A diagnostic algorithm to investigate pyrazinamide and ethambutol resistance in rifampicin resistant Mycobacterium tuberculosis isolates in a low incidence setting

Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is know to yield unreliable and inaccurate results especially in the context of multi-drug resistant tuberculosis (MDR-TB). In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for MDR-TB isolates. Sequencing results were validated using whole genome sequences (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. In contrast, phenotypic DST for ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance conferring mutations were found by sequencing the embB gene up to codon 497. In several isolates that tested ethambutol susceptible by phenotypic DTS, WGS identified known resistance conferring embB mutations. Thus, Sanger sequencing beyond he embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility results enabling clinicians to decide whether to include ethambutol as part of a MDR-TB regimen.