Departments of Medical Biotechnology, University of Gondar, Ethiopia and Addis Ababa Science and Technology University, Addis Ababa, Ethiopia.
Department of Medical Biotechnology, University of Gondar, Gondar, Ethiopia.
BMC Infect Dis. 2022 Dec 29;22(1):963. doi: 10.1186/s12879-022-07930-1.
Visceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples.
DNA extraction was performed on a total of 90 smear slide samples using phenol-chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme.
Of the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR-RFLP patterns.
In conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR-RFLP is more recommendable than a microscope.
内脏利什曼病是由利什曼原虫复合种引起的，如果不及时治疗，可传播到内部器官并导致死亡。利什曼病的诊断基于临床症状和体征、显微镜检查、血清学和分子技术。由于临床表现广泛多样，不同种属的反应相似，因此通常难以确定种属水平，这对患者的正确治疗和管理至关重要。因此，本研究旨在评估 ITS1 区 PCR-RFLP 分析用于鉴定来自临床涂片患者样本的利什曼原虫种属。
采用酚-氯仿法从 90 份涂片样本中提取 DNA。使用利什曼原虫参考株 DNA 确定 PCR 检测下限。使用 LITSR/L5.8S 属特异性引物扩增 ITS1 区，得到 320bp 的产物，然后用 HaeIII 限制性内切酶进行 ITS1-PCR 产物的 RFLP 分析，以确认利什曼原虫种属。
在总样本中，ITS1-PCR 分别显示 42 份（46.7%）、6 份（6.7%）、37 份（41.1%）和 5 份（5.6%）为真阳性、假阳性、真阴性和假阴性。以显微镜检查为金标准，ITS1-PCR 技术的灵敏度、特异性、阳性预测值和阴性预测值分别为 89.4%、86.0%、87.5%和 88.1%。所有 ITS1-PCR 阳性的临床样本经 PCR-RFLP 图谱均确认为利什曼原虫种属。
总之，ITS1-RFLP 方法对内脏利什曼病患者的阴性临床涂片样本中利什曼原虫种属的鉴定具有较高的灵敏度和特异性。两种方法之间也存在显著的相关性和一致性。对于直接从临床样本中鉴定利什曼原虫种属，无论属和种属水平，PCR-RFLP 比显微镜更具推荐价值。
