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

Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug resistant Mycobacterium tuberculosis complex 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. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance conferring mutations were found in the embB gene at codons 354, 406 and 497. In several isolates that tested ethambutol susceptible by phenotypic DTS, well-known resistance conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility as basis for rational design of MDR-TB regimens with or without ethambutol.

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