beneficiaries. This is particularly challenging providing the commonly used diagnostic techniques with low sensitivity in low transmission zones resulting in significant increase in number as far as programs are successful. Therefore, it is required to develop and validate alternative diagnostic techniques to address this issue.²⁵

Some other important challenges for the control of STHs infections are the sustainability of STH control programs, implementation of operational research, strengthening the institutional capacity and partnership, and the development of new drugs.⁶³ Therefore, the long term viability of control program is based on strengthening the health system. Additionally, the reinforcement of the capacity of community ownership and the school system is required for the efficient participation of the communities and schools in disease control. Finally, the support for drug discovery and development along with operational research is essential to fulfill gaps and improving the implementations of the control. For example, to understand some key issues like epidemiology, assessment of morbidity, efficacy testing of drugs, and drug resistance monitoring is of vital importance. Surveillance of STH infections, improvement in water supply, preventive chemotherapy, and health education may be helpful in minimizing these challenges and further spread of infection.⁶⁴

Strategies for control

The delegates of World Health Assembly crafted a resolution in 2001, which encouraged the member states to sustain and intensify their efforts for the elimination and control of STH and schistosomiasis infections. Furthermore, the Assembly also called the United Nations Organizations, Non Government Organizations, and bilateral agencies to strengthen their support to endemic countries for the control, prevention, and elimination of communicable diseases.

The control strategy of STH infection is geared towards controlling the morbidity throughout the series of deworming of people at risk in endemic areas. WHO considered the women of childbearing age (including breastfeeding women and women pregnant in the 2nd and 3rd trimesters), school and preschool age children, and adults involved in high risk occupations like teapickers, miners are vulnerable and at risk of STH infection.⁶⁵

There are three main interventions or strategies for the control of STH infection, such as treatment with anthelmintic drugs, health education, and improved sanitation.⁶⁶

Treatment with anthelmintic drug (also called deworming) aims in reducing the morbidity by decreasing the worm burden. Individuals residing in endemic areas and those without prior diagnosis are at risk and the recommended control strategy is the periodic medicinal treatment. WHO suggested once a year treatment administration in areas of more than 20% STH infection prevalence. However, twice a year treatment should be planned when the infection prevalence is more than 50%.⁶⁷

The control strategy for preschool and school age children can be integrated with school health programs or child health days for efficient treatment administration. Deworming activities allow easy provision in the propagation of hygiene and health education of public health programs like improving sanitation and promotion of hand washing practices in schools, thus proven to be advantageous and a good entry point. With such strategy, in 2013 more than 368 million school age children in endemic countries have been treated with anthelmintic medicines. Mebendazole (500 mg) and Albendazole (400 mg) are the WHO’s drug of choice used as medicines for STH infections and are affordable, effective, easily accessible, and safe. These medicines are made available to all endemic countries through their health ministries being donated by WHO.⁶⁵

In contrast to these, health education and improved sanitation aim to control the infection transmission and reinfection through reducing the water and soil contamination using the methods that usually cut across several sectors (e.g. infrastructure, health education, and veterinary services). These interventions have shown to be effective and sustainable.⁶⁸

Diagnostic tools for parasitic infections

Several diagnostic tools are available and being used for the investigation of parasitic infections particularly STHs. Since a single “gold standard” test (with 100% accuracy) does not exist for detection of intestinal parasites. Some diagnostic techniques are briefly described below.

Direct wet preparation

The examination of feces by direct microscopy through wet preparation is essential for the detection of parasitic elements like the motile larvae of Strongyloides stercoralis. It is also used for the detection of high concentration of eggs of helminthes infection with Ascaris lumbricoides. It is rapid and inexpensive but not often used in control programs because it is only semi-quantitative. However, lower sensitivity of the direct wet mount technique has been reported for the detection of low intensity infection.⁶⁹ It is routinely used for the detection of protozoan parasites like trophozoites of Giardia lamblia, Entamoeba histolytica, and rarely Balantidium coli. A thin smear of fecal sample is examined under light microscope for the detection of eggs/larvae/trophozoites of parasite species. An iodine or eosin preparation is also required to identify the oocytes/cysts of intestinal protozoa.^70,71 The wet mount method has been chosen for the routine diagnosis due to its easy use, cost effectiveness, and time saving compared to other techniques. The parasites detection rate using wet mount method in single stool examination is very limited because of its poor sensitivity.⁷²

