The Epidemiology of Enterobiasis and Molecular Characterization of Enterobius vermicularis in Children of Karachi, Sindh, Pakistan

Syeda Batool Zehra1, Abdullah G. Arijo2, Aly Khan3, Nasira Khatoon1* and Samina Waheed1

1Department of Zoology, University of Karachi, Karachi-75270, Pakistan; 2Department of Veterinary Parasitology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, Pakistan; 3CDRI, Pakistan Agricultural Research Council, University of Karachi, Karachi-75270, Pakistan.

Received | September 06, 2022; Accepted | October 12, 2022; Published | October 31, 2022

*Correspondence | Nasira Khatoon, Department of Zoology, University of Karachi, Karachi-75270, Pakistan; Email: [email protected]

Citation | Zehra, S.B., Arijo, A.G., Khan, A., Khatoon, N., and Waheed, S., 2022. The epidemiology of enterobiasis and molecular characterization of Enterobius vermicularis in children of Karachi, Sindh, Pakistan. Pakistan Journal of Nematology, 40(2): 86-91.

DOI | https://dx.doi.org/10.17582/journal.pjn/2022/40.2.86.91

Keywords | Epidemiology, Enterobiasis, Enterobius vermicularis, Molecular characterization, Oxyuridae

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Pinworms (Enterobius vermicularis) of the family Oxyuridae cause oxyuriasis/ enteterobiasis. The worm has been known since antiquity and Linnaeus was the first to describe it in 1758. Pinworms are common in birds, mammals, reptiles and amphibians but they are rare in fish and absent in dogs and cats, which can take up pinworm eggs from the environment and infect humans through their fur. This microscopic nematode is found all across the world especially in children. It is the most common parasitic infection in the world. Its entire life cycle was also described in detail (Leuckart, 1865).

E. vermicularis is one of the most common parasitic helminth in humans. It was estimated that over 209 million people are sick around the world, with children aged 5 to 10 making up more than a 30% of those affected (Cook, 1994; Kucik et al., 2004; Fan et al., 2019; Rawla and Sharma, 2021). Despite the fact that enterobiasis is unrelated to socioeconomic class, race, or culture, it is facilitated by factors such as poor personal or group hygiene as well as overcrowding (in preschools, schools, orphanages, and family groups) (Cook, 1994; St Georgiev, 2001; Burkhart and Burkhart, 2005). Pinworm eggs are transmitted from person to person in these conditions, either directly through the anus-to-mouth pathway, by finger contamination, or indirectly through contaminated objects (toys, food) (Cook, 1994; Vermund and Wilson, 2000). Majority of E. vermicularis infections are asymptomatic or produce ambiguous symptoms such as perianal pruritus, which leads to local epidermal irritation and bacterial infections. Other symptoms include abdominal discomfort, loss of appetite, weight loss, sleepiness, restlessness and irritability (Burkhart and Burkhart, 2005; Vermund and Wilson, 2000). Pinworm infection can cause enuresis (Çulha and Duran, 2006; Otu-Bassey et al., 2011) however it is rare. In the present study, the research findings were based on two primary parameters, molecular and statistical analyses, to be specific. Statistical analysis of obtained data based on age, gender, pinworm count, and infection severity. According to current research on pinworm, it is the most common condition in girls as compared to boys. Molecular research revealed that the specimens recovered during this study were strikingly comparable to previously collected E. vermicularis isolates from around the world. The present study has determined the severity of infection in the general public in order to design safe prophylactic strategies to protect individuals against pinworm also demonstrates that cleanliness and personal hygiene are major protective factors.

Materials and Methods

Samples were collected from the laboratory of Jinnah Hospital, Karachi, Sindh, Pakistan. From the recorded data, 60 children were found positive for pinworm-infection. These children were of various sexes and ages. Traditional pinworm diagnosis, PCR for pinworm detection has been performed. The findings in this research are based on two key factors: Molecular analysis and statistical analysis. When used with traditional morphological techniques, molecular biology technologies have proven to be effective in distinguishing closely related species according to the method of Zelck et al. (2011).

