Ghasemloo Hadi, Rasti Sima, Delavari Mahdi, Doroodgar Abbas
Department of Parasitology and Mycology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Pak J Biol Sci. 2016;19(3):136-142. doi: 10.3923/pjbs.2016.136.142.
Cutaneous leishmaniasis is a common skin disease caused by leishmania parasite. An accurate diagnosis of parasites species is possible using molecular techniques. This study was carried out to compare internal transcribed spacer (ITS1) and kinetoplast deoxyribonucleic acid (KDNA) genes for identifying Leishmania species by Polymerase Chain Reaction (PCR), furthermore, genetic diversity of isolates was studied. This research examined 130 patients who were suspected of cutaneous leishmaniasis and referred to Kashan's health centers from 2011-2014. After DNA extraction from serosity, PCR were performed using ITS1 and KDNA primers. Cutaneous Leishmaniasis was diagnosed by the observation of 320 bp band in the ITS1-PCR. The PCR products were digested with restriction enzyme HaeIII and then leishmania species were identified by patterns of enzymatic digestion. The diagnostic criteria of Cutaneous Leishmaniasis (CL) in KDNA-PCR were based on the observation of 760 and 650 bp for Leishmaniasis tropica and Leishmaniasis major, respectively. Twelve isolates of leishmania were sequenced and the phylogenetic tree was traced using the results of sequencing by Mega 4 software. Out of 130 suspected patients to CL, 70 (53.8%) and 98 (75.4%) isolates were positive by Restriction Fragment Length Polymorphism (RFLP) of ITS1 and KDNA, respectively. Using ITS1 PCR, 60 samples (85.7%) and 10 samples (14.3%) were identified as L. tropica and L. major, respectively, ITS1-PCR had 25.3% false negative, compare to microscopy. While, microscopy had false negative in 13 cases compare to KDNA-PCR. Due to the lower sensitivity of the PCR-RFLP of ITS1, KDNA-PCR is recommended for diagnosis of CL. The L. tropica and L. major are the causative agents of CL.
皮肤利什曼病是一种由利什曼原虫寄生虫引起的常见皮肤病。使用分子技术可以准确诊断寄生虫种类。本研究旨在比较内转录间隔区（ITS1）和动基体脱氧核糖核酸（KDNA）基因，通过聚合酶链反应（PCR）鉴定利什曼原虫种类，此外，还研究了分离株的遗传多样性。本研究检查了2011年至2014年期间怀疑患有皮肤利什曼病并转诊至喀山健康中心的130名患者。从浆液中提取DNA后，使用ITS1和KDNA引物进行PCR。通过观察ITS1-PCR中的320 bp条带诊断皮肤利什曼病。PCR产物用限制性内切酶HaeIII消化，然后通过酶切模式鉴定利什曼原虫种类。KDNA-PCR中皮肤利什曼病（CL）的诊断标准分别基于热带利什曼原虫和硕大利什曼原虫的760 bp和650 bp条带的观察。对12株利什曼原虫进行测序，并使用Mega 4软件的测序结果绘制系统发育树。在130名疑似CL患者中，分别有70株（53.8%）和98株（75.4%）分离株通过ITS1和KDNA的限制性片段长度多态性（RFLP）检测呈阳性。使用ITS1 PCR，分别有60份样本（85.7%）和10份样本（14.3%）被鉴定为热带利什曼原虫和硕大利什曼原虫，与显微镜检查相比，ITS1-PCR有25.3%的假阴性。而与KDNA-PCR相比，显微镜检查有13例假阴性。由于ITS1的PCR-RFLP敏感性较低，建议使用KDNA-PCR诊断CL。热带利什曼原虫和硕大利什曼原虫是CL的病原体。