Kato-Katz technique

This is most widely used technique to determine the prevalence and intensity of STHs infection, and is recommended for the quantification of STH eggs in stool of human by the WHO.⁷³ The advantages of this technique over the other copro-microscopic methods are high sensitivity, quantification of eggs and inexpensive with minimal infrastructure requirement.⁷⁴

Formol-ether concentration method

This method is commonly used for the diagnosis of STHs infection and is performed in specialized laboratories. The formol used in this method inactivates the organism, thus minimizing the risk of laboratory acquired infection from fecal pathogen. It is a rapid method.⁷⁵ This method is used for the diagnosis of STHs and intestinal protozoa. Its diagnostic sensitivity is greatly improved when used along with Kato-Katz method for helminthes infection.⁷⁶ Formol-ether concentration method (FEC) method is highly sensitive than Kato-Katz method to diagnose the soil transmitted helminthes and Taenia species infections.⁷⁷

Zinc sulfate flotation method

This method is used for concentrating the eggs of Trichuris trichiura but also cysts of Entamoeba histolytica and Giardia lamblia. The solution of zinc sulfate with specific gravity 1.180 to 1.200 is used in this technique. Fecal sample (1.0 gram) is emulsified in tap water followed by the removal of debris through straining. Then the sediment is suspended in ZnSO₄ solution (4 mL) following centrifugation. The cyst and eggs float on to the top after allowing the suspension for 30 to 45 min. The eggs/larvae are collected by placing the cover slip on the tube and then transferred to a glass slide for microscopic examination.⁷⁸ A published study revealed that zinc sulfate flotation technique is advantageous over the centrifugal sedimentation method to significantly detect the eggs of light helminths like those of E. vermicularis and T. trichiura in faeces.⁷⁸

Saturated sodium chloride flotation method

This is a very important and cost effective tool commonly practiced for concentrating the eggs of A. lumbricoides and Hook worm, particularly in field surveys. It works on the same principle as that of above described zinc sulfate technique but saturated solution of sodium chloride is used as flotation solution.⁷⁰

FLOTAC techniques

The use of FLOTAC technique in diagnosing STHs in humans have been suggested in recent studies and these techniques have been extensively used in the fields of veterinary sciences.⁷⁹ FLOTAC techniques are used for counting the helminthes egg in fecal sample both in veterinary and human.⁸⁰ It has the possibility of testing up to 83 days old preserved stool samples. It has less time consuming and takes only about 12 to 15 min from preparation to microscopic analysis for detecting helminthes eggs.⁸¹ This technique is highly sensitive, precise, accurate, and cost effective to diagnose the helminth and protozoan infections in animals and humans. However, centrifugation is required in this technique, and hence might be out of reach in resource limited settings. The modified FLOTAC technique called Mini-FLOTAC technique also has high sensitivity for the diagnosis of protozoan and helminth infections in animals and human fecal and urine sample.^82,83

Stoll’s dilution egg-counting method

Stoll’s technique is a rapid and inexpensive technique that offers the possibility of egg quantification. This technique uses 3 g of feces by making 15 times dilution with water in a screw cap container. In case of using formed stool sample, sodium hydroxide (0.1 mole/L) is recommended. The solution is homogenized in the screw capped container. With the help of Pasteur pipette, the suspension (0.15 mL) is transferred onto a glass slide and examined microscopically after placing cover slip over it. The number of eggs per gram feces is quantified by multiplying the eggs counted by 100.⁸⁴

Quantitative fecal examination through McMaster method

This technique is useful for the quantitative determination of nematode worm infection load which is expressed in eggs per gram feces. It is a fast method and can easily recover the debris free floating eggs that are loaded into a counting chamber.⁵⁹ Several studies evaluated the sensitivity of this method. McMaster was found to be more sensitive for the detection of STHs when compared to Kato-Katz technique providing more efficacy and accurate results.⁸⁵

