Rezaei Zahra, Azarang Eqlima, Shahabi Saeed, Omidian Mostafa, Pourabbas Bahman, Sarkari Bahador
Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
J Trop Med. 2020 Aug 28;2020:5351098. doi: 10.1155/2020/5351098. eCollection 2020.
It has been documented that the genotypic traits in symptomatic and asymptomatic cases of visceral leishmaniasis (VL) may be different. The current study aimed to find out and compare the genotype and intraspecies diversity of Internal Transcribed Spacer 1 (ITS1) from asymptomatic and symptomatic VL cases in southern Iran. . Buffy coat samples from seven VL patients, with clinical signs and symptoms, and seven asymptomatic VL cases, were evaluated in this study. Samples of asymptomatic individuals were obtained from children living in a VL endemic area in southern Iran, while the samples of symptomatic subjects were obtained from patients admitted to hospitals with a diagnosis of VL. DNA was extracted from the buffy coats of the samples and PCR-amplified, targeting the ITS1of . The PCR products were sequenced, and the consensus sequences were assembled and multiple-aligned with a set of strains retrieved from the GenBank, using Clustal W. The phylogenetic tree was rooted, using MEGAX software, and the diversities based on haplotype and nucleotides, as well as the number of polymorphic sites, were measured using DnaSP v5.0 software. The results of ITS1 sequencing in 5 out of 7 asymptomatic VL cases showed 99.25% to 100% similarity with the ITS1 sequence (accessed number: MN648746), and one isolate was considered as just sp. In one sample, 99.75% similarity was seen with the ITS1 sequence of . Of the symptomatic VL patients, the PCR product revealed a 340 bp band corresponding to in all of the samples. By analyzing the ITS1 sequences, all seven sequences formed a clade somewhat different from other species and considered as sp. Haplotype and nucleotide diversity were much more prevalent in symptomatic cases where six haplotypes were seen in the ITS1 of from symptomatic patients and only two haplotypes were observed in the samples from asymptomatic cases. The findings of the current study showed that the ITS1 from symptomatic VL and asymptomatic cases has significant genetic differences. Besides, infection with was reported, for the first time, in an asymptomatic case, which deserves further study.
有文献记载，内脏利什曼病（VL）有症状和无症状病例的基因型特征可能不同。本研究旨在找出并比较伊朗南部无症状和有症状VL病例的内部转录间隔区1（ITS1）的基因型和种内多样性。本研究对7例有临床体征和症状的VL患者以及7例无症状VL病例的血沉棕黄层样本进行了评估。无症状个体的样本取自伊朗南部VL流行地区的儿童，而有症状受试者的样本取自因VL诊断入院的患者。从样本的血沉棕黄层中提取DNA并进行PCR扩增，以靶向其ITS1。对PCR产物进行测序，将一致序列进行组装，并使用Clustal W与从GenBank检索的一组菌株进行多重比对。使用MEGAX软件构建系统发育树的根，并使用DnaSP v5.0软件测量基于单倍型和核苷酸的多样性以及多态性位点的数量。7例无症状VL病例中有5例的ITS1测序结果显示与ITS1序列（登录号：MN648746）的相似度为99.25%至100%，有一个分离株被认为只是某种利什曼原虫。在一个样本中，与某ITS1序列的相似度为99.75%。在有症状的VL患者中，PCR产物在所有样本中均显示出对应于某利什曼原虫的340 bp条带。通过分析ITS1序列，所有7个序列形成了一个与其他利什曼原虫物种有些不同的进化枝，并被认为是某种利什曼原虫。单倍型和核苷酸多样性在有症状病例中更为普遍，有症状患者的ITS1中有6种单倍型，而无症状病例的样本中仅观察到2种单倍型。本研究结果表明，有症状和无症状VL病例的利什曼原虫ITS1存在显著的遗传差异。此外，首次在无症状病例中报告了某种利什曼原虫感染，这值得进一步研究。
