Gomal Centre of Biochemistry and Biotechnology, Gomal University, Dera Ismail Khan, Khyber Pakhtunkhwa, Pakistan; Programmatic Management of Drug resistant TB Unit, TB Culture Laboratory, Mufti Mehmood Memorial Teaching Hospital, Dera Ismail Khan, Khyber Pakhtunkhwa, Pakistan.
Department of Mathematic, University of Science and Technology, Bannu, Khyber Pakhtunkhwa, Pakistan.
Infect Genet Evol. 2019 Jul;71:42-46. doi: 10.1016/j.meegid.2019.03.007. Epub 2019 Mar 16.
Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.
在发展中国家，如巴基斯坦，结核病（TB）的耐药性是一个重大的公共卫生挑战。多重等位基因特异性聚合酶链反应（MAS-PCR）是一种 DNA 扩增方法，有助于快速检测和控制巴基斯坦的耐药性结核病（DR-TB）。本研究的目的是测试 MAS-PCR 在巴基斯坦检测耐药性的效用。药物敏感性测试（DST）用于鉴定来自巴基斯坦结核病病例的利福平耐药和敏感临床分离株。MAS-PCR 用于检测 213 株耐药和 37 株敏感分离株中 rpoB 基因中最常见的突变。在 213 株临床分离株中，MAS-PCR 鉴定出 24 株（11.3%）突变 D435Y（天冬氨酸 435 突变为酪氨酸）、14 株（6.6%）突变 H445Y（组氨酸 445 突变为酪氨酸）、124 株（58.2%）突变 S450L（丝氨酸 450 突变为亮氨酸）和 18 株（8.4%）突变 S450W（丝氨酸 450 突变为色氨酸）。MAS-PCR 未在 33 株（15.5%）病例中检测到已知突变。在 12 株中，在 rpoB 基因中检测到密码子 434（蛋氨酸 434 突变为异亮氨酸）的新突变和密码子 435（天冬氨酸 435 突变为酪氨酸）的常见变异，表明存在双重突变。在 4 株中，鉴定出密码子 432（谷氨酰胺 432 突变为脯氨酸）的新突变。在另外 4 株中，鉴定出突变蛋氨酸 434 缬氨酸和组氨酸 445 天冬酰胺。此外，在 5 株中发现 rpoB 突变（亮氨酸 452 突变为脯氨酸）。DNA 测序证实了其余 8 株 rpoB 无突变。MAS-PCR 的敏感性为 88.3%，特异性为 100%，以 DST 为参考，这表明该方法可作为巴基斯坦初筛耐多药结核病（MDR-TB）的初步标志物。
