COCCIDIAN INTESTINAL PARASITES AMONG CHILDREN IN AL-TORBAH CITY IN YEMEN: IN COUNTRY WITH HIGH INCIDENCE OF MALNUTRITION

Emad Najeeb Ali Shamsan¹, CAO De-ping², Hassan A. Al-Shamahy³, Manal Mutaher Ali Al- Hajj⁴, JIANG Bo-fan⁵, Zhang Yaogang⁶

¹Immunology and Parasitology, Faculty of Medicine, Qinghai University, China.

²Medical Microbiology, Faculty of Medicine, Qinghai University, China.

³Medical Microbiology and Clinical Immunology, Faculty of Medicine and Health Sciences, Sana’a University, Yemen.

⁴Biology Department, Faculty of Sciences, Sana'a University, Republic of Yemen.

⁵Pathogen Biology, Faculty of Medicine, Qinghai University, China.

⁶Pathogen Biology, Faculty of Medicine, Qinghai University, China.

DOI: https://doi.org/10.22270/ujpr.v4i4.301

ABSTRACT

Diarrhoea is an important cause of malnutrition, morbidity and mortality among children in Yemen. Coccidian parasitic infections are an important cause of diarrhea in children particularly malnutrition and immune-compromised patients, but their investigations are rarely required by the treating physicians in apparently immunocompetent children. This study was aimed to find the prevalence of intestinal coccidian parasites in country with high incidence rate of malnutrition. Between May 2016 and October 2016, 228 fecal samples from 228 selected school children in Al Turbah city, Taiz governorate, Yemen, aged between 6 and 15 years were examined using wet-mount preparations and formal concentration method then films stained by modified acid-fast staining. Also data of children were collected including demographic data, and sources of water. Findings of positive intestinal coccidian parasites were analyzed in relation with demographic data, and sources of water.  The prevalence of Cryptosporidium species, Cyclospora species and Isospora belli were 75.9%, 45.6% and 1.75% respectively. There was significant association between positive of Cryptosporidium species and females (OR= 2.1 times, P=0.01), and spring water source (OR=4 times, P=0.04), while there was no significant association between positive of Cryptosporidium species and others factors studied. Also there was no significant association between positive of Cyclospora species and Isospora belli and children sex, age groups, or different sources of water. In conclusion the study highlights the high prevalence of coccidian parasites among immunocompetent school children in Yemen. The clinicians in Yemen need to be aware that coccidian parasites are a potential cause of childhood diarrhea even in immunocompetent children.

Keywords: Children, Coccidian, Cryptosporidium, Cyclospora, Isospora, Yemen.

INTRODUCTION

Coccidian is a primary microscopic parasite that infects the intestinal system of most human and animal organisms. These organisms are one of the main concerns of doctors, especially with increasing the rate of HIV. Coccidian parasites (Cryptosporidium spp., Isospora belli, and Cyclospora spp.) are the most common intestinal parasites in immunocompromised patients that can usually lead to fatal acute diarrhea while causing moderate and limited gastrointestinal disorders in individuals with a natural immune system^1,2,3.  Cryptosporidium and Isospora have been identified as important and widespread causes of diarrheal disease in both immunocompromised individuals and those with immunodeficiency causes such as acute malnutrition as well as people who move from a clean environment to a contaminated environment such as tourists so it has been described as traveler disease^4,5.  Acute or chronic diarrhea syndromes caused by these parasites are usually accompanied by weight loss, dehydration, abdominal pain and malabsorption syndrome in immunocompromised patients⁶. Chronic diarrhea in malnourished children can also increase the incidence of these parasites and mortality in these patients⁶. Coccidiosis diarrhea has been observed with fluid loss of 25l/day in infected patients, which can last for weeks in immunocompromised patients⁷.

Isospora belli is one of the opportunistic coccidian parasites that affects HIV⁺/AIDS patients, especially in developing countries of Africa, Asia, and Latin America with low levels of hygiene. It is always considered as a neglected parasite and there is lack of enough investigation, particularly in immune-compromised patients, so the previous factors lead to underestimate this infection⁸. Various risk factors for coccidian parasites such as use of contaminated drinking water, exposure to animals, lack of sewage, poverty, etc., have been reported to be associated with coccidian gastrointestinal infections⁹.  In Yemen, the prevalence of coccidian parasites has not been studied, not among immunocompromised patients as well as children with diarrhea or malnutrition in which the malnutrition rate among children in Yemen raised due to the Saudi-Emirati aggression against Yemen¹⁰.  Therefore, it may not be out of context to suggest that identification of these so-called opportunistic pathogens should be an essential part of investigations for childhood diarrhea, particularly with the rise of malnutrition rate among children in Yemen which affect the immune status of the children. With this aim, this study was conducted to find the prevalence of intestinal coccidian parasites among children in country with high incidence of malnutrition and determine some risk factors associated with this infection in Al Turbah city, Taiz governorate, Yemen.