Antigen detection methods

The so far described methods are based on detecting the parasitic elements (eggs/cysts/larvae) in stool sample. However, the use of enzyme linked immunosorbent (ELISA) assay capturing coproantigens has been reported in some published studies. The assay is based on the principle of assay is based on the principle of capturing the parasites excretory/secretory proteins by using specific polyclonal antibodies. Antigen detection methods are effective for the diagnosis of hookworm and S. stercoralis infections but have not been explored for the diagnosis of STH. However, immunological tests have been successfully used for the diagnosis of various protozoan and food borne helminth infections.^86,87

Polymerase chain reaction

Currently the use of molecular methods with higher diagnostic precision, such as PCR, is recommended for morphologically indistinguishable species.⁸⁸ PCR is a DNA based detection method with tremendous success in bacteriology and virology, and now efforts are made toward the use of PCR as the first line diagnostic tool for parasitic infection. PCR reactions involves the repetitive cycle of three steps including denaturation, annealing/primer binding, and amplification/extension by Taq DNA Polymerase.⁸⁹ Molecular methods have high sensitivities and specificities and require very small amount of DNA as the starting material and with the major requirement is to know the target DNA sequence in order to design the primers of interest for amplification. DNA damage in stool samples, contaminants amplification, requiring well trained personnel, and the unavailability of infrastructure in low resource settings are some of the drawbacks of molecular methods.⁹⁰ Studies have reported that both the qPCR and digital PCR have the ability to detect very small amount of A. lumbricoides with higher sensitivities. So, these techniques are very useful to control transmission in detecting STH infections particularly in low intensity infection settings. The multiplex qPCR method allows the simultaneous detection and quantification of several target DNA sequences. These methods use the combination of multiple primer sets for the amplification of DNA in real time. Several multiplex qPCR assays have been developed for STH infection detection in past decade.⁹¹ Global efforts toward the control and elimination of STH infection in LMIC urged the need of alternative diagnostic assays demonstrating the excellent reproducibility, consistency and high throughput⁹² or with high specificity and sensitivity but potentially inexpensive and can be used in limited resource settings.⁹³

Prevention of worms infection

Many species of the intestinal helminths can be killed by treating with Albendazole or Mebendazole.⁹⁴ This can only control the disease for a short time period in a target population and re infection occurs soon. For long term prevention, measures should be adopted to stop the transmission of intestinal worms.⁹⁵ Proper washing of hands with soaps after defecation and before feeding the children should be done. Properly cooked and washed vegetables should be used. Proper water supply and improving the sanitation is not possible in short time span so patient should adopt those measures which can be practiced on personal behalf to protect themselves from intestinal worm infection.⁹⁶ Soil-transmitted infections are among the most common infections causing abdominal pain, diarrhea, general malaise, weakness, and chronic intestinal blood loss. Literature review shows that the people with poor hygiene and the school going children are usually developing intestinal worms.⁹⁷ Intestinal worm infestation is a matter of great concern especially for third world countries because these countries are overcrowded with improper and poor sanitation system and water supply which are the root cause for this disease and favors the transmission of this infection.⁹⁸ Almost 27% of entire school and preschool age children in the world need treatment with anti-helminthic drugs.⁹⁹ Maintaining hygiene may decrease the rate of intestinal worms.¹⁰⁰ According to World Health Organization, all those areas in which this disease is endemic, their people must adopt preventive therapy.⁹⁴ If the risk of this infection is up to 20%, they should use preventive chemotherapy with anti-helminthic drug once a year and if the risk is more than 50%, they should adopt twice a year preventive chemotherapy.¹⁰⁰ This review makes a case for standard school-based plan to administer chemotherapy to decrease the burden of intestinal worm infestation in school going children in Pakistan.

Conclusion and suggestions

Accurate diagnosis of parasitic infections is crucial for both the management of individual patient and population based studies like trials for drug efficacy and the surveillance programs for the control and elimination of parasitic diseases in human public health. The decision made for the diagnosis is dependent on clinical laboratory testing in clinical medicine. Several available techniques for the detection of STH infections differ significantly in their sensitivity, field applicability, simplicity, and the cost but still no gold standard technique with 100% accuracy exist. This is the need of the hour to make efforts in developing a gold standard test for the diagnosis of parasitic infections. The detection and identification of protozoans and helminthes can be challenging in developing countries; however, the occurrence of parasitic infection and higher positivity rate provide the basis for the enhancement of expertise in developing countries. Artifacts in stool samples show close resemblance with parasites, particularly to an inexperienced microscopist in reporting such structures as parasites. A successful control of STHs infections across high endemic areas call to strengthen health system and interventions, to ensure the access of drugs against helminthes in all health services, co-implementation, and the synchronized use of different anthelminthic drugs, health education, adequate sanitation, promotion of access to safe water, and the resource mobilization to sustain control activities.

