Prevalence of cryptosporidium species in paediatric patients in Eastern Nepal

Introduction

Cryptosporidiosis is an important cause of childhood diarrhoea worldwide. Various studies ^1–4 in South East Asia have reported the prevalence of cryptosporidiosis ranging from 3% ¹ – 13% ² in the paediatric population.

It is a zoonotic disease and infection occurs following the ingestion of the oocyst, which is readily infectious upon excretion. ⁵ Chlorination fails to destroy the oocysts of cryptosporidium and transmission through drinking water has been proven. ⁶

In 2006, only 89% of Nepalese had access to safe drinking water and even fewer had improved sanitation facilities. Human cryptosporidiosis is expected in this region as it is an agricultural area with domestic animal rearing being a common practice. At present, cryptosporidium is not routinely looked for when diarrhoea is investigated. We therefore decided to determine the prevalence and seasonality of cryptosporidium in paediatric diarrhoea and assess the need to search for this organism in such cases.

Materials and methods

This is a descriptive cross-sectional study carried out over a period of one year from October 2007 to September 2008. We evaluated 863 stool samples from children below 15 years of age who sought medical help for diarrhoea at the BP Koirala Institute of Health Sciences, a tertiary care hospital situated in Dharan in eastern Nepal.

All the stool samples were examined the same day for parasitic ova and cysts by preparing physiological saline mount and Lugol's iodine mount. Modified Ziehl-Neelsen staining was done for three smears of each specimen in order for us to look for coccidian parasites. All stained stool smears were examined by two independent observers. The specimens were considered positive for cryptosporidium oocysts if bright pink structures between 4 µm and 6 µm in diameter were identified. Similarly, acid-fast spherical structures 8 µm–10 µm in size with crumpled cellophane paper appearance were identified as oocysts of Cyclospora cayetanensis. We also looked for Isospora oocysts, acid-fast structures 20–30 µm long and 10–20 µm wide. All negative samples were confirmed by repeating the mounts and smears after formol ether concentration of the specimen.

A control group of 100 age-matched children who had not experienced diarrhoea in the preceding two weeks were processed in a similar manner.

Results

Eight hundred and sixty-three diarrhoeal stool samples were evaluated for parasitic infection. Intestinal parasitic infection was seen in 79 (9.2%). Coccidian oocysts were detected in 38 (4.4%) of the diarrhoeal stools. Of these, 36 (4.1%) were oocysts of cryptosporidium and two were oocysts of cyclospora. Other pathogenic protozoan parasitic cysts encountered were: Giardia lamblia in 33 (3.8%) stool specimens; and Entamoeba histolytica/dispar in four samples. The helminthic ova were those of Hymenolepis nana in two specimens and one each of Trichuris trichiura and Enterobius vermicularis. The majority (62.3%) of the positive cases, including cases of cryptosporidiosis (63.8%), were from the 6–10 years age group. Seven cases of cryptosporidiosis had concurrent infection with G. lamblia. One child had mixed infection with G. lamblia and H. nana. The cases of cryptosporidiosis were concentrated (72% of the positive cases) during the months June–August.

The control children were negative for coccidian parasites.

Discussion

Only a few studies have been published which focus on the role of coccidian parasites in childhood diarrhoea in Nepal. Epidemiological studies show a higher rate of cryptosporidiosis in children from underdeveloped and developing countries than from the developed countries. ³

The prevalence seen in our study (4.4%) is close to those seen in other parts of Nepal ³ and elsewhere in Asia. ^1,7,8 A previous study done in our centre in 2001 had shown the incidence of cryptosporidiosis among children below five years to be 5.6%. ⁹ It is interesting to note that in our study, cryptosporidium was more frequent than G. lamblia and E. histolytica. Moreover, 29 of the diarrhoeal stools yielded solely cryptosporidium and two solely cyclospora as diarrhoeagen. The absence of coccidia in the control group also suggests a high pathogenic potential of this parasite.

The age group most affected by intestinal parasitosis, including cryptosporidiosis, in the current study was the 6–10-year-olds. A similar age predominance was seen in a study by Hahid et al. ¹⁰ The high rates of cryptosporidiosis in children and those under five years old seen in previous studies from Nepal ^3,9 emphasize the need for more comprehensive, community-based prospective studies in order to define the role of cryptosporidiosis in childhood diarrhoea, malnutrition, growth stunting and cognitive impairment reported by other studies. ^10–12

The rainy season (June-August) in Dharan is usually hot and humid. The case clustering of cryptosporidiosis during this season is similar to those observed in other parts of the world. ^1,13 This can be attributed to the increased chance of drinking water sources being contaminated from surface water containing both animal and human excreta bearing the infectious oocysts. The contaminated river and well waters used to clean the local vegetable products before bringing them to the markets may well be a potential source of cryptosporidiosis. Lack of sanitation and poor personal hygiene further confound the situation.

We could have expected a higher infection rate in our study if more than one stool specimen had been collected from each child because of the intermittent nature of the oocyst excretion. More sensitive techniques such as Auramine phenol and immunofluorescence also would have detected more cases. ¹³ Species identification would have given the epidemiological information of the source and subsequent measures of prevention.

Conclusion

This study demonstrates that cryptosporidiosis is a relatively common but ignored cause of diarrhoea among young children in Nepal. The incorporation of modified Ziehl-Neelsen staining in routine stool examination in paediatric patients would have established definitive aetiological diagnosis and averted the often unnecessary use of antibiotics, as cryptosporidiosis in immunocompetent, healthy children is self-limiting with symptomatic treatment. The importance of routine screening for these coccidian parasites is further heightened by the current HIV/AIDS epidemic. It would be worthwhile to conduct detailed epidemiologic studies in order to explore the magnitude of the problem posed by these coccidian parasites and to examine their significance for public health in Nepal.