INTRODUCTION

Cryptosporidiosis is an opportunistic diarrhoeal disease caused by an intracellular spore forming protozoan parasite known as Cryptosporidia. About 21 species of Cryptosporidium have been identified but the main specie responsible for human infection is C. hominis formerly known as C. parvum human genotype or C. parvum type 1 (Loureiro et al., 1989; NiChito et al., 1998). The infection is transmitted by ingestion of the oocyst in faecally contaminated food and water from animals to humans and through person to person contact. The probability of transmission from just a small amount of contamination is fairly high. It is often transmitted in day care setting to care givers, older children and adults who come in contact with infected children at home (Alpert et al., 1984) or hospital acquired (Baxby, 1983; Koch et al., 1985). A recent study has shown that the 50% infective dose (LD₅₀) of C. parvum is only 132 oocysts for healthy persons with no previous serological immunity to cryptosporidiosis (O’Donoghue et al., 1995)

The commonest symptom of cryptosporidiosis is diarrhea, which is usually watery stool. It is accompanied by abdominal cramps, nausea, vomiting, fever, headache and loss of appetite (Khan, 2000). The symptoms differ greatly in immunocompetent and immunocompromised individuals. In the former the infection is acute, yet a self limiting diarrhoeal illness whereas the illness is much more severe in the later. The incubation period may range from 1 to 12 days (Juranek, 1995).

Cryptosporidiosis is endemic in the tropics with high prevalence in less developed countries (Gatei et al., 2003) Children are more vulnerable in developing Cryptosporidium-associated diarrhoea (Abdel-Messih et al., 2005). It has been shown that children less than 12 months of age are 2.4 times more likely to be infected with Cryptosporidium while children aged between 12 and 23 months are 1.9 times more likely to be infected as compared with older ones (Abdel-Messih et al., 2005). Molbak et al. (1997) reported increased carriage of cryptosporidiosis in malnourished children. Studies conducted in Brazil, Peru and Guinea-Bissau have associated cryptosporidiosis with impairment of growth, physical fitness and cognitive function (Checkley et al., 1998; Guerrant et al., 1999; Molbak et al., 1997).

There are isolated studies on the epidemiology of cryptosporidiosis in Nigeria. In one of these studies, Useh and Jonah (1998) reported a prevalence of 4.4% for C. parvum amongst diarrhoeal subjects of all ages in Calabar, South Eastern Nigeria. In the same setting, Alaribe et al. (1998) reported a prevalence of 5.6% among diarrhoeic patients. Infection was not reported among healthy control subjects with no history of diarrhoea. Elsewhere in North Central and South Western Nigeria, higher prevalence of cryptosporidiosis of 4.8 and 14% were reported among undernourished children and diarrhoeic patients by Ikeh et al. (2006) and Nwabuisi (1998) respectively. Although these studies have confirmed the presence of cryptosporidiosis in Nigeria, this organism is not sought for routinely even in Teaching Hospitals like ours. It is likely that this organism may be playing a very important role particularly as an opportunistic pathogen among the HIV/AIDS patients in Nigeria. The paucity of information on the prevalence and significance of Cryptosporidium parvum as an agent of infantile diarrhoea necessitated this study. It is envisaged that the findings of this study may herald some radical decisions on the diagnosis and control of diarrhoea of Cryptosporidium origin.

MATERIALS AND METHODS

Study area: The subjects were Children who presented with diarrhoea and were attended to at the Children Emergency ward (CHER) of the University of Calabar Teaching Hospital (UCTH) and the Out-Patient Department (OPD) of the General Hospital, all in Calabar between April and September, 2006. The UCTH is a tertiary health institution which caters principally for residents of Cross River and Akwa Ibom states and others referred from other states in Nigeria and the neighboring Republic of Cameroon. The states mentioned above are located in the South-eastern rainforest belt of Nigeria which experiences a typical tropical climate. Calabar and its immediate environs lack adequate supply of portable water. The drainage system is poor. There is inadequate management of domestic waste. These may provide effective media for the transmission of parasitic diseases.

Subjects and consent: Two hundred children aged between 2 months and 8 years diagnosed of diarrhoea and 100 healthy non-diarrhoiec control subjects of the same age range who lived within the University community were examined. Diarrhoea was defined as the presence of three or more loose or watery stools within a 24 h period (Sherries, 1989). Stool samples were collected from both patients and test subjects in clean universal containers and processed within 30 min in the Parasitology Department of the UCTH.

A written permission was sought and obtained from the Ethical Committee in the two hospitals. Verbal consent was sought and obtained from parents to enable their children participate in the study.

Examination of samples: The standard method of stool examination as described by WHO (1991) was adopted for the study. These included macroscopy, microscopic examination of wet mounts in saline and Lugol’s iodine and the formal ether concentration technique.

Formal ether concentration technique: All the samples were concentrated using Formal ether concentration technique. One milliliter of a well mixed stool sample was put in a tube containing 4 mL of 10% formalin. Three milliliter of the 10% formalin was again added and mixed by shaking. The suspension was sieved using a coffee strainer into a centrifuge tube. Three milliliter of diethyl ether was added and stoppered. It was then mixed vigorously for 1 min. The stopper was removed and the suspension centrifuged for 1 min at 400 g. The entire column of the fluid below the faecal debris and ether was carefully removed using a pasteur pipette and transfer into another centrifuge tube. Ten percent formalin was added to the transferred suspension to make up to 10 mL. It was then centrifuge at 1000 g for 10 min. The supernatant was decanted and the bottom of the tube tapped to re-suspend the deposit. The deposit was examined using the 10 and 40x objectives for the presence of cyst or ova of parasite.

Modified Ziehl Nelson Staining Technique for Oocyst: The modified Ziehl Nelson technique was used for the demonstration of oocyst of Cryptosporiduim. A methanol fixed thin smear of faeces was stained in cold carbol fuchsin for 10 min. Differentiation was done with 1% hydrochloric acid-ethanol while 0.25% methylene blue was utilized as counterstain. Crypotosporidium oocyst appeared as bright rose-pink spheres on a pale blue background (Current, 1989).

Data analysis: The data obtained from the study were analysed using Chi-square test to determine the influence of age and sex on the prevalence of infection.