Dueñas Eva, Nakamoto Jose A, Cabrera-Sosa Luis, Huaihua Percy, Cruz María, Arévalo Jorge, Milón Pohl, Adaui Vanessa
Laboratory of Biomolecules, Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas (UPC), Lima, Peru.
Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.
Front Microbiol. 2022 Sep 15;13:958693. doi: 10.3389/fmicb.2022.958693. eCollection 2022.
Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus , is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the . () subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10 (kDNA) or 5 × 10 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan- detection, whereas the kDNA PCR/CRISPR assay was specific for . () detection. No cross-reaction was observed with strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of infections at the genus and . () subgenus levels.
皮肤利什曼病是由利什曼原虫属的原生动物寄生虫引起的一种疾病，在拉丁美洲的许多地区是一个主要的公共卫生问题。鉴于在同一流行地区存在其他与利什曼病病变相似且同时出现的病症，其诊断较为困难。寄生虫学和分子方法相结合可实现准确诊断，传统上分子方法在集中式参考和研究实验室进行，因为它们需要专门的基础设施和操作人员。成簇规律间隔短回文重复序列/CRISPR相关蛋白（CRISPR/Cas）系统最近推动了用于核酸检测的创新工具的发展，这些工具具有高特异性、灵敏度和速度，并且易于适用于即时检测。在此，我们利用CRISPR-Cas12a系统对利什曼原虫属进行分子检测，重点关注在秘鲁和拉丁美洲其他流行地区传播的具有医学相关性的寄生虫物种，其中巴西利什曼原虫是皮肤和黏膜利什曼病的主要病原体。我们开发了两种针对利什曼病分子诊断中常用的多拷贝靶点的检测方法：18S核糖体RNA基因（18S rDNA），在利什曼原虫物种中高度保守，以及动基体DNA（kDNA）微小环的一个在Viannia（V.）亚属中保守的区域。我们基于CRISPR的检测方法在进行PCR预扩增后能够检测低至5×10（kDNA）或5×10（18S rDNA）个寄生虫基因组当量/反应。18S PCR/CRISPR检测方法实现了对利什曼原虫属全检测，而kDNA PCR/CRISPR检测方法对V.（V.）具有特异性。未观察到与Y菌株或人类DNA的交叉反应。我们将49份临床样本与作为参考检测的kDNA实时PCR检测方法进行比较，评估了这些检测方法的性能。kDNA PCR/CRISPR检测方法与参考检测方法表现相当，阳性和阴性百分比一致性均为100%。18S PCR/CRISPR检测方法的阳性和阴性百分比一致性分别为82.1%和100%。这些发现支持了新开发的基于CRISPR的分子工具在利什曼原虫属和V.（V.）亚属水平上对利什曼原虫感染进行一线诊断的潜在适用性。
