Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots

BackgroundZoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. MethodsAn established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA ShieldTM) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. ResultsThe use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was [≤]0.0002 parasites/{micro}L for P. knowlesi and 0.002 parasites/{micro}L for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/{micro}L); Divis et al. real-time 18S rRNA (0.0002 parasites/{micro}L); Lubis et al. hemi-nested SICAvar (1.1 parasites/{micro}L) and Lee et al. nested 18S rRNA (11 parasites/{micro}L). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/{micro}L respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. ConclusionOur findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections. Author SummaryThe monkey malaria parasite Plasmodium knowlesi has been found to increasingly infect humans across Southeast Asia via the bite of its anopheline mosquito vectors. Human infections with a similar monkey parasite, Plasmodium cynomologi, have also been reported. The diagnostic tools commonly used to detect these malaria species are often unable to detect very low-level infections. We aimed to to improve surveillance detection tools and blood sample collection methods to detect these zoonotic malaria species and understand the extent of transmission and the burden of disease. This study validated and compared the use of molecular laboratory assays targeting these species. We found that with the use of reverse transcription, large improvements in the limit of detection were possible, by up to 10,000-fold for initial malaria screening, and up to 2759-fold for specific P. knowlesi detection. Findings from this study support the use of ultrasensitive detection tools to improve surveillance approaches to emerging zoonotic malaria species.