Azam Mudsser, Singh Saurabh, Gupta Ratan, Mayank Mayank, Kathuria Sushruta, Sharma Shruti, Ramesh V, Singh Ruchi
ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi 110029, India.
Department of Dermatology, Venereology, Leprology, All India Institute of Medical Sciences, Jodhpur 342005, India.
Pathogens. 2024 Sep 4;13(9):759. doi: 10.3390/pathogens13090759.
The accurate diagnosis and identification of species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using , , and standard strain DNA. The assay was applied on 93 clinical samples with confirmed infection, including VL ( = 30), PKDL ( = 50), and CL ( = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for and up to 0.1 pg for and . The detection limit for the 7SL-RNA HRM-PCR was 1 pg for and 10 pg for and . The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL ( = 0.007), PKDL ( = 0.0002), and CL ( = 0.03) samples. The ITS-1 HRM-PCR assay could identify spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions.
准确诊断和鉴定利什曼原虫物种对于利什曼病的治疗选择和有效治疗至关重要。本研究旨在开发和评估高分辨率熔解曲线分析（HRM）-PCR在印度次大陆用于鉴定引起内脏利什曼病（VL）、黑热病后皮肤利什曼病（PKDL）和皮肤利什曼病（CL）的物种的应用。选择了两个多拷贝靶标（ITS-1和7SL-RNA基因），并使用杜氏利什曼原虫、婴儿利什曼原虫和硕大利什曼原虫标准菌株DNA建立了HRM-PCR检测方法。该检测方法应用于93例确诊利什曼原虫感染的临床样本，包括VL（n = 30）、PKDL（n = 50）和CL（n = 13）病例。ITS-1 HRM-PCR检测方法对杜氏利什曼原虫可检测低至0.01 pg的模板DNA，对婴儿利什曼原虫和硕大利什曼原虫可达0.1 pg。7SL-RNA HRM-PCR的检测限对杜氏利什曼原虫为1 pg，对婴儿利什曼原虫和硕大利什曼原虫为10 pg。ITS-1 HRM-PCR在93例利什曼病病例中鉴定出68例（73.11%），而7SL-RNA HRM-PCR仅能检测出93例中的18例（19.35%）。在VL（r = 0.007）、PKDL（r = 0.0002）和CL（r = 0.03）样本中，基于kDNA的低Ct值与ITS-1 HRM-PCR阳性之间观察到显著相关性。ITS-1 HRM-PCR检测方法可鉴定印度次大陆引起不同临床形式利什曼病的利什曼原虫物种，提供快速准确的结果，可指导临床管理和治疗决策。
