Drug-Resistant Tuberculosis on the Balkan Peninsula: Determination of Drug Resistance Mechanisms with Xpert MTB/XDR and Whole-Genome Sequencing Analysis

ABSTRACT 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 ethA 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 oxyR-ahpC 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. IMPORTANCE 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 ethA 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.