Centro de Investigaciones en Microbiología y Biotecnología-UR (CIMBIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
Animal Hospital of Smithson Valley, 286 Singing Oaks, Ste 113, Spring Branch, TX, 78070, USA.
Parasit Vectors. 2023 Dec 18;16(1):458. doi: 10.1186/s13071-023-06073-9.
Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp.
A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform.
The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection.
This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases.
利什曼病是一种由利什曼属的专性细胞内原生动物引起的寄生虫病。这种感染的临床表现广泛，症状在很大程度上取决于致病寄生虫的种类。在这里，我们提出了一种新的 70-kDa 热休克蛋白基因（hsp70）标记物的设计和应用，该标记物长 771bp（HSP70-Long）。我们评估了其敏感性、特异性和诊断性能，采用基于扩增子的 MinION™ DNA 测序检测来鉴定来自人类和有皮肤利什曼病（CL）和内脏利什曼病（VL）的临床样本中的不同利什曼物种。我们还对我们的新型标记物与之前发表的 HSP70 标记物 HSP70-Short 进行了比较分析，后者跨越 330bp。
我们收集了来自哥伦比亚、委内瑞拉和美国的 27 个样本的数据集，其中 26 个样本来自患有 CL 的人和狗和猫，一个样本来自美国患有 VL 的狗（但最初来自希腊）。从每个样本中提取 DNA，并使用两种不同的 HSP70 标记物（HSP70-Short 和 HSP70-Long）进行常规 PCR 扩增。随后，使用 MinION™测序平台对产物进行测序。
结果突出了新设计的 HSP70-Long 引物的独特特征，展示了该引物的显著特异性，尽管其敏感性低于 HSP70-Short 标记物。值得注意的是，这两种标记物都具有很强的区分能力，不仅可以区分利什曼属内的不同物种，还可以识别样本中的合并感染。
本研究强调了基于 HSP70 的 MinION™ 测序在区分不同利什曼物种和识别来自利什曼病病例样本中的合并感染事件方面的出色特异性和有效性。
