Nested qPCR assay to detect Babesia duncani infection in hamsters and humans

Human babesiosis is caused by Babesia duncani that is transmitted through tick bites, blood transfusions, and transplacental transmission. Despite its health burden, diagnostic assays for this pathogen are either unsuitable for clinical applications or have a low detection efficiency; therefore, it remains undetected during transfusion and utilization of blood and blood-component transfusions. This study used a molecular approach via nested quantitative polymerase chain reaction (qPCR) by designing primers and probes corresponding to the variable regions of B. duncani 18S rRNA gene to specifically detect B. duncani DNA in experimentally infected LVG Golden Syrian hamster (n = 70) and human (n = 492; tick bite patients from Gansu Province, China) blood samples. Moreover, comparative analyses of this technique with previously reported nested PCR and microscopy were conducted. The newly optimized diagnostic technique exhibited no cross-reactivity with genomic DNA or plasmids containing the 18S rRNA gene of other zoonotically important Babesia spp., including B. microti, B. divergens, B. crassa, and B. motasi Hebei. The detection limit of nested qPCR was approximately one plasmid copy in 20 μL or one infected red blood cell in 200 μL whole blood. The specificity and sensitivity of the method were 100% and 98.6%, respectively. Comparative analyses revealed that nested qPCR detected B. duncani had relatively higher efficacy and specificity than microscopic examination and nested PCR. The 492 human blood samples were negative for B. duncani infection. Thus, the present study provides an improved diagnostic assay for the efficient and effective detection and analysis of B. duncani infections and its prevalence in infection-prone areas.