优势标记（ISSR-PCR）与共显性标记（RLEP-PCR）用于麻风病实验室诊断的比较评估。

Dominant marker (inter-simple sequence repeat-polymerase chain reaction) versus codominant marker (RLEP-polymerase chain reaction) for laboratory diagnosis of leprosy: A comparative evaluation.

机构信息

Department of Epidemiology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India.

Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research, New Delhi, India.

出版信息

Int J Mycobacteriol. 2020 Jan-Mar;9(1):18-23. doi: 10.4103/ijmy.ijmy_190_19.

DOI:10.4103/ijmy.ijmy_190_19

PMID:32474483

Abstract

BACKGROUND

Leprosy is a contagious disease and was eliminated globally in 2002. Since then, new cases were continuously detected from different parts of the world. Untreated leprosy cases shed millions of bacteria and are the main cause of dissemination of the disease. Currently, leprosy is detected by acid-fast bacilli (AFB) microscopy and has a low sensitivity ranging from 10% to 50%. The correlation between clinical findings and microscopy is unable to provide a conclusive case detection. Thus, in the present study, we compared to molecular methods, namely RLEP-polymerase chain reaction (RLEP-PCR) and inter-simple sequence repeat-PCR (ISSR-PCR) taking AFB microscopy as a gold standard for the detection of leprosy.

METHODS

A total of 168 clinically diagnosed leprosy patients were recruited in this study including 58 multibacillary and 110 paucibacillary patients. Slit-skin smear samples were taken for both microscopy and molecular study. Primers for RLEP-PCR were taken from the previous reports. The primers for ISSR-PCR were designed by screening the whole genome of Mycobacterium leprae TN strain (GenBank accession AL450380) for the presence of simple sequence repeats. One primer (TA)CAwas synthesized and used for molecular amplification of ISSR-PCR.

RESULTS

We found that the efficacy of the AFB microscopy was 24.40%, whereas the efficacy of RLEP-PCR and ISSR-PCR was 63.09% and 73.21% (P = 0.000, 0.000, and 0.469), respectively. The area under the curve of receiver operating characteristic curve for the comparison of three diagnostic methods was 0.845. An enhancement of 48.81% in the case detection rate by ISSR-PCR over AFB microscopy and 10.12% over RLEP-PCR was also found. Our study clearly reveals that ISSR-PCR is a better tool for diagnosis of leprosy than AFB microscopy and RLEP-PCR. Interestingly, both the PCR techniques RLEP-PCR and ISSR-PCR are able to detect samples which were negative for AFB microscopy.

CONCLUSION

Thus, the demonstration of ISSR-PCR in SSS samples can provide a better sensitive and confirmative tool for early diagnosis of leprosy.

摘要

背景

麻风病是一种传染性疾病，已于 2002 年在全球范围内被消除。自那时以来，世界各地不断发现新的病例。未经治疗的麻风病患者会排出数百万细菌，是疾病传播的主要原因。目前，麻风病通过抗酸杆菌（AFB）显微镜检查发现，其灵敏度范围为 10%至 50%，较低。临床发现与显微镜检查之间的相关性无法提供明确的病例检测。因此，在本研究中，我们比较了分子方法，即 RLEP-聚合酶链反应（RLEP-PCR）和简单重复序列间 PCR（ISSR-PCR），以 AFB 显微镜检查作为麻风病检测的金标准。

方法

本研究共招募了 168 例临床诊断为麻风病的患者，包括 58 例多菌型和 110 例少菌型患者。对每位患者的皮损刮片样本同时进行显微镜检查和分子研究。RLEP-PCR 的引物取自先前的报道。ISSR-PCR 的引物通过筛选麻风分枝杆菌 TN 株（GenBank 登录号 AL450380）的全基因组中简单重复序列的存在情况而设计。合成了一条引物（TA）CA 并用于 ISSR-PCR 的分子扩增。

结果

我们发现 AFB 显微镜检查的效果为 24.40%，而 RLEP-PCR 和 ISSR-PCR 的效果分别为 63.09%和 73.21%（P=0.000、0.000 和 0.469）。三种诊断方法比较的受试者工作特征曲线下面积为 0.845。与 AFB 显微镜检查相比，ISSR-PCR 可将病例检出率提高 48.81%，与 RLEP-PCR 相比提高 10.12%。我们的研究清楚地表明，ISSR-PCR 是一种比 AFB 显微镜检查和 RLEP-PCR 更好的麻风病诊断工具。有趣的是，两种 PCR 技术 RLEP-PCR 和 ISSR-PCR 都能够检测到 AFB 显微镜检查为阴性的样本。