Molecular diagnosis of schistosomiasis: application of real-time PCR for pre- and post- treatment evaluation

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Schistosomiasis, a major human helminthic disease in terms of mortality and morbidity infects over 200 million people worldwide. It is a major health problem due to its high prevalence in affected communities and its ability to manifest into severe clinical forms. New diagnostics for detection of infections are essential considering that the present parasitological methods, though specific, are not very sensitive and not always able to judge accurately the performance of Praziquantel (PQZ) in terms of adult worm death but rather in terms of cessation of egg excretion. A highly sensitive real-time PCR (RT-PCR) developed for the detection of Schistosoma sp. DNA in both urine and faeces sample was compared with microscopy detection of schistosome eggs. Both urine and stool samples were collected from study participants aged between 3 and 20 years before and after (3 weeks and 8 weeks) treatment with PQZ. Study participants were found to have either single (S. mansoni or S. haematobium) or mixed S. mansoni and S. haematobium infections by microscopic examination of urine and stool samples. Utilising the two diagnostic methods, microscopy detected both S. haematobium and / or S. mansoni eggs in 93% (133) urine and/ or stool samples collected before treatment whereas RT-PCR amplified DNA in 97.2% (139) of same samples. A significant reduction in prevalence and intensity of infection was observed after PZQ treatment with no significant differences between sexes and age groups using both detection methods. Exhibiting a higher sensitivity, RT-PCR detected Schistosoma DNA in 67% and 69.3% urine and/ or stool samples tested at 3 and 8 weeks post treatment respectively whereas microscopy detected schistosome eggs in 25% and 30.7% at 3 and 8 weeks post treatment respectively. RT-PCR therefore provided a lower estimation of the cure rate than the parasitological technique (microscopy detection of eggs) since it detected more positive cases at all post-treatment surveys. RT-PCR amplified 35.4% (75/212) of urine egg negative samples and 67.1% (94/140) of the stool egg negative samples when comparing both microscopy and RT-PCR for the entire study population over all three time periods. However RT-PCR failed to amplify schistosme DNA in 20 individuals with light egg intensity infection in urine and 3 individuals with light egg intensity in stool. Results of this study show RT-PCR to be significantly more sensitive than microscopy in detecting and evaluating infection prevalence, an important aspect of epidemiological studies. Most probably, RT-PCR for evaluation of treatment resulted in lower cure rate due to its ability to detect DNA from schistosome eggs (devitalised or containing a living miracidium), including cell-free DNA released from damaged eggs and any juvenile or adult worm DNA breakdown products in the faecal and urine samples even if the drug is killing the vast majority of worms. Thus, RT-PCR technique can be especially useful in circumstances of lower intensity or prevalence of infection, a condition for which the parasitological examination shows a well-documented limitation of its sensitivity.
A thesis submitted to the Department of Theoretical and Applied Biology, College of Science, Kwame Nkrumah University of Science and Technology in partial fulfilment of the requirements for the award of Master of Philosophy degree.