Detection of pyrazinamide heteroresistance in Mycobacterium tuberculosis

Abstract

AimPyrazinamide (PZA) is a first-line key agent in the effective treatment of tuberculosis (TB), including PZA susceptible multidrug-resistant tuberculosis (MDR-TB). Occasionally, TB patients might have mixed infections with both drug-sensitive and -resistant strains. This is termed heteroresistance. If 10% of the bacterial population is resistant to PZA, there is an increased risk for poor treatment outcome. The aim of this study is to evaluate the ability of the three established drug susceptibility testing (DST) techniques – BACTEC MGIT 960, Wayne’s pyrazinamidase test and Sanger sequencing of the pncA gene – to detect 10% PZA heteroresistance. MethodsMixed cultures of the fully drug susceptible Mycobacterium tuberculosis H37Rv reference strain and two laboratory-generated isogenic H37Rv mutants (with C475G and T254C pncA mutations, respectively) were made in proportions of 100%, 10%, 5% and 1% of the PZA-resistant (PZA-R) strain. Corresponding mixed cultures were also made using one drug-susceptible and one PZA-resistant MDR clinical isolate with the T62G pncA mutation, both belonging to one specific MIRU cluster. Additional mixes of 50%, 75%, 90% and 99% of the PZA-R strains were prepared for the Wayne’s test. Tests were for all methods performed in duplicates at two separate occasions. ResultsUsing the MGIT system, the in vitro-generated PZA-R strains were generally detected at a 5% proportion while the clinical PZA-R isolate only was detected at the critical 10% proportion, except for one test occasion. Sanger sequencing was unable to detect 10% PZA heteroresistance. Wayne’s test also failed to detect the critical level of 10% PZA resistance; instead it displayed misguiding results determining highly resistant samples as susceptible. ConclusionHeteroresistance is caused by present drug-resistance development and/or dual infections with one resistant and one susceptible strain. Mixed infections with resistant strains may occur in up to 20% of all TB cases in high burden areas, according to limited data. This study showed that only the phenotypic BACTEC MGIT system was capable in determining the critical proportion of 10% PZA resistance, whereas neither the Sanger nor the Wayne’s test were successful in this respect. This indicates a need for diagnostic tools with increased sensitivity to determine heteroresistance in M. tuberculosis.