Evaluation of a pan-LeishmaniaSL-RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts
Authors: Pareyn Myrthe , Hendrickx Rik , Girma Nigatu , Hendrickx Sarah , Van Bockstal Lieselotte , Van Houtte Natalie , Shibru Simon , Maes Louis , Leirs Herwig , Caljon Guy 
Published: January 2020

AbstractBackgroundIn eco-epidemiological studies,Leishmaniadetection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-LeishmaniaSYBR Green quantitative PCR (qPCR) assay which specifically detects the conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively assessed the SL-RNA assay performance for the detection ofLeishmaniain field and laboratory infected sand flies and in tissue samples from hyraxes as reservoir hosts.Principal findingsThe 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. Copy numbers of kDNA 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 1.7). The theoretical limit of detection and quantification of the SL-RNA qPCR respectively reached down to 10−3and 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.ConclusionThis study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable for the detection and quantification ofLeishmaniain sand flies. The assay provides complementary information to the standard kDNA assays, since it is pan-Leishmaniaspecific and detects viable parasites, a prerequisite for identification of vectors and reservoirs.Author summaryIn order to identify vectors and reservoirs ofLeishmania, a large number of sand fly and animal tissue samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-cost methods are required for nucleic acid isolation andLeishmaniadetection. Most approaches amplify DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among variousLeishmaniaspecies and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible with a low-cost, crude extraction approach and performs equally well on laboratory and field infected sand fly samples as kDNA qPCR assays. The assay can detect 10−3parasite equivalent in sand flies and enablesLeishmaniaquantification down to 10 parasites. We found that the copy number of SL-RNA is 3-fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in hyrax tissue samples appeared less efficient, which is presumably due to long-term storage without RNA stabilizing reagents. Overall, our assay is complementary to kDNA assays as it can identify viableLeishmaniastages, which provides pivotal information for identification of reservoirs and vectors and their transmission capacity.