Truden Sara, Sodja Eva, Žolnir-Dovč Manca
University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia.
Microbiol Spectr. 2023 Mar 6;11(2):e0276122. doi: 10.1128/spectrum.02761-22.
The new molecular assay Xpert MTB/XDR (Cepheid, Sunnyvale, CA, USA) was launched in 2021 to detect Mycobacterium tuberculosis (MT) complex with mutations conferring resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolone (FQ), and second-line injectable drugs (SLIDs). The aim of our study was to evaluate the performance of the Xpert MTB/XDR rapid molecular assay on rifampicin-resistant, multidrug-resistant, and pre-extensively resistant tuberculosis (TB) isolates in a clinical laboratory in the Balkan Peninsula compared to a phenotypic drug susceptibility test (pDST). Xpert MTB/XDR was used to test positive Bactec MGIT 960 (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) cultures or DNA isolates. In the case of discrepant results between Xpert MTB/XDR and pDST, the usefulness of whole-genome sequencing (WGS) was emphasized. In our study, 80 MT isolates from different Balkan countries were selectively chosen from the National Mycobacterial Strain Collection in Golnik, Slovenia. Isolates were tested with the Xpert MTB/XDR assay, conventional pDST, and WGS. Xpert MTB/XDR showed high sensitivities of 91.9%, 100%, and 100% for detecting INH, FQ, and SLID resistance, respectively, compared to pDST. In contrast, low sensitivity (51.9%) for ETH resistance was achieved because isolates harbored widespread mutations across the gene. The specificity of Xpert MTB/XDR was 100% for all drugs except for INH (66.7%). Further investigation with WGS revealed -57c→t mutations in the region marked with uncertain significance, which caused the low specificity for detecting INH resistance with the new assay. Xpert MTB/XDR can be used in clinical laboratories for the rapid detection of INH, FQ, and SLID resistance. Moreover, it can be used to rule in resistance to ETH. Additional use of WGS is recommended in cases of discrepant results between pDST and Xpert MTB/XDR. Future improvements of Xpert MTB/XDR with the inclusion of additional genes may increase the usefulness of the assay. The Xpert MTB/XDR was tested on drug-resistant Mycobacterium tuberculosis complex isolates from the Balkan Peninsula. Positive Bactec MGIT 960 cultures or DNA isolates were tested as starting material. According to the results of our study with Xpert MTB/XDR, sensitivities for the detection of SLID, FQ, and INH resistance were sufficient (>90%) for the assay to be implemented into diagnostic algorithms. In our study, WGS revealed lesser-known mutations in genes conferring INH and ETH resistance, and their impact on resistance is still unknown. Mutations in the gene causing resistance to ETH were scattered along structural gene without high-confidence markers for resistance. Therefore, resistance to ETH should be reported based on a combination of methods. Because the Xpert MTB/XDR assay was found to have good performance, we propose that it should be the method of choice for confirming resistance to INH, FQ, and SLID and conditionally for resistance to ETH.
新型分子检测方法Xpert MTB/XDR（美国加利福尼亚州森尼韦尔市的塞菲德公司）于2021年推出，用于检测结核分枝杆菌（MT）复合群中对异烟肼（INH）、乙硫异烟胺（ETH）、氟喹诺酮（FQ）和二线注射用药物（SLIDs）具有耐药性的突变。我们研究的目的是在巴尔干半岛的一家临床实验室中，与表型药物敏感性试验（pDST）相比，评估Xpert MTB/XDR快速分子检测方法对耐利福平、耐多药和广泛耐药前结核病（TB）分离株的检测性能。使用Xpert MTB/XDR检测Bactec MGIT 960（美国新泽西州富兰克林湖市的BD公司）培养阳性的菌株或DNA分离株。如果Xpert MTB/XDR和pDST结果不一致，则强调全基因组测序（WGS）的作用。在我们的研究中，从斯洛文尼亚戈尔尼克的国家分枝杆菌菌株保藏中心中选择性地选取了来自巴尔干不同国家的80株MT分离株。对分离株进行Xpert MTB/XDR检测、传统pDST检测和WGS检测。与pDST相比，Xpert MTB/XDR检测INH、FQ和SLID耐药性的敏感性分别高达91.9%、100%和100%。相比之下，由于分离株在该基因上存在广泛的突变，检测ETH耐药性的敏感性较低（51.9%）。除INH外（66.7%），Xpert MTB/XDR对所有药物的特异性均为100%。WGS进一步研究发现，在意义不明确的区域存在-57c→t突变，这导致了该新检测方法检测INH耐药性的特异性较低。Xpert MTB/XDR可用于临床实验室快速检测INH、FQ和SLID耐药性。此外，它可用于判定ETH耐药性。当pDST和Xpert MTB/XDR结果不一致时，建议额外使用WGS。未来通过纳入更多基因对Xpert MTB/XDR进行改进，可能会提高该检测方法的实用性。对来自巴尔干半岛的耐多药结核分枝杆菌复合群分离株进行了Xpert MTB/XDR检测。以Bactec MGIT 960培养阳性的菌株或DNA分离株作为起始材料进行检测。根据我们使用Xpert MTB/XDR的研究结果，检测SLID、FQ和INH耐药性的敏感性足以满足（>90%）将该检测方法纳入诊断算法的要求。在我们的研究中，WGS揭示了赋予INH和ETH耐药性的基因中鲜为人知的突变，其对耐药性的影响仍不清楚。导致ETH耐药性的该基因中的突变分散在结构基因上，没有耐药性的高可信度标记。因此，应结合多种方法报告ETH耐药性。由于发现Xpert MTB/XDR检测方法性能良好，我们建议它应作为确认INH、FQ和SLID耐药性以及有条件确认ETH耐药性的首选方法。
