Detection of multidrug-resistance in Mycobacterium tuberculosis by phenotype- and molecular-based assays.

BACKGROUND: The whole-genome sequencing (WGS) is becoming an increasingly effective tool for rapid and accurate detection of drug resistance in Mycobacterium tuberculosis complex (MTBC). This approach, however, has still been poorly evaluated on strains from Central and Eastern European countries. The purpose of this study was to assess the performance of WGS against conventional drug susceptibility testing (DST) for the detection of multi-drug resistant (MDR) phenotypes among MTBC clinical strains from Poland and Lithuania. METHODS: The study included 208 MTBC strains (130 MDR; 78 drug susceptible), recovered from as many tuberculosis patients in Lithuania and Poland between 2018 and 2021. Resistance to rifampicin (RIF) and isoniazid (INH) was assessed by Critical Concentration (CC) and Minimum Inhibitory Concentration (MIC) DST as well as molecular-based techniques, including line-probe assay (LPA) and WGS. The analysis of WGS results was performed using bioinformatic pipeline- and software-based tools. RESULTS: The results obtained with the CC DST were more congruent with those by LPA compared to pipeline-based WGS. Software-based tools showed excellent concordance with pipeline-based analysis in prediction of RIF/INH resistance. The RIF-resistant strains demonstrated a relatively homogenous MIC distribution with the mode at the highest tested MIC value. The most frequent RIF-resistance conferring mutation was rpoB S450L. The mode MIC for INH was two-fold higher among double katG and inhA mutants than among single katG mutants. The overall rate of discordant results between all methods was calculated at 5.3%. Three strains had discordant results by both genotypic methods (LPA and pipeline-based WGS), one strain by LPA only, three strains by MIC DST, two strains by both MIC DST and pipeline-based WGS, and the remaining two strains showed discordant results with all three methods, compared to CC DST. CONCLUSIONS: Considering MIC DST results, current CCs of the first-line anti-TB drugs might be inappropriately high and may need to be revised. Both molecular methods demonstrated 100% specificity, while pipeline-based WGS had slightly lower sensitivity for RIF and INH than LPA, compared to CC DST.

Spoligotyping of Mycobacterium tuberculosis - Comparing in vitro and in silico approaches.

Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches. The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet. Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively. All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.

Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study.

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical isolates in Lithuania.

Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation -15C-->T in the promoter of the mabA-inhA operon.