Significance of intestinal protozoan parasites as diarrhoea-causing infectious agents in children presenting to the Agogo Presbyterian Hospital

Diarrhoeal disease is one of the leading causes of morbidity and mortality in young children in developing countries. This study assessed the use of multiplex real-time PCR assay and microscopy to determine the prevalence and significance of intestinal protozoan parasites as diarrhoeal-causing infectious agents in children in the Ashanti Akyem North District of the Ashanti Region, Ghana. Between June 2007 and May 2008, fresh stool samples were obtained from 1373 children up to 13 years of age attending the child welfare clinic of the Agogo Presbyterian Hospital. 0.20g of each stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex real time Polymerase Chain Reaction (RT-PCR) for pathogenic protozoan parasites. Of the 1373 children examined, 60.5% were clinically declared symptomatic whilst 39.5% were asymptomatic. The prevalence rates of the enteric protozoa detected by microscopy from the symptomatic and asymptomatic children were Giardia lamblia, 13.1% and 20.5%, Cryptosporidium species, 4.9% and 4.9%, Entamoeba histolytica/dispar, 0.4% and 1.2%, Blastocystis hominis, 10.1% and 20.3% Entamoeba coli, 4.0% and 12.7%, Chilomastix mesnili, 2.6% and 6.2%, Endolimax nana 1.8% and 8.7%, Entamoeba hartmani 0.68% and 3.32% respectively. The prevalence rates of helminths detected in both symptomatic and asymptomatic children were Ascaris lumbricoides 0.16%, Hookworm 0.6%, Strongyloides stercolaris 1.0%, Hymenolepis nana 0.4% and Metagonimus species 0.5%. Multiplex real time PCR detected 31.2% Giardia lamblia and 5.7% Cryptosporidium parvum in the symptomatic whilst 40.3% Giardia lamblia, 2.4% Cryptosporidium parvum and only one case of Entamoeba histolytica were detected in the asymptomatic children. By using an expanded gold standard the sensitivity and specificity of PCR for Giardia lamblia detection was 96.9% and 81.6% respectively, whilst 65.2% sensitivity and 98.7% specificity was observed with microscopy. Sensitivities of 76.2% and 80% and specificities of 98.2% and 97.2% for microscopy and PCR respectively for the detection of Cryptosporidium parvum were also observed. This present study showed low rates of helminths and relatively high rates of protozoa infections in the study children. However, protozoa pathogens detected amongst the symptomatic and asymptomatic children were found to be similar; thus the significance of these pathogens as diarrhoeal causing agents in the district is therefore unclear. This present study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites.
A Thesis Submitted in Fulfillment of the Requirements for the Degree Of MASTER OF PHILOSOPHY in the Department of Clinical Microbiology, School of Medical Sciences,2010.