Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand.
PLoS One. 2024 Aug 26;19(8):e0307601. doi: 10.1371/journal.pone.0307601. eCollection 2024.
Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the Leishmania genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving Leishmania (Mundinia) martiniquensis and novel Leishmania (Mundinia) orientalis underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two Leishmania species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10-2 parasite/μL for L. martiniquensis and 10-4 parasite/μL for L. orientalis. Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic Leishmania species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for L. martiniquensis and 83.3% for L. orientalis, and an excellent agreement (κ value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these Leishmania species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications.
利什曼病是一种由利什曼原虫属的寄生虫原生动物引起的被忽视的热带病，仍然是一个全球性的健康关注问题，具有很高的发病率和死亡率。在泰国，涉及利什曼原虫（Mundinia）martiniquensis 和新型利什曼原虫（Mundinia）orientalis 的本土利什曼病病例的发病率上升，突显了准确诊断和有效控制策略的迫切需要。本研究提出了一种灵敏和特异的核酸侧向流动免疫分析（NALFIA），该方法将双重 PCR 分析与侧向流动装置（LFD）条带格式相结合。该方法针对内部转录间隔区 1（ITS1）区域，该区域因其保守和可变序列的独特组合而闻名，使用标记有生物素、地高辛和异硫氰酸荧光素（FITC）标记物的引物，能够精确鉴定物种并区分这两种利什曼原虫。值得注意的是，该检测方法的灵敏度超过了琼脂糖凝胶电泳，能够检测到低至 10-2 个寄生虫/μL 的 L. martiniquensis 和 10-4 个寄生虫/μL 的 L. orientalis。值得注意的是，该检测方法具有可靠的特异性，与其他主要内脏利什曼原虫物种或参考生物没有交叉扩增。使用 62 个临床样本进行的评估进一步证实了 PCR-LFD 检测方法的有效性，对于 L. martiniquensis 的灵敏度为 100%，对于 L. orientalis 的灵敏度为 83.3%，与嵌套 PCR 的一致性非常好（κ 值=0.948）。这种集成检测方法代表了诊断工具的一个有希望的进展，提供了快速和准确的结果，可以为有效疾病管理和控制做出重大贡献。鉴于这些利什曼原虫物种在当前公共卫生情况下的相关性增加，该检测方法是诊断和研究应用的有价值工具。