SUBJECTS AND METHODS

The study conducted in the Faculty of Sciences, Taiz University, Taiz city, Yemen. Between May 2016 and October 2016, 228 fecal samples from 228 selected school children in Al Turbah city, Taiz governorate, aged between 6 and 15 years were examined. As a standard protocol, after receiving the samples in the laboratory, stool samples were processed and wet-mount preparations with both saline and iodine were prepared and screened within 2 h of sample collection to look for motile Trophozoites, larvae, ova, and cyst. Stool samples were also processed by the formalin-ether concentration technique and reexamined with saline and iodine wet-mount preparations as well as stained with Modified acid-fast stain³ to look for Cryptosporidium, Cyclospora and Isospora oocysts. Each wet-mount preparation and stained fecal smears were examined by a clinical microbiologist and the researcher (ENAS) independently and findings were cross-checked. After those children demographic data and sources of water were collected in standard questionnaire. Next, findings of positive intestinal coccidian parasites were analyzed in relation with demographic data, and sources of water.

RESULTS

The study results illustrated in Table 1 to Table 7. The prevalence of Cryptosporidium spp, Cyclospora spp and Isospora belli were 75.9%, 45.6% and 1.75% respectively. There was significant association between positive of Cryptosporidium species and females (OR= 2.1 times, CI=1.1-3.9, P=0.01), and spring water source (OR=4 times, CI=1.0-17 P=0.04), while there was no significant association between positive of Cryptosporidium spp and others factors studied. Also there was no significant association between positive of Cyclospora spp, Isospora belli and children sex, age groups, or different sources of water.

DISCUSSION

Cryptosporidium, Isospora and Cyclospora have become increasingly prevalent in patients with immune deficiency and normal immunity people. Humans can infect Coccidian infections through fecal-oral route, through direct person-to-person or animal-to-person contact in addition to consuming contaminated water or food¹¹ while no animal reservoir for human Isospora has been identified¹². In the current study the prevalence of Cryptosporidium spp was 75.9%, while Cyclospora spp was next commonest coccidian pathogen (45.6%).  The results of this study are higher than that reported in general population of developing and developed countries in which the rate of Cryptosporidium oocysts was recorded from 6.1 and 2.1%, respectively¹³. Also the current study rate of Cryptosporidium spp (75.9%) was even higher than the prevalence rates of Cryptosporidiosis among HIV⁺/AIDS diarrheic patients which ranged from 10% to 33.4%^13,14 or among diarrheic children with normal immunity (7%)¹⁵.  In the current study, there was significant association between positive of Cryptosporidium species and females (OR= 2.1 times, CI=1.1-3.9, P=0.01) (Table 2). The current result is different from other studies carried out in developed and developing countries in which the rate of  Cryptosporidium species infections are roughly equal in both sexes^13,15,16.

Although animals are known to be the potential source of Cryptosporidiosis, aquatic sources are also known as one of the major sources of Cryptosporidium.¹³ In the current study, there was a significant correlation between positive Cryptosporidium species and the source of spring water (OR = 4 times, CI = 1.0-17, P = 0.04) (Table 3). This association can be explained by that Cryptosporidium spp. which can be found in surface water and groundwater resources through fecal contamination, which can affect drinking water resources¹⁷. Interestingly, Cryptosporidium oocysts are able to pass through the water treatment process because of their resistance to routine disinfectants and their small size¹⁸.

