Evaluation of a pan-Leishmania SL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

Background In eco-epidemiological studies,  Leishmania  detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan- Leishmania  SYBR Green quantitative PCR (qPCR) assay has recently been developed, which specifically detects the conserved spliced-leader RNA (SL-RNA) sequence. This study comparatively assessed the SL-RNA assay performance for detection of  Leishmania  in field and laboratory infected sand flies and in tissue samples from hyraxes, as reservoir hosts.  Principal findings The qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL-RNA and kDNA detection. kDNA copy numbers were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels were approximately 3-fold lower in sand fly promastigotes (ΔCt of 1.7). The theoretical limit of detection and quantification of the SL-RNA qPCR respectively reached down to 10 -3  and 10 parasite equivalents. SL-RNA detection in stored hyrax samples was less efficient with some false negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.  Conclusion Collectively, this study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable for detection and quantification of  Leishmania  in sand flies. The assay provides complementary information to the standard kDNA assays, as it is pan- Leishmania  specific and detects viable parasites, which is a prerequisite for identification of vectors and reservoirs.