National TB Control Program, National TB Reference Laboratory, Islamabad 44000, Pakistan.
Department of Microbiology, Quaid-i-Azam University, Islamabad 44000, Pakistan.
J Med Microbiol. 2024 Sep;73(9). doi: 10.1099/jmm.0.001884.
The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms. This study aims to compare RIF DST in using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in and was also screened in the current and previous RIF resistance genomic isolates to find their compensatory role. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in , including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.
利福平（RIF）药敏试验（DST）表型和分子方法之间的不匹配在 中构成了重大挑战，可能导致误诊和不适当的治疗。比较 RIF 表型和分子方法，包括全基因组测序（WGS），可能有助于更好地了解耐药机制。本研究旨在通过两种表型和分子方法（包括 GeneXpert RIF 检测（GX）和 WGS）比较 中的 RIF DST，以更好地理解。该研究评估了两种表型液体培养基方法[Lowenstein-Jensen（LJ）和分枝杆菌生长指示剂管（MGIT）]、一种靶向分子方法（GX）和一种 WGS 方法。此外，还在当前和以前的 RIF 耐药 基因组分离物中筛选了 中的突变频率，以发现其补偿作用。总共 25 株 RIF 耐药分离株，包括 9 株来自治疗失败和复发病例，这些病例在 LJ、MGIT 和 GX 上的 DST 结果不一致和一致，均进行了 WGS。表型 DST 结果表明，11 株分离株（44%）在 LJ 和 MGIT 上敏感，但在 GX 上耐药。这些分离株在 中显示出多个突变，包括 Thr444>Ala、Leu430>Pro、Leu430>Arg、Asp435>Gly、His445>Asn 和 Asn438>Lys。相反，在 GX 和 MGIT 上敏感但在 LJ 上耐药的 4 株分离株在 WGS 中为 野生型。然而，这些分离株在 PonA1 基因中具有几个新的突变，包括 10 个核苷酸插入和两个非同义突变（Ala394>Ser、Pro631>Ser），以及 PonA2 中的一个非同义突变（Pro780>Arg）。与 WGS 相比，MGIT 上的 RIF DST 不匹配率高于 LJ 和 GX。德里/CAS 谱系中的这些不匹配主要与治疗失败和复发病例有关。RIF 耐药的 WGS 相对昂贵，但对于 MGIT、LJ 和 GX 上 DST 结果不一致的分离株，可能需要考虑进行 WGS，以确保准确的诊断和适当的治疗选择。