The prevalence of Isospora spp in the current study was very low (1.75%) comparing with high rates of Cryptosporidium spp (75.9%) and Cyclospora spp (45.6%). Although, Isosporiasis has worldwide distribution especially in tropical and subtropical regions, but there are rare reports of this infection¹⁹. Isospora belli is considered as an opportunistic infection in immunocompromised individuals, mainly AIDS patients, all over the world. Moreover, Isosporiasis has been reported as the most prevalent intestinal parasitic disease among AIDS patients^20,21.  Assis and colleagues reported the frequency rate 10.1% and 6.7% in HIV-positive patients for Cryptosporidium spp and Isospora belli, respectively²². In another study, the prevalence rate of Isosporiasis in Nigeria was reported 3.1% in HIV-positive patients while no Isospora infection was observed in the healthy controls¹⁹.  As well, the current study finding is in agreement with other studies that have suggested low prevalence rate of Isosporiasis in immunocompetent or immunocompromised patients. However, treatment of Isosporiasis usually is successful in all types of cases but recurrence cases are common²⁰.  It is known that detection of Isospora in direct examination of stool samples in most of laboratories is unusual. Alternatively, cases of Isosporiasis has being raised up together with increase of HIV-infected subjects that can increase gastrointestinal complications in immunocompromised patients. Isosporiasis is generally transmitted through ingestion of sporulated oocysts from contaminated food and water¹². Although some cases of homosexuality have been reported to have Isosporiasis more than other individuals²³, but because of the fact that Isospora oocysts require to mature and become infectious in the environment, direct contact with faeces is unlikely to be the usual course of transmission²⁴. Therefore, sanitation for water and food is very important in prevention programs. However, as explained, although reports of Isosporiasis cases are low, but this infection should be considered a neglected disease in Yemen, especially in people with immune disorders. The results of this study highlight the fact that coccidian parasites should not be overlooked by the clinical microbiologists, while investigating cases of diarrhea, even if these are not requested by the physician.

The fact that 75.9% of Cryptosporidium cases were seen among immunocompetent cases shows the existence of this pathogen even in non immune compromised pediatric population. Cryptosporidium is an important etiological agent and its diagnosis is of utmost importance as this is a useful guide for the prompt treatment of such cases. More importantly, it can be diagnosed by relatively simple and inexpensive techniques such as acid-fast staining, which can also help avoid invasive procedures such as colonoscopy and intestinal biopsies in cases of persistent diarrhea^25,26. We would also like to point out the fact that the data we have presented here shows the actual burden of this parasitic infection and perhaps less than the real one due to parasitic oocysts are shed intermittently, which may not necessarily correspond to periods of clinical symptoms. Correspondingly, oocysts may not be eliminated during the first stage of infection when the asexual stage of the life cycle predominates and clinical symptoms become apparent²⁷.

CONCLUSION

The study highlights the high prevalence of coccidian parasites among immunocompetent school children in Yemen. The clinicians in Yemen need to be aware that coccidian parasites are a potential cause of childhood diarrhea even in immunocompetent children.

ACKNOWLEDGMENTS

The authors would like to acknowledge Faculty of Medicine, Qinghai University in the Republic of China and the National Center of Public Health Laboratories in Taiz (NCPHL), Yemen for support and provided working space and materials.

CONFLICT OF INTEREST

"No conflict of interest associated with this work”.