Footnotes

Acknowledgements

Authors are highly thankful to University of Sargodha, Government College University Faisalabad and the Higher Education Commission of Pakistan for providing the access to published literature in various data bases, and acknowledged their support.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Muhammad Riaz

Rida Zainab

Muhammad Irfan Ullah

Muhammad Akram

References

Liao

Kao

, et al. (2016) Prevalence of intestinal parasitic infections among school children in capital areas of the Democratic Republic of São Tomé and Príncipe, West Africa. African Health Sciences 16(3): 690–697.

Mohammadi

Hosseini-Safa

Ardakani

MJE

, et al. (2015) The relationship between intestinal parasites and some immune-mediated intestinal conditions. Gastroenterology and Hepatology from Bed to Bench 8(2): 123–131.

Siddiqui

(2017) An overview of parasitic infections of the gastro-intestinal tract in developed countries affecting immunocompromised individuals. Journal of Parasitic Diseases 41(3): 621–626.

Hart

(2018) How mammals stay healthy in nature: The evolution of behaviours to avoid parasites and pathogens. Philosophical Transactions of the Royal Society B: Biological Sciences 373: 20170205.

Anastasiou

Papathanasiou

Schepartz

, et al. (2018) Infectious disease in the ancient Aegean: Intestinal parasitic worms in the Neolithic to Roman period inhabitants of Kea, Greece. Journal of Archaeological Science: Reports 17: 860–864.

Lirio

GAC

Labana

Bernardo

IRA

, et al. (2018) Survey of intestinal parasites including associated risk factors among food vendors and slaughterhouse workers in Metro Manila, Philippines. KnE Social Sciences 3(6): 493–505.

Strunz

Addiss

Stocks

, et al. (2014) Water, sanitation, hygiene, and soil-transmitted helminth infection: A systematic review and meta-analysis. PLoS Medicine 11: e1001620.

Khadka

Maharjan

(2018) Pinworm (Enterobius vermicularis) infection among primary level government school children of Chhampi, Lalitpur District, Nepal. National Journal of Health Sciences 3(2): 46–50.

Kumar

Jain

(2014) A study of prevalence of intestinal worm infestation and efficacy of anthelminthic drugs. Medical Journal Armed Forces India 70: 144–148.

10.

Salam

Azam

(2017) Prevalence and distribution of soil-transmitted helminth infections in India. BMC Public Health 17(1): 201.

11.

Sah

Bhattarai

Yadav

, et al. (2013) A study of prevalence of intestinal parasites and associated risk factors among the school children of Itahari, Eastern Region of Nepal. Tropical Parasitology 3(2): 140–141.

12.

Tchakounté

Nkouayep

Poné

(2018) Soil contamination rate, prevalence, intensity of infection of Geohelminths and associated risk factors among residents in Bazou (West Cameroon). Ethiopian Journal of Health Sciences 28(1): 63–72.

13.

Nxasana

Baba

Bhat

, et al. (2013) Prevalence of intestinal parasites in primary school children of Mthatha, Eastern Cape Province, South Africa. Annals of Medical and Health Sciences Research 3: 511–516.

14.

Blitz

Riddle

Porter

(2018) The risk of chronic gastrointestinal disorders following acute infection with intestinal parasites. Frontiers in Microbiology 9: 17.

15.

Efstratiou

Ongerth

Karanis

(2017) Waterborne transmission of protozoan parasites: Review of worldwide outbreaks-an update 2011–2016. Water Research 114: 14–22.

16.

Erismann

Diagbouga

Schindler

, et al. (2017) School children’s intestinal parasite and nutritional status one year after complementary school garden, nutrition, water, sanitation, and hygiene interventions in Burkina Faso. The American Journal of Tropical Medicine and Hygiene 97(3): 904–913.

17.

Müller

Krawinkel

(2005) Malnutrition and health in developing countries. CMAJ: Canadian Medical Association Journal 173(3): 279–286.

18.