Pinworms were found in toddler excrement that had been magnified, cleaned in tap water, and kept at 20°C. Then, to extract DNA, pinworms from 37 kids who lived in various parts of Germany were thawed and mechanically homogenised (Qiamp DNA minikit, Qiagen, Hilden, Germany). Using universal primers, the ribosomal DNA (5S rDNA) of E. vermicularis was amplified and sequenced as partly overlapping fragments of %S. Each primer was 100 nM, and each deoxynucleoside triphosphate (dNTP) was 50 M. The amplification reaction mixtures (50 µl) also contained 2.5 mM MgCl2, 0.5 units of polymerase, and 10 µl of template. The following steps were used in the PCR amplification process: (i) denaturation at 95°C for 5 min; (ii) 40 cycles, with each cycle consisting of 1 cycle that lasted 60 s at 94°C, 60 s at 50 to 60°C, and 2 min at 72°C for 2 min; and (iii) a final extension step lasting 10 min at 72°C. Under otherwise comparable circumstances, 5S rDNA fragments were amplified for diagnostic purposes at 55°C using the Enterobius-specific primers Ev18S.F1 and Ev18S.R1. On agarose gels stained with ethidium bromide, products were found. PCR products were sequenced using a BigDye terminator cycle sequencing kit and an ABI Prism 310 genetic analyzer either directly or after gel extraction (QIAquick gel extraction kit; Qiagen, Hilden, Germany) and cloning (TOPO-TA; Invitrogen, Karlsruhe, Germany) (Applied Biosystems, Warrington, United Kingdom). When the heights of the current alternative nucleotide peaks were equal, or when a minor peak greatly outperformed the background level and made up 50% of the primary peak, site polymorphisms were assessed.

Statistical analysis is used to determine the intensity of infection in relation to age and gender. The core data, which included 60 children, was entered into a Microsoft Excel spreadsheet, for constructing graphs, whereas chi-square test calculated by using the statistical program Statistical Package for Social Students (SPSS version 18.0).

Results and Discussion

Molecular analysis

The total gDNA from individual pinworms (14 specimens) was separately extracted and the PCR amplification was performed using the extracted gDNA of the pinworms with primers and PCR conditions as specified. By utilizing the Nanodrop ND-200 instrument to measure the gDNA content in E. vermicularis samples, 260/280 absorbance measurement peaks were calculated for the accuracy of gDNA concentrations, the amount of DNA was represented as ng/µl Figure 2. The first round of PCR yielded the 122 bp band on agarose gel for some specimens (EV1, EV2, EV3, EV7, EV8, EV10, EV11, EV12, EV13 and EV14) and also in the second round of PCR using the same primers and PCR outcomes of the first round of PCR as a template for DNA reaction yielded the 122 bp band for all specimens (Figure 3A and B).

 

 

 

The PCR products were purified as described and sequenced (4 specimens) by using 5S rRNA gene both forward and reverse primers for E. vermicularis. The results indicate that all the sequences aligned which belong to the same species with 100% similarity (Figure 4) whereas the sequences aligned was also estimated for E. vermicularis with other related species (Figure 5).

 

 

The 5S rRNA gene fragment of E. vermicularis was amplified and sequenced and the DNA sequences obtained were compared for similarity check. In the MEGA 6.0 programme, the phylogenetic tree was built using nucleotide sequences from various helminths, with partial deletion for gaps and missing data, Poisson correction for nucleotide substitutions, and uniform rates among sites, were bootstrapped 10,000 times with bootstrap values of branches shown in percentages. Results indicate that the specimens collected during the present study were highly similar with already studied isolates from different regions of the world. Furthermore, the phylogenetic tree analysis shows that the E. vermicularis falls in a separate clade from other pinworms (Syphacia obvelata, Syphacia muris and Aspiculuris tetraptera) indicating that the E. vermicularis is genetically distinct from other pinworms (Figure 6).

 

Statistical analysis

Statistical analysis of obtained data based on age, gender, pinworm count, and infection severity. According to present research on pinworm, it is the most common condition amongst girls as compared to boys during the years 2019 to 2020, with the help of the frequency distribution of children by gender. The prevalence was estimated using percentages. Table 1 and Figure 1 are showing mean intensity of pinworms in children by gender. Figures depict that, the mean intensity in boys than in girls. However, the association between gender and number of pinworms was calculated by chi-square test of value 12.87. Since, the p<0.05 which is statistically significant showing that there was an association between gender and number of pinworms.

