Real-time PCR primers used in the study.

Leishmaniasis is a significant public health concern in large parts of the world including Israel, with limited diagnostic tools available for effective surveillance and control. Traditional methods for sand fly species identification, Leishmania detection, and blood meal analysis are time-consuming and prone to errors. To address these challenges, this study aimed to develop PCR-high resolution melt (HRM) assays to accurately determine sand fly species, Leishmania infection and blood meal sources in sand flies. Field-collected sand flies from all regions of Israel were used for the validation of three PCR-HRM assays. These included 254 sand fly males and females identified morphologically for species verification; 1,120 unfed females for Leishmania detection, and 538 engorged females for blood meal identification. PCR products were subjected to HRM curve analysis, and results were compared to nucleotide sequencing and sand fly morphology. Eleven sand fly species, 25 different host species blood meals and four Leishmania species were discerned and each presented a specific HRM pattern. Of the 1,658 analyzed females, 16 (1%) were positive for Leishmania, and the species identified were: Leishmania major, L. tropica, L. infantum and L. donovani. Blood meal source was identified in 520 (96.7%) engorged females. Blood from four animal species (domestic cat, rock hyrax, European hare, cow) accounted for 53% of the sand fly blood meals and the remaining 47% came from 21 other animal species. The sand fly species distribution showed that L. major and L. donovani vectors were mostly prevalent in arid southern Israel while L. tropica and L. infantum vectors were abundant in central and northern Israel. These results present the current knowledge of the different Leishmania species life cycles, vectors, and host species present in Israel and substantiate the utility of the assays developed herein which combine the advantages of PCR and the discriminatory power of HRM.利什曼病（Leishmaniasis）是全球广大区域内的重大公共卫生问题，以色列亦受其波及，当前可用的诊断工具难以满足有效监测与防控需求。传统的白蛉（sand fly）物种鉴定、利什曼原虫检测以及血源分析方法不仅耗时冗长，且易出现检测误差。为应对上述挑战，本研究旨在开发聚合酶链反应-高分辨率熔解（PCR-high resolution melt, HRM）检测方法，以精准判定白蛉物种、白蛉体内的利什曼原虫感染状态以及白蛉的吸血宿主来源。 本研究使用以色列全境野外采集的白蛉样本对三种PCR-HRM检测方法进行验证，样本涵盖三类：经形态学鉴定以验证物种正确性的254只雌雄白蛉；用于利什曼原虫检测的1120只未吸血雌蛉；以及用于血源鉴定的538只已吸血雌蛉。对扩增得到的PCR产物进行HRM曲线分析，并将检测结果与核苷酸测序（nucleotide sequencing）及白蛉形态学鉴定结果进行比对。 本次研究共区分出11种白蛉、25种不同宿主的血源以及4种利什曼原虫，每一类对象均具备独特的HRM熔解图谱。在1658只被分析的雌蛉中，有16只（占比1%）利什曼原虫检测结果呈阳性，所鉴定出的病原物种为：硕大利什曼原虫（Leishmania major）、热带利什曼原虫（L. tropica）、婴儿利什曼原虫（L. infantum）以及杜氏利什曼原虫（L. donovani）。在520只（占比96.7%）已吸血雌蛉中成功鉴定出血源信息。其中家猫、岩蹄兔、欧洲野兔与牛四种动物的血液占白蛉吸血来源的53%，剩余47%的血源则来自另外21种动物。 白蛉物种的分布特征显示：硕大利什曼原虫与杜氏利什曼原虫的传播媒介主要流行于以色列南部干旱区域，而热带利什曼原虫与婴儿利什曼原虫的传播媒介则大量分布于以色列中部与北部地区。本研究结果阐明了以色列境内目前已知的各类利什曼原虫的生活周期、传播媒介以及宿主物种分布情况，同时证实了本研究开发的整合了聚合酶链反应优势与高分辨率熔解鉴别能力的检测方法具有良好的实用价值。