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

Abstract

13Background 14 In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently 15 accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast 16 DNA (kDNA). A pan-Leishmania SYBR Green quantitative PCR (qPCR) assay which specifically detects the 17 conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively 18 assessed the SL-RNA assay performance for the detection of Leishmania in field and laboratory infected 19 sand flies and in tissue samples from hyraxes as reservoir hosts. 20 Principal findings 21 The qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite 22 preservation and extraction under presumed unfavorable conditions for downstream RNA detection. 23 Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly 24 compatible with downstream SL-RNA and kDNA detection. Copy numbers of kDNA were found to be 25 identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels 26 were approximately 3-fold lower in sand fly promastigotes (ΔCt 1.7). The theoretical limit of detection 27 and quantification of the SL-RNA qPCR respectively reached down to 10 -3 and 10 parasite equivalents. SL-28 RNA detection in stored hyrax samples was less efficient with some false negative assay results, most 29 likely due to the long-term tissue storage in absence of RNA stabilizing reagents. 30 Conclusion 31 This study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable 32 for the detection and quantification of Leishmania in sand flies. The assay provides complementary 33 information to the standard kDNA assays, since it is pan-Leishmania specific and detects viable parasites, 34 a prerequisite for identification of vectors and reservoirs. 3 35 Author summary 36 In order to identify vectors and reservoirs of Leishmania, a large number of sand fly and animal tissue 37 samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-38 cost methods are required for nucleic acid isolation and Leishmania detection. Most approaches amplify 39 DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that 40 detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among various Leishmania 41 species and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible 42 with a low-cost, crude extraction approach and performs equally well on laboratory and field infected 43 sand fly samples as kDNA qPCR assays. The assay can detect 10 -3 parasite equivalent in sand flies and 44 enables Leishmania quantification down to 10 parasites. We found that the copy number of SL-RNA is 3-45 fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in 46 hyrax tissue samples appeared less efficient, which is presumably due to long-term sto...

Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients

“…Positive detection following blood digestion, especially of low parasite load, may signify only the persistence or insufficient clearing of parasite-cell-free DNA that resulted from collapsed infection in sand fly during any stage of parasite establishment. One approach to circumvent this limitation can be by targeting parasite RNA as an indirect marker of cellular viability, as RNA degrades fast following cellular death [48]. Since tissue specimens in general are more challenging than liquid samples for the extraction of nucleic acids, further investigation is also needed to standardize a field feasible and rapid method for extraction of nucleic acids from sand fly midgut specimens for the implementation of RPA.…”

“…Recently we compared magnetic bead-based rapid extraction method coupled with LD-RPA assay for the detection of L. donovani in skin biopsy samples of PKDL patients; this however was found underperforming when compared with the reference DNA extraction (column) method using Qiagen kit [49]. In another approach, an extraction method developed by using in-house crude extraction buffer and ethanol precipitation showed similar sand fly DNA extraction efficiencies to column method [48]; however, enhanced centrifugation speed and time could be a limiting factor in its field implementation. Further optimization in nucleic acid extraction and detection parameters would thus be critical for assay sensitivity and accurate estimation of transmissible parasite load in sand flies.…”