Establishment of TaqMan Probe-based Molecular Detection and Quantification of Cryptosporidium Species in Ghana: Cohort Study in School-aged Children

<ns3:p> Background Cryptosporidiosis, caused by <ns3:italic>Cryptosporidium</ns3:italic> spp., causes diarrhoeal disease in both humans and animals globally. However, research and treatment for diarrhoeal disease in Africa are generally focused on viral and bacterial pathogens. We adopted quantitative PCR (qPCR) for improved identification of <ns3:italic>Cryptosporidium</ns3:italic> parasites from clinical and non-clinical samples in Ghana. Methodology In a cross-sectional surveillance study, human stool samples obtained from five study sites across Ghana were analysed using gel-based nPCR-RFLP and/or TaqMan probe-based qPCR for detection, genotyping, and quantitation of cryptosporidiosis. Results We analysed two sets of samples collected from children of both sexes ages ≤12 years. The first set of 168 samples was analysed by nPCR-RFLP (nested PCR-Restriction Fragment Length Polymorphism). We detected 60 <ns3:italic>Cryptosporidium</ns3:italic> spp <ns3:italic>.</ns3:italic> infections (35.7%) of which 39 were clinical patients (representing 23.2% of total sample size: 14.9% <ns3:italic>C. hominis</ns3:italic> ; 7.1% <ns3:italic>C. parvum;</ns3:italic> 0.6% each of <ns3:italic>C. meleagridis</ns3:italic> and <ns3:italic>C. baileyi</ns3:italic> ) and 21 were non-clinical (12.5% of sample size: 6.0% <ns3:italic>C. hominis</ns3:italic> and 6.5% <ns3:italic>C. parvum</ns3:italic> ). The second set of samples were collected from non-clinical settings from 92 children and analysed by TaqMan qPCR. We detected <ns3:italic>Cryptosporidium</ns3:italic> spp. infections in 28.3% of this population. This consisted of 7.6% <ns3:italic>C. parvum</ns3:italic> (approximately 10 <ns3:sup>3</ns3:sup> oocysts/g faeces), 16.3% <ns3:italic>C. hominis</ns3:italic> (10 <ns3:sup>3</ns3:sup> –10 <ns3:sup>6</ns3:sup> , oocysts/g faeces), and 4.4% with <ns3:italic>C. parvum</ns3:italic> and <ns3:italic>C. hominis</ns3:italic> co-infections. More males were infected than females (17.4% vs 10.9%). The highest infections were among ages 5–8 years (18.5%) and the lowest 0–4 years (4.4%) <ns3:italic>.</ns3:italic> Conclusion From our studies, TaqMan qPCR emerged as more efficient than nPCR-RFLP in detecting <ns3:italic>C. parvum</ns3:italic> and <ns3:italic>C. hominis.</ns3:italic> Confirmation of other zoonotic <ns3:italic>Cryptosporidium</ns3:italic> spp. detected by nPCR-RFLP requires DNA sequencing. It is, therefore, recommended that the TaqMan qPCR system be adapted to enhance molecular epidemiological studies on cryptosporidiosis in Africa <ns3:italic>.</ns3:italic> </ns3:p>