Lim

Brooker

Belizario

Jr , et al. (2018) Diagnostic tools for soil-transmitted helminths control and elimination programs: A pathway for diagnostic product development. San Francisco, CA: Public Library of Science.

19.

Garcia

Arrowood

Kokoskin

, et al. (2018) Laboratory diagnosis of parasites from the gastrointestinal tract. Clinical Microbiology Reviews 31(1): e00025.

20.

Organization

(2017) Guideline: Preventive Chemotherapy to Control Soil-Transmitted Helminth Infections in at-Risk Population Groups. Geneva: World Health Organization .

21.

Padmaja

Swaroop

Nageswararao

(2014) Prevalence of intestinal parasitic infections among school children in and around Amalapuram. Journal of Public Health Medical Research 2(2): 36–38.

22.

Helminthiases

St (2012) Eliminating soil-transmitted helminthiases as a public health problem in children: Progress report 2001–2010 and strategic plan 2011–2020. France: World Health Organization 1(1): 19–29.

23.

Gadisa

Jote

(2019) Prevalence and factors associated with intestinal parasitic infection among under-five children in and around Haro Dumal Town, Bale Zone, Ethiopia. BMC Pediatrics 19(1): 385.

24.

Bundy

Walson

Watkins

(2013) Worms, wisdom, and wealth: Why deworming can make economic sense. Trends in Parasitology 29: 142–148.

25.

Tchuente

(2011) Control of soil-transmitted helminths in sub-Saharan Africa: Diagnosis, drug efficacy concerns and challenges. Acta Tropica 120: S4–S11.

26.

Strunz

Suchdev

Addiss

(2016) Soil-transmitted helminthiasis and vitamin A deficiency: Two problems, one policy. Trends in Parasitology 32(1): 10–18.

27.

Saki

Khademvatan

Foroutan-Rad

, et al. (2017) Prevalence of intestinal parasitic infections in Haftkel County, southwest of Iran. International Journal of Infection Control 4(4): e15593.

28.

Doni

Gurses

Simsek

, et al. (2015) Prevalence and associated risk factors of intestial parasites among children of farm workers in the southeastern Anatolian region of Turkey. Annals of Agricultural and Environmental Medicine 22(3): 438–442.

29.

Pullan

Smith

Jasrasaria

, et al. (2014) Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasites & Vectors 7(1): 37.

30.

Ngwese

Manouana

Moure

PAN

, et al. (2020) Diagnostic techniques of soil-transmitted helminths: Impact on control measures. Tropical Medicine and Infectious Disease 5(2): 93.

31.

Haswell-Elkins

Elkins

Anderson

(1989) The influence of individual, social group and household factors on the distribution of Ascaris lumbricoides within a community and implications for control strategies. Parasitology 98: 125–134.

32.

Gilles

(1985) Selective primary health care: Strategies for control of disease in the developing world. XVII. Hookworm infection and anemia. Reviews of Infectious Diseases 7(1): 111–118.

33.

Bethony

Brooker

Albonico

, et al. (2006) Soil-transmitted helminth infections: Ascariasis, trichuriasis, and hookworm. The Lancet 367(9521): 1521–1532.

34.

Mazigo

Ambrose

Zinga

, et al. (2010) Prevalence of intestinal parasitic infections among patients attending Bugando Medical Centre in Mwanza, north-western Tanzania: A retrospective study. Tanzania Journal of Health Research 12(3): 178–182.

35.

Gelaw

Anagaw

Nigussie

, et al. (2013) Prevalence of intestinal parasitic infections and risk factors among schoolchildren at the University of Gondar Community School, Northwest Ethiopia: A cross-sectional study. BMC Public Health 13(1): 304.

36.

Cañete

Díaz

García

, et al. (2012) Intestinal parasites in children from a day care centre in Matanzas City, Cuba. PLoS One 7(12): e51394.

37.

Hegazy

Younis

Aminou

, et al. (2014) Prevalence of intestinal parasites and its impact on nutritional status among preschool children living in Damanhur City, El-Behera Governorate, Egypt. Journal of the Egyptian Society of Parasitology 44(2): 517–524.

38.

Wegayehu

Tsalla

Seifu

, et al. (2013) Prevalence of intestinal parasitic infections among highland and lowland dwellers in Gamo area, South Ethiopia. BMC Public Health 13(1): 151.

