In this prospective study from April 2012 to March 2013, we included children under 5 years of age suffering from acute or persistent diarrhea. The study was approved by the Ethical committee of the institution. Assuming the prevalence of Cryptosporidium parvum as 13% [4], a margin of error of 5% with a 95% confidence interval, the calculated sample size was 175 children. Acute diarrhea was defined as three or more loose stools per day over a 72 hours period; diarrhea persisting for more than 14 days was defined as persistent diarrhea [5,6]. Children with known immunosuppression, history of receiving antibiotics or antiparasitic drugs for current episode of diarrhea, known food allergies, history of recurrent hospitalization due to infections, history of intake of steroids in last three months or history of infection with unusual organisms, were excluded from our study. The parents of these children were interviewed using a pre-designed form for demographic, epidemiological and clinical history. The stool sample was collected in a clean, leak-proof container, and examined on the same day by preparation of wet mount with lugol’s iodine and by Kinyoun’s acid-fast staining for the presence of C. parvum oocyst, after concentrating the sample by formalin-ether sedimentation [7]. Rest of the stool specimen was divided in two parts. One part was processed for culture for detection of other enteric pathogens and the other was preserved in 10% formalin for antigen detection by commercially available Cryptosporidium surface antigen detection ELISA kit (DRG international Inc. USA). Data were analyzed by SPSS 17.0. Qualitative data were compared by chi-square test or Fisher exact test, as applicable.

Out of 175 stool samples collected in our study, seven were positive for oocyst of C. parvum using Kinyoun’s acid-fast staining, and 48 (27.4%) were positive for cryptosporidium antigen by ELISA. All seven patients harbouring cryptosporidium oocyst were positive for cryptosporidium antigen in the stool.

Among the cryptosporidium-positive children, 66.7% used municipal water supply for drinking; 10.4% and 8.3% were using water from well and tube well, respectively. Only four children presented with severe dehydration. History of contact with domestic animal was present in only five children positive for cryptosporidium antigen in their stool samples.

The relationship between the nutritional status and cryptosporidiosis is summarized in Table I. Out of 48 cryptosporidium-positive patients, five and six children had weight-for-age Z score between -2 to -3 and <-3, respectively.

TABLE I   Clinical and Socio-Demographic Profile of Children with and without Cryptosporidiosis

In our study group, cryptosporidium-positive samples were co-infected with Vibrio cholerae, 01 ogawa (2 cases), Salmonella choleraesuis (1 case) and Giardia lambia cyst (1 case). Cryptosporidium-negative five cases were culture positive for Vibrio cholerae and one for Salmonella-typhimurium.

This prospective study in urban Northern Indian immunocompetent under-five children with diarrhea reported a high prevalence rate (27.4%) of Cryptosporidium infection. Sensitivity of microscopy was poor in comparison to antigen detection by ELISA.

Similar high prevalence of this parasite in children with diarrhea has been reported earlier from India [8] and Bolivia [9]. Low sensitivity of microscopy may be explained due to presence of less number of parasites in children with intact immune response, which inhibits further proliferation of parasites.

Contact with domestic animals as a significant risk factor [10] was not seen in our study, as also reported from Mexico [11] and Brazil [12]. Infection rates in our study were not influenced by lack of breastfeeding or source of drinking water. Unsafe drinking water has earlier been reported as a risk factor in a large outbreak of cryptosporidiosis in Milwaukee Wisconsin in 1993 [13]. Although malnutrition, especially stunting, has been signi-ficantly associated with cryptosporidiosis, in our study it was not observed as a risk factor. Due to lack of follow-up in our study, it is difficult to conclude as to whether Cryptosporidium infection leads to malnutrition or vice versa. Our study had limitations of being hospital-based and without follow-up.

To conclude, infection with Cryptosporidium species is prevalent in a significant proportion of immunocompetent children suffering from diarrea in urban Northern India. This parasite is an important etiological agent of acute gastroenteritis and diarrheal illness among children. Prompt identification of this agent by antigen detection should be an essential part of studies investigating etiology of childhood diarrhea. Efforts must be initiated to develop facilities for cryptosporidium antigen detection at all levels for proper diagnosis and management of childhood diarrhea.

Funding: None; Competing interests: None stated

1. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrheal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): A prospective, case-control study. Lancet. 2013;382:209-22.

2. Checkley W, Gilman RH, Epstein LD, Suarez M, Diaz JF, Cabrera L, et al. Asymptomatic and symptomatic cryptosporidiosis: their acute effect on weight gain in Peruvian children. Am J Epidemiol. 1997:156-63.

3. Sunnotel O, Lowery CJ, Moore JE, Dooley JS, Xiao L, Millar BC, et al. Cryptosporidium. Lett Appl Microbiol. 2006:43;7-16.

4. Mathan MM, Venkatesan S, George R, Mathew M, Mathai V. Cryptosporidium and diarrhoea in Southern Indian children. Lancet. 1985:2;1172-5.

5. Walker CLF, Bhutta ZA, Bhandari N, Teka Telahun, Shahid F, Taneja S, et al. Zinc supplementation for the treatment of diarrhea in infants in Pakistan, India and Ethiopia. J Pediatr Gastroenterol Nutr. 2006:43;357-63.

6. Bhatnagar S, Bahl R, Sharma PK, Kumar GT, Saxena SK, Bhan MK. Zinc with oral rehydration therapy reduces sool output and duration of diarrhea in hospitalized children: A randomized controlled trial. J. Pediatr Gastroenterol Nutr. 2004;38:34-40.

7. Garcia LS. Diagnostic Medical Parasitology. In: Macroscopic and Microscopic Examination of Fecal Specimens. 4th ed. Vol 2. Washington, DC: ASM Press; c2001. p. 741-85.

8. Kaur R, Rawat D, Kakkar M, Uppal B, Sharma VK. Intestinal parasites in children with diarrhoea in Delhi India. Southeast Asian J Top Med Public Health. 2002;33:4.

9. Esteban JG, Aguirre C, Flores A, Strauss W, Angles R, Mascoma S. High Cryptosporidium prevalence in healthy Aymara children from the Northern Bolivian Altiplano. Am J Trop Med Hyg. 1998;50:50-1.

10. Stantic-Pavlinic M, Xiao L, Glaberman S, Lal AA, Orazen T, Rataz- Verglez A, et al. Cryptosporidiosis associated with animal contacts. Wein Klin Wochenschr. 2003;115:125-7.

11. Javier Enriquez F, Avila CR, Ignacio Santos J, Tanaka-Kido J, Vallejo O, Sterling CR. Cryptosporidium infections in Mexican children: clinical, nutritional, enteropathogenic, and diagnostic evaluations. Am J Trop Med Hyg. 1997;56:254-57.

12. Pereira MD, Atwill ER, Barbosa AP, Silva SA, Garcia-Zapata MT. Intra-familial and extra-familial risk factors associated with Cryptosporidium parvum infection among children hospitalized for diarrhea in Goiania, Goias, Brazil. Am J Trop Med Hyg. 2002;66:787-93.