Optimisation and analytical assessment of a TaqMan™ probe-based real-time PCR assay designed to diagnose infection with Schistosoma japonicum.

backgroundIntestinal schistosomiasis, caused by infection with Schistosoma japonicum, remains a zoonotic neglected tropical disease (NTD) of significant public health importance in parts of China, the Philippines, and Indonesia. Ongoing schistosomiasis control programmes in these areas have achieved significant reductions in disease prevalence, transmission, and morbidity associated with infection; however, they have also presented new challenges, particularly in detecting and monitoring low-intensity and low-prevalence S. japonicum infections. Highly sensitive and highly specific diagnostic assays are therefore needed for effective disease diagnosis and transmission monitoring in low-endemicity areas, and the development of such assays has been recommended by the World Health Organisation.

methodsWe first aimed to determine whether a Schistosoma spp. genus-specific real-time PCR assay routinely used to diagnose infections with African schistosome species (namely Schistosoma mansoni and Schistosoma haematobium), as well as two more recently developed S. japonicum species-specific real-time PCR assays, can accurately diagnose low-intensity S. japonicum infections. We also sought to optimise one of the S. japonicum species-specific real-time PCR assays by adding the same DNA extraction and PCR internal positive control target commonly used with the Schistosoma spp. genus-specific real-time PCR assay. This was done using in silico analyses and endpoint PCR with Sanger sequencing and real-time PCR using genomic DNA isolated from Schistosoma spp. adult worms, H

resultsWe demonstrate that the Schistosoma spp. genus-specific real-time PCR assay and one of the S. japonicum species-specific real-time PCR assays described here are likely incapable of reliably diagnosing low-intensity S. japonicum infections. We also demonstrate that the other S. japonicum species-specific real-time PCR assay can be performed using an additional DNA extraction and PCR internal positive control target and may reliably diagnose low-intensity S. japonicum infections.

conclusionsWhilst clinical assessment of the optimised S. japonicum species-specific real-time PCR assay described here is needed, it is our hope that this assay will become a standard and routinely used method to diagnose low-intensity S. japonicum infections and will be used support future S. japonicum transmission surveillance and elimination programmes.