39.

Asrat

Tewodros

Alemayehu

(2011) Prevalence and risk factors of IPs among Delgi school children, northern Gonder, Ethiopia. Journal of Parasitology and Vector Biology 3: 75–81.

40.

Ayalew

Debebe

Worku

(2011) Prevalence and risk factors of intestinal parasites among Delgi school children, North Gondar, Ethiopia. Journal of Parasitology and Vector Biology 3: 75–81.

41.

Hailu

(2014) Current prevalence of intestinal parasites emphasis on hookworm and schistosoma mansoni infections among patients at Workemeda Health Center, Northwest Ethiopia. Clinical Microbiology: Open Access 3(4): 1–4.

42.

Wani

Ahmad

(2009) Intestinal helminths and associated risk factors in children of district Pulwama, Kashmir, India. Indian Journal of Medical Microbiology 27(1): 81–82.

43.

Rayan

Verghese

McDonnell

(2010) Geographical location and age affects the incidence of parasitic infestations in school children. Indian Journal of Pathology and Microbiology 53(3): 498–502.

44.

Wani

Ahmad

Zargar

, et al. (2007) Prevalence of intestinal parasites and associated risk factors among schoolchildren in Srinagar City, Kashmir, India. Journal of Parasitology 93(6): 1541–1543.

45.

Al-Taie

(2009) Prevalence of intestinal parasitic infection in Baghdad city. Journal of the Faculty of Medicine 51(2): 187–191.

46.

Mwanthi

Kinoti

Wamae

, et al. (2008) Prevalence of intestinal worm infections among primary school children in Nairobi City, Kenya. East African Journal of Public Health 5(2): 86–89.

47.

Chandrashekhar

Joshi

Gurung

, et al. (2005) Prevalence and Distribution of Intestinal Parasitic Infestations Among School Children in Kaski District, Western Nepal. Journal of Biomedical Sciences 4(1): 78–82.

48.

Awolaju

Morenikeji

(2009) Prevalence and intensity of intestinal parasites in five communities in south-west Nigeria. African Journal of Biotechnology 8(18): 4542–4546.

49.

Chaudhry

Afzal

Malik

(2004) Epidemiological factors affecting prevalence of intestinal parasites in children of Muzaffarabad district. Pakistan Journal of Zoology 36(4): 267–271.

50.

Khan

Jehan

Akram

, et al. (2012) Prevalence of intestinal protozoan and worm infestation in primary school going children of 5-10 years of age in district Bannu. Annals of Pakistan Institute of Medical Sciences 8: 243–248.

51.

Ullah

Sarwar

Aziz

, et al. (2009) Intestinal worm infestation in primary school children in rural Peshawar. Gomal Journal of Medical Sciences 7(2): 132–136.

52.

Ahsan-ul-Wadood

Rhman

Qasim

(2005) Frequency of intestinal parasite infestation in Children Hospital Quetta. Pakistan Journal of Medical Research 44(2): 87–88.

53.

Shaikh

Begum

Hussain

, et al. (2009) Prevalence of intestinal protozoan and helminth parasites in Sukkur, Sindh. Sindh University Research Journal-SURJ (Science Series) 41(2): 53–58.

54.

Gabbad

Elawad

(2014) Prevalence of intestinal parasite infection in primary school children in Elengaz area, Khartoum, Sudan. Academic Research International 5(2): 86.

55.

Dash

Al-Zarouni

Anwar

, et al. (2010) Prevalence of intestinal parasitic infections in Sharjah, United Arab Emirates. Human Parasitic Diseases 2: 21–24.

56.

Verle

Kongs

, et al. (2003) Prevalence of intestinal parasitic infections in northern Vietnam. Tropical Medicine & International Health 8(10): 961–964.

57.

Mehraj

Hatcher

Akhtar

, et al. (2008) Prevalence and factors associated with intestinal parasitic infection among children in an urban slum of Karachi. PLoS One 3(11): e3680.

58.

Tomono

Anantaphruti

Jongsuksuntigul

, et al. (2003) Risk factors of helminthiases among schoolchildren in southern Thailand. Southeast Asian Journal of Tropical Medicine and Public Health 34: 264–268.

59.

Organization

(2019) Bench Aids for the Diagnosis of Intestinal Parasites. Geneve: World Health Organization.

