Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis

IF 4.6 2区医学 Q1 MICROBIOLOGY

Annals of Clinical Microbiology and Antimicrobials Pub Date : 2024-04-05 DOI:10.1186/s12941-024-00687-2

Yu Jeong Choi, Yoonjung Kim, Hye Jung Park, Dokyun Kim, Hyukmin Lee, Young Ah Kim, Kyung-A Lee

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引用次数: 0

Abstract

The prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial. A multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes. The analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12–100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment. We developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

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开发用于检测和监测结核分枝杆菌和耐药结核病的多重液滴数字 PCR 方法

韩国结核病患者的耐多药结核病（MDR-TB）发病率约为 4.1%，高于经合组织 2.6%的平均水平。药物使用不足和患者依从性差会通过选择性压力增加 MDR-TB 的流行。因此，在诊断时及时发现结核病患者的耐药性并在治疗过程中对这些耐药菌株进行定量监测至关重要。我们开发了一种多重液滴数字 PCR（ddPCR）检测方法，并使用从韩国国家结核病协会购买的 9 株已知突变状态的结核分枝杆菌 DNA 材料进行了评估。我们共收集了 18 份转入 PCR 分析的 MDR-TB 残留样本。从样本中提取总 DNA 并进行四重 ddPCR 分析。其结果与已知耐药表型的结果进行了比较。多重 ddPCR 检测法检测 INH、RIF、EMB、FQ 和 SM 耐药性突变的分析灵敏度和特异性分别为 71.43% 至 100%，94.12% 至 100%。后续样本结果表明，即使在开始治疗后，四重 ddPCR 检测法也能灵敏地检测到 IS6110 和其他突变。我们开发了一种灵敏而准确的多重 ddPCR 检测方法，可定量检测结核病的存在，并同时检测耐药性突变。这一工具可帮助临床医生诊断和监测结核病的治疗。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Annals of Clinical Microbiology and Antimicrobials MICROBIOLOGY-

CiteScore

8.60

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： Annals of Clinical Microbiology and Antimicrobials considers good quality, novel and international research of more than regional relevance. Research must include epidemiological and/or clinical information about isolates, and the journal covers the clinical microbiology of bacteria, viruses and fungi, as well as antimicrobial treatment of infectious diseases. Annals of Clinical Microbiology and Antimicrobials is an open access, peer-reviewed journal focusing on information concerning clinical microbiology, infectious diseases and antimicrobials. The management of infectious disease is dependent on correct diagnosis and appropriate antimicrobial treatment, and with this in mind, the journal aims to improve the communication between laboratory and clinical science in the field of clinical microbiology and antimicrobial treatment. Furthermore, the journal has no restrictions on space or access; this ensures that the journal can reach the widest possible audience.