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

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.


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 patients. 6 Chronic diarrhea in malnourished children can also increase the incidence of these parasites and mortality in these patients. 6 Coccidiosis diarrhea has been observed with fluid loss of 25 l / day in infected patients, which can last for weeks in immunocompromised patients. 7 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 immunecompromised patients, so the previous factors lead to underestimate this infection. 8 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 infections. 9 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 Yemen. 10 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 stain 3 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 tables 1 to 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 food 11 while no animal reservoir for human Isospora has been identified. 12 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%, respectively. 13 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 resources. 17 Interestingly, Cryptosporidium oocysts are able to pass through the water treatment process because of their resistance to routine disinfectants and their small size. 18 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 infection. 19 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, respectively. 22 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 controls. 19 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 common. 20 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 water. 12 Although some cases of homosexuality have been reported to have Isosporiasis more than other individuals 23 , 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 transmission. 24 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-immunocompromised 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 apparent. 27

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.