60.

Novianty

Dimyati

Pasaribu

, et al. (2018) Risk factors for soil-transmitted helminthiasis in preschool children living in farmland, North Sumatera, Indonesia. Journal of Tropical Medicine 2018: 1–6.

61.

Fobi

Yameogo

Noma

, et al. (2015) Managing the fight against onchocerciasis in Africa: APOC experience. PLoS Neglected Tropical Diseases 9(5): e0003542.

62.

Kiani

Haghighi

Rostami

, et al. (2016) Prevalence, risk factors and symptoms associated to intestinal parasite infections among patients with gastrointestinal disorders in Nahavand, Western Iran. Revista do Instituto de Medicina Tropical de São Paulo 58: 42–46.

63.

Khan

Pullan

Okello

, et al. (2019) “For how long are we going to take the tablets?” Kenyan stakeholders’ views on priority investments to sustainably tackle soil-transmitted helminths. Social Science & Medicine 228: 51–59.

64.

Tian

Luo

Zhong

, et al. (2019) Challenges in the control of soil-transmitted helminthiasis in Sichuan, Western China. Acta Tropica 199: 105132.

65.

Organization

(2013) Schistosomiasis: Progress Report 2001-2011, Strategic Plan 2012-2020. Geneva, Switzerland: World Health Organization.

66.

Hotez

Bundy

Beegle

, et al. (2006) Helminth infections: Soil-transmitted helminth infections and schistosomiasis. In: Disease Control Priorities in Developing Countries 2nd edition. Washington, DC: The International Bank for Reconstruction and Development/The World Bank.

67.

Organization

(2006) Preventive Chemotherapy in Human Helminthiasis. Coordinated Use of Anthelminthic Drugs in Control Interventions: A Manual for Health Professionals and Programme Managers. Geneva: World Health Organization.

68.

Mascie-Taylor

Alam

Montanari

, et al. (1999) A study of the cost effectiveness of selective health interventions for the control of intestinal parasites in rural Bangladesh. The Journal of Parasitology 85(1): 6–11.

69.

Levecke

De Wilde

Vandenhoute

, et al. (2009) Field validity and feasibility of four techniques for the detection of Trichuris in simians: A model for monitoring drug efficacy in public health? PLoS Neglected Tropical Diseases 3(1): e366.

70.

Cheesbrough

(2006) District Laboratory Practice in Tropical Countries, Part 2. Cambridge, UK: Cambridge university press.

71.

Ndao

(2009) Diagnosis of parasitic diseases: Old and new approaches. Interdisciplinary Perspectives on Infectious Diseases 2009: 278246.

72.

Yimer

Hailu

Mulu

, et al. (2015) Evaluation performance of diagnostic methods of intestinal parasitosis in school age children in Ethiopia. BMC Research Notes 8: 1–5.

73.

Organization

(2002) Prevention and Control of Schistosomiasis and Soil-Transmitted Helminthiasis: Report of a WHO expert committee. Geneva, Switzerland: World Health Organization.

74.

Katz

Chaves

Pellegrino

(1972) A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni. Revista do Instituto de Medicina Tropical de Sao Paulo 14: 397–400.

75.

Richardson

Gross

Smith

(2008) Comparison of Kato–Katz Direct Smear and Sodium Nitrate Flotation for Detection of Geohelminth Infections. Comparative Parasitology 75: 339–341.

76.

Steinmann

Utzinger

Z-W

, et al. (2010) Multiparasitism: A neglected reality on global, regional and local scale. Advances in Parasitology 73: 21–50.

77.

Santos

FLN

Cerqueira

Soares

(2005) Comparison of the thick smear and Kato-Katz techniques for diagnosis of intestinal helminth infections. Revista da Sociedade Brasileira de Medicina Tropical 38(2): 196–198.

78.

Inês

EdJ

Figueiredo Pacheco

Carneiro Pinto

, et al. (2016) Concordance between the zinc sulphate flotation and centrifugal sedimentation methods for the diagnosis of intestinal parasites. Biomédica 36(4): 519–524.

79.

Knopp

Glinz

Rinaldi

, et al. (2009) FLOTAC: A promising technique for detecting helminth eggs in human faeces. Transactions of the Royal Society of Tropical Medicine and Hygiene 103(12): 1190–1194.

