University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia.
Omega d.o.o., Ljubljana, Slovenia.
J Glob Antimicrob Resist. 2022 Jun;29:507-512. doi: 10.1016/j.jgar.2021.09.019. Epub 2021 Nov 21.
Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (DST) in clinical TB isolates from two countries of the Balkan Peninsula.
Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel.
Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS.
Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.
下一代测序（NGS）可以对与吡嗪酰胺（PZA）耐药性最相关的遗传改变进行全面分析。然而，目前尚无研究报道巴尔干半岛分枝杆菌结核（TB）分离株中 PZA 耐药的分子背景。我们旨在比较 Ion Torrent 技术与表型 BACTEC MGIT 960 药敏试验（DST）检测两种巴尔干半岛国家临床 TB 分离株中与 PZA 耐药相关基因（pncA）全长分析的可行性。
1996 年至 2017 年，我们回顾性选择了 61 株 TB 分离株。为了在从 TB 分离株中提取的基因组 DNA 中鉴定与耐药相关的基因变异，使用 AmpliSeq™试剂盒自动生成 AmpliSeq 文库，用于 Chef DL8 和 Ion AmpliSeq TB Research Panel。
在 61 株 TB 分离株中，有 56 株对任何抗生素均表型耐药。其中，38/56 株（67.9%）对吡嗪酰胺表型耐药，34/38 株（89.5%）检测到 pncA 突变。pncA 启动子区的突变是最常见的遗传改变，在 8 株 TB 分离株中均有发现。将 NGS 数据与传统的 BACTEC MGIT 960 DST 进行比较，两种方法在识别 PZA 耐药性方面具有非常强的一致性（91.8%），NGS 的敏感性（89.5%）和特异性（95.7%）均较高。
使用 NGS 检测 PZA 耐药性似乎是监测巴尔干半岛 TB 耐药性的一种有价值的工具，至少比表型 DST 提前 1 周提供有用信息的潜力巨大。
