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

Background: Zoonotic and 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.

Methods: An established ultra-sensitive genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for , and using total nucleic acid preserved (DNA/RNA Shield) isolates and archived dried blood spots (DBS). LODs for selected specific assays, and reference and -specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls.

Results: The use of reverse transcription improved species detection by up to 10,000-fold ( genus), 2759-fold (), 1000-fold () and 10-fold (). The median LOD with RT for the Kamau et al. genus RT-qPCR assay was ≤0.0002 parasites/μL for and 0.002 parasites/μL for both and . The LODs with RT for -specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/μL); Divis et al. real-time 18S rRNA (0.0002 parasites/μL); Lubis et al. hemi-nested (1.1 parasites/μL) and Lee et al. nested 18S rRNA (11 parasites/μL). The LOD for and specific assays with RT were 0.02 and 0.20 parasites/μL respectively. For DBS samples the median LOD for the 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 genus and -assays were 100% specific for species and detection, respectively, from 190 clinical infections and 48 healthy controls. Reference specific primers demonstrated known cross-reactivity with .

Conclusion: Our findings support the use of an 18S rRNA genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human species infections.