REFERENCES

1. Bera P, Das S, Saha R, Ramachandran VG, Shah D. Cryptosporidium in children with diarrhea: A hospital-based study. Indian Pediatr 2014; 51:906–8.
2. Kaur R, Rawat D, Kakkar M, Uppal B, Sharma VK. Intestinal parasites in children with diarrhea in Delhi, India. Southeast Asian J Trop Med Public Health 2002; 33:725-9. 
3. Gupta S, Narang S, Nunavath V, Singh S. Chronic diarrhoea in HIV patients: prevalence of coccidian parasites. Indian J Med Microbiol 2008; 26(2):172-5.
4. Ryan ET, Wilson ME, Kain KC. Illness after international travel. N Engl J Med 2002; 347(7):505-16.
5. Ud Din N, Torka P, Hutchison RE, Riddell SW, Wright J, Gajra A. Severe Isospora (Cystoisospora) belli diarrhea preceding the diagnosis of human T-cell-leukemia-virus-1-associated T-cell lymphoma. Case Rep Infect Dis 2012:640104. 
6. De Oliveira-Silva MB, De Oliveira LR, Resende JC, Peghini BC, Ramirez LE, Lages-Silva E, Correia D. Seasonal profile and level of CD4+ lymphocytes in the occurrence of cryptosporidiosis and cystoisosporidiosis in HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Rev Soc Bras Med Trop 2007; 40( 5):512-5.
7. Baqai R, Anwar S, Kazmi SU. Detection of Cryptosporidium in immunosuppressed patients. J Ayub Med Coll Abbottabad. 2005; 17( 3):38-40.
8. Certad G, Arenas-Pinto A, Pocaterra L, Ferrara G, Castro J, Bello A, et al. Isosporiasis in Venezuelan adults infected with human immunodeficiency virus: clinical characterization. Am J Trop Med Hyg 2003; 69(2):217-22.
9. Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, Baveja UK. Enteric opportunistic parasites among HIV infected individuals: associated risk factors and immune status. Jpn J Infect Dis 2007; 60(2–3):76-81.
10. High Incidence rate of Severe Acute Malnutrition (SAM) among children in Yemen. https://www.unicef.org/.../malnutrition-amongst-children-yemen-all-t. Last Accessed 2019 August.
11. Wang L, Xiao L, Duan L, Ye J, Guo Y, Guo M, et al. Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficilein children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Negl Trop Dis 2013; 7(9): e2437. 
12. Rodriguez-Morales AJ, Castañeda Hernandez DM. Protozoa: Cystoisospora belli(Syn. Isospora belli). In: Encyclopedia of Food Safety; Editors: Motarjemi Y, Moy GG, Todd ECD, editors. Academic Press 2014:45-8.
13. Lee JK, Song HJ, Yu JR. Prevalence of diarrhea caused by Cryptosporidium parvum in non-HIV patients in Jeollanam-do, Korea. Korean J Parasitol 2005; 43( 3):111-4.
14. Nahrevanian H, Assmar M. Cryptosporidiosis in immunocompromised patients in the Islamic Republic of Iran. J Microbiol Immunol Infect 2008; 41(1):74-7.
15. Hamedi Y, Safa O, Haidari M. Cryptosporidium infection in diarrheic children in southeastern Iran. Pediatr Infect Dis J 2005; 24(1):86-8.
16. Sangani GS,  Mirjalali H, Farnia S, and Rezaeian Prevalence of intestinal coccidial infections among different groups of immunocompromised patients. Iran J Parasitol 2016; 11(3):332-338.
17. Peletz R, Mahin T, Elliott M, Montgomery M, Clasen T. Preventing cryptosporidiosis: the need for safe drinking water. Bull World Health Organ 2013; 91(4):238-8A.
18. Cho EJ, Yang JY, Lee ES, Kim SC, Cha SY, Kim ST, et al. A Waterborne Outbreak and Detection of Cryptosporidium Oocysts in Drinking Water of an Older High-Rise Apartment Complex in Seoul. Korean J Parasitol 2013; 51(4):461-6. 
19. Olusegun AF, Okaka CE, Luiz Dantas Machado R. Isosporiasis in HIV/AIDS Patients in Edo state, Nigeria. Malays J Med Sci 2009; 16(3):41-44.
20. Lindsay DS, Dubey J, Blagburn BL. Biology of Isospora spp. from humans, nonhuman primates, and domestic animals. Clin Microbiol Rev 1997; 10(1):19-34. 
21. Kumar SS, Ananthan S, Lakshmi P. Intestinal parasitic infection in HIV infected patients with diarrhoea in Chennai. Indian J Med Microbiol 2002; 20(2):88-91.
22. Assis DC, Resende DV, Cabrine-Santos M, Correia D, Oliveira-Silva MB. Prevalence and genetic characterization of Cryptosporidium  and Cystoisospora belli in HIV-infected patients. Rev Inst Med Trop Sao Paulo 2013; 55(3):S0036-46652013000300149.
23. Forthal DN, Guest SS. Isospora bellienteritis in three homosexual men. Am J Trop Med Hyg 1984; 33(6):1060-4.
24. Lumb R, Hardiman R. Isospora belli infection. A report of two cases in patients with AIDS. Med J Australia 1991; 155(3):194-6.
25. Fathy MM, Abdelrazek NM, Hassan FA, El-Badry AA. Molecular copro-prevalence of Cryptosporidium in Egyptian children and evaluation of three diagnostic methods. Indian Pediatr 2014; 51:727-9.
26. Shoaib S, Tauheed S, Hafiz A. Frequency of Cryptosporidium in childhood diarrhoea – Importance of modified acid fast technique. J Ayub Med Coll Abbottabad 2003; 15:3-5.
27. Mirdha BR, Kabra SK, Samantray JC. Isosporiasis in children. Indian Pediatr 2002; 39:941-4.

Table 1: The prevalence of coccidian intestinal parasites among 228 children in Al-Torbah city in Yemen.

Table 2: The prevalence rate of Cryptosporidium species in different sex and age of tested children.

Table 3: The association between Cryptosporidium species infections and income and water sources for tested children.

Table 4: The prevalence rate of Cyclospora species in different sex and age of tested children.

Table 5: The association between Cyclospora infections and income and water sources for tested children.

Table 6: The prevalence rate of Isospora belli in different sex and age of tested children.

Table 7: The association between Isospora belli infections and income and water sources for tested children.