Department of Medical Microbiology, School of Biomedical Sciences, College of Health Sciences, Makerere University, P,O Box 7072, Kampala, Uganda.
BMC Infect Dis. 2014 Sep 4;14:481. doi: 10.1186/1471-2334-14-481.
Introduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR.
A total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM).
Xpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant.
MDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.
Xpert® MTB/RIF 检测的引入通过同时检测细菌和利福平（rif）的耐药性，彻底改变了结核病（TB）的诊断，利福平是耐多药结核病（MDR-TB）的替代标志物，也是一线抗结核药物之一。一般来说，rpoB 突变可在全球 96.1%的利福平耐药结核分枝杆菌（MTB）菌株中发现，这些突变通常位于 MTB rpoB 基因的 507-533 位氨基酸残基（81bp）的区域，即利福平耐药决定区（RRDR）。在这项研究中，我们使用 Xpert® MTB/RIF 与琼脂比例法（Middlebrook 7H11）比较，确定了 Kampala 的 MDR-TB 频率，并进一步使用 Xpert® MTB/RIF 检测 RRDR 中 81bp 区的不同 rpoB 基因突变的探针频率。
2011 年 5 月至 2014 年 5 月期间，共对在 Makerere 大学分枝杆菌实验室接受 Xpert 检测的 1501 份标本进行了分析，采用 Xpert® MTB/RIF 检测。对 MTB 和利福平均阳性的标本进一步采用 Middlebrook 7H11 琼脂比例法（APM）进行完整的一线抗分枝杆菌药物敏感性检测。
Xpert® MTB/RIF 检测出 313 份 MTB 阳性标本，其中 12 份标本在 MTB 的 RRDR 81bp 区有四个不同的 rpoB 基因突变，同时具有 MTB 和利福平耐药性，进一步检测发现其中一个（1/12）标本对 APM 中的利福平单耐药，其余 11 个标本为 MDR-TB。与观察到的 rif 耐药相关的探针如下：E（7/12）、B（3/12）、A（1/12）、D（1/12），与探针 C 无关。没有一个标本产生与一个以上探针失效（突变组合）相关的 rif 耐药。探针 D 与利福平单耐药相关。
在研究人群中，MDR-TB 为 3.5%。与探针 E（58%）相关的突变，也称为密码子 531 和 533，是 Xpert® MTB/RIF 检测在这一环境中发现的最常见的 rpoB 基因突变，通过琼脂比例法抗菌药物敏感性试验，该检测的探针 E 鉴定出的突变实际上是 MDR-TB 株。未检测到密码子 522 的突变。