80.

Cringoli

Rinaldi

Maurelli

, et al. (2010) FLOTAC: New multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. Nature Protocols 5(3): 503–515.

81.

Glinz

Silué

Knopp

, et al. (2010) Comparing diagnostic accuracy of Kato-Katz, Koga agar plate, ether-concentration, and FLOTAC for Schistosoma mansoni and soil-transmitted helminths. PLoS Neglected Tropical Diseases 4(7): e754.

82.

Cringoli

Maurelli

Levecke

, et al. (2017) The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals. Nature Protocols 12(9): 1723.

83.

Barda

Rinaldi

Ianniello

, et al. (2013) Mini-FLOTAC, an innovative direct diagnostic technique for intestinal parasitic infections: Experience from the field. PLoS Neglected Tropical Diseases 7(8): e2344.

84.

Singh

Chanu

Dutta

(2018) Comparative evaluation of harada–Mori and agar plate culture for the identification of hookworm species under limited resources. Journal of Natural Science, Biology and Medicine 9(2): 127.

85.

Levecke

Behnke

Ajjampur

, et al. (2011). A comparison of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz thick smear methods for soil-transmitted helminths. PLoS Neglected Tropical Diseases 5(6): e1201.

86.

Sykes

McCarthy

(2011) A coproantigen diagnostic test for Strongyloides infection. PLoS Neglected Tropical Diseases 5(2): e955.

87.

Rehman

Ullah

Khan

, et al. (2020) Glutathione-S-transferase: An important diagnostic antigen of liver amphistome Gigantocotyle explanatum, infecting the Indian water buffalo? Acta Tropica 205: 105400.

88.

Soares

Benitez

AdN

Santos

BMd

, et al. (2020) A historical review of the techniques of recovery of parasites for their detection in human stools. Revista da Sociedade Brasileira de Medicina Tropical 53: 1–9.

89.

Verweij

Brienen

Ziem

, et al. (2007) Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. The American Journal of Tropical Medicine and Hygiene 77(4): 685–690.

90.

Lorenz

(2012) Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments 63: e3998.

91.

Mejia

Vicuña

Broncano

, et al. (2013) A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. The American Journal of Tropical Medicine and Hygiene 88(6): 1041–1047.

92.

Cunningham

Stothard

Osei-Atweneboana

, et al. (2018) Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: A pilot study in detection and discrimination of soil-transmitted helminth and schistosome species. Parasitology 145(13): 1733–1738.

93.

Stracke

Clarke

Awburn

, et al. (2019) Development and validation of a multiplexed-tandem qPCR tool for diagnostics of human soil-transmitted helminth infections. PLoS Neglected Tropical Diseases 13(6): e0007363.

94.

Albonico

Smith

Ercole

, et al. (1995) Rate of reinfection with intestinal nematodes after treatment of children with mebendazole or albendazole in a highly endemic area. Transactions of the Royal Society of Tropical Medicine and Hygiene 89(5): 538–541.

95.

Bath

Eneh

Bakken

, et al. (2010) Combating evolving pathogens: The impact of perception and knowledge on the treatment and prevention of intestinal worms in the Manikganj district of Bangladesh. The Yale Journal of Biology and Medicine 83(4): 171–184.

96.

Mascie-Taylor

Karim

, et al. (2003) The cost-effectiveness of health education in improving knowledge and awareness about intestinal parasites in rural Bangladesh. Economics & Human Biology 1(3): 321–330.

97.

Walker

Wachs

Gardner

, et al. (2007) Child development: Risk factors for adverse outcomes in developing countries. The Lancet 369(9556): 145–157.

98.

Pokhrel

Viraraghavan

(2004) Diarrhoeal diseases in Nepal vis-à-vis water supply and sanitation status. Journal of Water and Health 2(2): 71–81.

99.

Taylor-Robinson

Maayan

Soares-Weiser

, et al. (2015) Deworming drugs for soil-transmitted intestinal worms in children: Effects on nutritional indicators, haemoglobin, and school performance. Cochrane Database of Systematic Reviews 2015(7): CD000371.

100.

Savioli

Bundy

Tomkins

(1992) Intestinal parasitic infections: A soluble public health problem. Transactions of the Royal Society of Tropical Medicine and Hygiene 86(4): 353–354.