Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani

Abstract

Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.

Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

“…The number of droplets with amplification product is then measured, allowing for an estimate of template density without the need for a standard curve. ddPCR has been shown to yield more precise results than qPCR with less variation among technical replicates19, and has been successfully applied for the diagnosis and quantification of a number of human pathogens, including vector-borne infections202122.…”

Molecular Diagnostics in the Diagnosis of Parasitic Infection

“…Assays may be species specific (Chan et al, 2013;Teal et al, 2012;Wilson et al, 2014) or use subsequent sequence analysis or electrospray ionisation MS for differentiation to the species level (Eshoo et al, 2014;Herwaldt et al, 2003;Liu, 2013;Persing et al, 1992). The described assays have similar or improved sensitivity compared to conventional thick film examination.…”

Human Babesiosis: Pathogens, Prevalence, Diagnosis, and Treatment

“…Adaptations to the qPCR method include the droplet digital PCR (ddPCR), where the amplification reaction containing the DNA sample, fluorescently-labeled probe, is divided into many microscopic reaction droplets, each containing one or less copies of the target DNA. This technology has recently been applied to the detection of B. microti and B. duncani , and were shown to discriminate B. microti from B. duncani at a limit of limits of detection of ~10 gene copies [90]. …”