Molecular Mycobacteriology, Research Center Borstel, Germany.
BMC Microbiol. 2012 May 30;12:90. doi: 10.1186/1471-2180-12-90.
Drug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls.
The majority of isoniazid and rifampin resistant strains had mutations in katG315 (71.9%) and rpoB531 (50%). However, rpoB mutations in codons 511, 516 and 533 were also detected in five rifampin susceptible strains. MIC determinations revealed low-level rifampin resistance for those strains. Thus, the sensitivity and specificity of sequencing of katG for detection of drug resistance were 86.7% and 100% and for sequencing of rpoB 100% and 93.8%, respectively.Strikingly, none of the streptomycin resistant strains had mutations in rrs, but 47.5% harboured mutations in rpsL. Further changes were detected in gidB. Among ethambutol resistant strains 46.7% had mutations at embB306. Pyrazinamide resistant strains displayed a variety of mutations throughout pncA. The specificities of sequencing of rpsL, embB and pncA for resistance detection were high (96-100%), whereas sensitivities were lower (48.8%, 73.3%, 70%).
Our study reveals a good correlation between data from molecular and phenotypic resistance testing in this high-incidence setting. However, the fact that particular mutations in rpoB are not linked to high-level resistance is challenging and demonstrates that careful interpretation of molecular resistance assays is mandatory. In addition, certain variations, especially in gidB, appear to be phylogenetically informative polymorphisms rather than markers for drug resistance.
耐药性是结核分枝杆菌感染治疗的一个问题。对于分子耐药性检测，精确了解耐药性发展相关的基因组变异是至关重要的。然而，来自高发地区的数据却很少。因此，我们进行了系统的研究，分析了来自塞拉利昂的 97 株既往治疗过的结核分枝杆菌菌株，这些菌株在 katG、rpoB、rrs、rpsL、gidB、embB、pncA 中发生了突变，inhA 和 ahpC 中如果适用的话也进行了突变分析。在所研究的菌株中，50 株对异烟肼、利福平、链霉素、乙胺丁醇和吡嗪酰胺呈单药或多药耐药，或为 MDR，47 株完全敏感的菌株作为对照。
大多数异烟肼和利福平耐药菌株的 katG315（71.9%）和 rpoB531（50%）有突变。然而，在 5 株利福平敏感的菌株中也检测到 rpoB 密码子 511、516 和 533 的突变。MIC 测定显示这些菌株对利福平的耐药程度较低。因此，katG 测序检测耐药性的敏感性和特异性分别为 86.7%和 100%，rpoB 测序的敏感性和特异性分别为 100%和 93.8%。令人惊讶的是，没有一株链霉素耐药的菌株在 rrs 中有突变，但 47.5%的菌株 rpsL 有突变。在 gidB 中也检测到了进一步的变化。在乙胺丁醇耐药的菌株中，46.7%的菌株 embB306 有突变。吡嗪酰胺耐药的菌株在整个 pncA 中显示出多种突变。rpsL、embB 和 pncA 测序检测耐药性的特异性很高（96-100%），而敏感性较低（48.8%、73.3%、70%）。
在这个高发地区，我们的研究显示了分子耐药性检测和表型耐药性检测之间的良好相关性。然而，rpoB 中的某些突变与高水平耐药性无关，这一事实具有挑战性，表明需要仔细解释分子耐药性检测。此外，某些变异，特别是 gidB 中的变异，似乎是系统发生上有信息的多态性，而不是耐药性的标记。
