Integrating molecular tools into leishmaniosis surveillance: evaluation of a commercial qPCR kit in sand fly vectors

Abstract Background Leishmaniosis is a zoonotic disease caused by protozoan parasites of the Leishmania genus, primarily transmitted through the bites of infected female phlebotomine sand flies. Leishmania infantum is the most prevalent Leishmania species in the Mediterranean Region, affecting both humans and animals, mainly dogs. Integrated surveillance strategies, including xenomonitoring, are essential for early detection and cost-effective control in endemic areas. In 2021, an increase in human leishmaniosis cases was reported in the Region of Murcia (Spain), prompting an intensified entomological surveillance effort aimed at assessing Leishmania prevalence in vector populations. Phlebotomine sand flies were collected and analysed to determine the circulation of Leishmania spp. in the area. Subsequently, these field-collected samples were used to evaluate the diagnostic performance of a commercial real-time PCR (qPCR) kit designed for the detection of Leishmania DNA. Methods A comparative–retrospective analysis was carried out at the Parasitology Laboratory of the Department of Animal Pathology, University of Zaragoza. A total of 314 DNA extracts from pooled female sand fly samples, collected in urban and peri-urban areas of 29 municipalities in the Region of Murcia near recently reported human leishmaniosis cases (2021–2022), were analysed. Sampling was conducted in June and September 2023 using CO 2 -baited light traps to investigate the presence of Leishmania spp. in sand flies. The performance of the VIASURE qPCR assay (CerTest Biotec, Zaragoza, Spain) was assessed against a structured reference method consisting of an in-house qPCR assay, conventional PCR followed by Sanger sequencing and a SYBR Green qPCR used to resolve discordant results. Results Of the 314 pooled samples analysed, 21 tested positive for Leishmania spp. using the reference methods, while the VIASURE assay identified 23 Leishmania spp.-positive pooled samples. After resolving discrepancies, the obtained analytical sensitivity and specificity were 0.95 (95% confidence interval [CI] 0.76–0.99) and 0.99 (95% CI 0.97–0.99), respectively. Conclusions The commercial VIASURE qPCR assay showed high concordance with established molecular methods and demonstrated reliable performance for detecting Leishmania DNA in sand fly vectors. To our knowledge, this is the first study to specifically validate a commercially available qPCR kit in phlebotomine sand flies. These findings support its potential utility in entomological surveillance programs and public health interventions in leishmaniosis-endemic regions.