Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: 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.