Alvaro Alessandro, Cattaneo Giulia Maria, Varotto-Boccazzi Ilaria, Molteni Riccardo, Mendoza-Roldan Jairo Alfonso, Brilli Matteo, Conconi Matilde Silvia, Giovagnoli Virginia, Manenti Alessandro, Otranto Domenico, Bandi Claudio, Epis Sara
Department of Biosciences, University of Milan, 20133, Milan, Italy.
Department of Veterinary Medicine, University of Bari, Valenzano, 70010, Bari, Italy.
Parasit Vectors. 2025 Jul 1;18(1):243. doi: 10.1186/s13071-025-06871-3.
Leishmaniases, caused by protozoan parasites of the genus Leishmania, are vector-borne diseases occurring mainly in the tropics and subtropics of the world, as well as in the Mediterranean Basin. In this area, the mammalian pathogen Leishmania infantum is endemic, along with the reptile-associated Leishmania tarentolae. The two species occur in sympatry, and there is evidence that the exposure to L. tarentolae in mammalian hosts may elicit a protective immune response towards pathogenic Leishmania species. Accurate detection methods for both species are therefore crucial for gathering comprehensive information on the epidemiology of leishmaniases. In microbiological diagnosis, limits in detection performance imply the risk of false negatives and other issues, which highlights the need for sensitive methods.
Here, we developed a droplet digital polymerase chain reaction assay targeting the kinetoplast minicircle DNA, for the simultaneous and differential detection of L. infantum and L. tarentolae. The assay features primers designed to bind to both species and species-specific probes. The assay was validated on three cultured isolates for each species, whose cells were spiked into Leishmania-negative dog blood, and on Leishmania-positive sand flies. Sensitivity was assessed with testing serial dilutions, and specificity was evaluated by assessing the cross-reactivity of the probes with the controls of Leishmania-free dog blood and male sand fly DNA.
The assay demonstrated high sensitivity, with a limit of detection corresponding to one Leishmania cell in the reaction mix for isolates of both L. infantum and L. tarentolae. Limited cross-reaction of the L. tarentolae-targeting probe was observed on L. infantum isolates. No cross-reaction was observed with the controls of Leishmania-free dog blood and male sand flies.
The protocol can represent a valuable method for comprehensive surveillance in both canine hosts and sand flies in areas in which L. infantum and L. tarentolae occur in sympatry.
利什曼病由利什曼原虫属的原生动物寄生虫引起，是主要发生在世界热带和亚热带地区以及地中海盆地的媒介传播疾病。在该地区，哺乳动物病原体婴儿利什曼原虫是地方病，同时还有与爬行动物相关的塔兰托利什曼原虫。这两个物种同域分布，并且有证据表明哺乳动物宿主接触塔兰托利什曼原虫可能引发针对致病性利什曼原虫物种的保护性免疫反应。因此，针对这两个物种的准确检测方法对于收集利什曼病流行病学的全面信息至关重要。在微生物诊断中，检测性能的局限性意味着存在假阴性和其他问题的风险，这凸显了对敏感方法的需求。
在此，我们开发了一种针对动基体小环DNA的液滴数字聚合酶链反应检测方法，用于同时和鉴别检测婴儿利什曼原虫和塔兰托利什曼原虫。该检测方法的特点是设计了与两个物种结合的引物和物种特异性探针。该检测方法在每个物种的三个培养分离株上进行了验证，这些分离株被添加到利什曼原虫阴性的犬血中，同时也在利什曼原虫阳性的白蛉上进行了验证。通过测试系列稀释评估敏感性，并通过评估探针与无利什曼原虫犬血和雄性白蛉DNA对照的交叉反应来评估特异性。
该检测方法显示出高敏感性，婴儿利什曼原虫和塔兰托利什曼原虫分离株在反应混合物中的检测限均对应一个利什曼原虫细胞。在婴儿利什曼原虫分离株上观察到靶向塔兰托利什曼原虫的探针有有限的交叉反应。与无利什曼原虫犬血和雄性白蛉对照未观察到交叉反应。
该方案可成为在婴儿利什曼原虫和塔兰托利什曼原虫同域分布地区对犬宿主和白蛉进行全面监测的有价值方法。