 

Table 1: Prevalence of pinworms in children by Gender During 2019 to 2020.

Gender	Pinworms	Infected	Mean intensity	Chi-square	P value
Boy	43	21	2.04	12.87	0.045
Girl	129	39	3.30

 

E. vermicularis commonly known as pinworm (Ridley, 2012), has a worldwide distribution and is one of the most prevalent helminth infections in children in the developed world (Cook and Zumla, 2008). Over one billion people are infected worldwide with a significant prevalence among children aged 5 to 6. Adults were less likely to be affected, implying that resistance develops with age, may be due to acquired immunity. Depending on age and race, infection rates increased up to 40% (Bowman and Lynn, 2009). Infection occurs when eggs were consumed or inhaled.

The small-subunit ribosomal DNA sequences were used to construct the molecular phylogeny of Nematoda where E. vermicularis had been included Nadler et al. (2007). Thus, presently 5S rDNA has been analyzed and compared for sequence together with other nematode sequences used in previous studies. The prevalence of enterobiasis is thought to be lower in the tropics (WHO, 1981), although this finding appears to be based on weak survey methodology (Haswell-Elkins et al., 1987). The prevalence of enterobiasis has shown to diminish with increasing age in various investigations (Hayashi et al., 1959; Rahman, 1991), implying that it is mostly a childhood infection. However, in surveys conducted in India, the illness was found to be prevalent across all age categories (Haswell-Elkins et al., 1987). School students in a shantytown in Lima, Peru, had a prevalence of 42 percent (Gilman et al., 1991), which was equivalent to the 40 percent rate recorded in a similar population in Indonesia (Norhayati et al., 1994). Enterobiasis is much less common in blacks than it is in whites; the explanation for this disparity is unknown (Cram, 1943; Cherubin and Shookhoff, 1963). The incidence of E. vermicularis was 55 percent in an orthopaedic unit of a children’s hospital in Liverpool, England (Ashford et al., 1988). Extrapolating these figures to the entire country, at least 4.5 million children in the United States are infected with enterobiasis (Wagner and Eby, 1983). It was reported that, in tropical countries the percentage of incidence in children is surprisingly low as compared with the abundance of the other helminths. In warm countries rural life, scant loose clothing, sunshine dry heat, bathing facilities and toilet habits may provide conditions unfavorable for the existence of ova and transmission of infection. In cold countries crowded association in school and homes, lack of bathing and wearing of soiled clothing favor the transmission of the disease (Cram, 1941).

Random amplified polymorphic DNA (RAPD)-based assays based on polymerase chain reaction (PCR) have been successfully employed for the rapid and simple detection of genomic differences among parasites. The use of PCR provides a complementary strategy for parasite identification, and the genetic variability of various species can be examined using the RAPD technique, which generates an aenornic fingerprint. Although there is a lot of material in the literature regarding employing molecular approaches for nematode identification and phylogenic analysis, there are just a few studies on pinworms. Some of these research used DNA sequence analysis to uncover pinworm evolutionary links (Nakano et al., 2006; Okamoto et al., 2007). A high-fidelity PCR was utilized in another work to simplify a major section of the ribosomal gene complex of S. obvelata, S. inuris, A. tetraptera, and Passalurus ambiguus collected from laboratory rodents and rabbits (Feldman and Bowman, 2007).

Conclusions and Recommendations

The oxyurides produce health problems under unhygienic conditions especially in children. It is imperative that the general public is made aware of the hazards of oxyurides and also of the methods of developing and maintaining hygienic conditions. Medication is the main medical therapy for pinworm infections but keeping the house clean and practicing proper hygiene is also vital throughout therapy.

Novelty Statement

The specimens of present study indicated high similarity with already collected isolates of E. vermicularis from different regions of the world but phylogenetic tree analysis shows it is genetically distinct and falls in a separate clade form other pinworms (Syphacia obvelata, Syphacia muris and Aspiculuris tetraptera).

Author’s Contribution

Syeda Batool Zehra: Collected the samples.

Abdullah G. Arijo: Did molecular analysis.

Aly Khan: Did statistical analysis.

Nasira Khatoon: Provided literature.

Samina Waheed: Prepared the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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