Performance assessment of a duplex quantitative PCR assay for detecting <i>Leishmania infantum</i> and <i>Leishmania tarentolae</i> using three qPCR devices

ABSTRACT Among Leishmania spp. transmitted by phlebotomine sand flies, Leishmania infantum is regarded as the most widely distributed species worldwide and the primary causative agent of canine leishmaniasis. However, in regions where L. Infantum is endemic the sympatric occurrence of the herpetophilic Leishmania tarentolae in dogs, cats, and humans has prompted the development of sensitive and specific diagnostic assays. To evaluate the clinical and analytical sensitivities of a quantitative PCR (qPCR) assay targeting the minicircle kDNA fragment of both species, three devices with different technologies (lab-on-chip real-time PCR applied on the portable Q3-Plus V2 platform, CFX96 Real-Time System, and droplet digital PCR [ddPCR]) were tested. The sensitivity (limit of detection [LOD]) and specificity of the assay were assessed by processing DNA samples from cultures of both Leishmania spp. and from biological samples of Sergentomyia minuta ( n = 120; midgut), of lizards ( Podarcis siculus ; n = 29; blood, eggs, heart, intestine, kidneys, liver, lungs, and spleen), and of dogs ( n = 101; blood, bone marrow, conjunctival swab, lymph node, and skin). The performance of the assay applied on all devices was evaluated by comparing the quantification cycle values obtained by qPCR with the DNA copy number determined by ddPCR. The LOD of serial dilutions of DNA from each Leishmania spp., spiked DNA, and biological samples were overlapping across the devices, whereas receiver operating characteristic analysis showed a shift in sensitivity for ddPCR (AUC, 0.9858) compared to CFX96 and Q3 qPCRs (AUC, 0.9517). The assay developed is an efficient and sensitive tool regardless of the device tested, though ddPCR showed higher sensitivity in detecting low quantities of DNA in all samples, as supported by the highest AUC value. IMPORTANCE The development of sensitive and specific molecular tools for detecting Leishmania infantum and Leishmania tarentolae has been evaluated in a duplex qPCR assay applied on three devices with different technologies (lab-on-chip real-time PCR applied on the portable Q3-Plus V2 platform, CFX96 Real-Time System, and droplet digital PCR). Both Leishmania spp. were tested in different tissues of various animal hosts, and the clinical sensitivity and specificity were evaluated. The duplex qPCR assay developed herein proved to be an efficient tool, regardless of the device used, and will be useful in improving current knowledge on the distribution of L. infantum and of L. tarentolae and in better understanding the potential role of new hosts/reservoirs or vectors for these Leishmania spp.