Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
PLoS One. 2012;7(7):e40456. doi: 10.1371/journal.pone.0040456. Epub 2012 Jul 6.
The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.
先前尚未在中美洲（许多移民到美国的人都来自那里）研究导致结核分枝杆菌（Mycobacterium tuberculosis）耐药（Drug Resistance，DR）的个体基因突变的频率。目前，检测耐多药结核病（Multidrug-Resistant Tuberculosis，MDR-TB）的金标准是表型药物敏感性测试（Phenotypic Drug Susceptibility Testing，DST），这种方法资源密集且缓慢，会导致社区中 MDR-TB 的传播增加。我们评估了多重等位基因特异性聚合酶链反应（Multiplex Allele-Specific Polymerase Chain Reaction，MAS-PCR）作为一种快速分子工具，用于在巴拿马检测 MDR-TB。根据 DST，从存档中鉴定并培养了 67 株 MDR-TB 和 31 株药敏临床分离株。设计引物以针对导致一线药物异烟肼和利福平耐药的五个突变热点，然后进行 MAS-PCR。全基因组测序证实了 MAS-PCR 鉴定的 DR 突变，并提供了基因突变的频率。DNA 测序显示，70.1%的 MDR 株在 katG 基因的 315 密码子处发生点突变，19.4%在 mabA-inhA 启动子内，98.5%在 rpoB 的三个热点处。MAS-PCR 检测到了这些突变中的每一个，相对于 DST，异烟肼耐药的灵敏度为 82.8%，特异性为 100%，利福平耐药的灵敏度为 98.4%，特异性为 100%。巴拿马 MDR 株中个体 DR 突变的频率与其他结核病流行国家相似。MAS-PCR 的性能表明，它可能是一种相对廉价且技术上可行的方法，用于在发展中国家快速检测 MDR-TB。
