A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination

Abstract

Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 agar proportion (AP) critical concentrations and also at higher dilutions. The epidemiological cutoff value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Sanger sequencing was performed (atpE and Rv0678 genes) for any isolate testing resistant. The composite reference standard (CRS) defined susceptibility or resistance as is if all pDST methods agreed. If the pDST result was discordant, sequencing results were used for final classification. Geographically diverse and bedaquiline-unexposed isolates were tested (n = 495). The epidemiological cutoff value for BMD was confirmed to be 0.12 μg/ml. The majority of isolates were determined to be susceptible by all methods (467/495; 94.3%), and 28 were determined to be resistant by at least one method; 4 of these were determined to be resistant by all methods. Of the 28 resistant isolates, 12 harbored Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be determined to be resistant by more than one method (5/7) compared to isolates with a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4 to 3.4%, depending on the method used. All methods performed well for bedaquiline susceptibility determination; however, resistance detected should be investigated by a second, alternative method.

Keywords: Mycobacterium tuberculosis; bedaquiline; drug resistance; drug susceptibility testing; tuberculosis.

FIG 1

BDQ MIC distribution for BMD frozen plates by particular laboratory (a) and by all laboratories (n = 494) (b). The total number of isolates was 494 (Lab 1, 104; Lab 2, 100; Lab 3, 100; Lab 4, 91; and Lab 5, 99). The arrow indicates the ECOFF determined by visual inspection.

FIG 2

BDQ MIC distribution for BMD dry plates by particular laboratory (a) and by all laboratories (n = 494) (b). The total number of isolates was 494 (Lab 1, 104; Lab 2, 100; Lab 3, 100; Lab 4, 91; and Lab 5, 99). The arrow indicates the ECOFF determined by visual inspection.

FIG 3

ECOFF plots and values for BMD frozen (a) and dry (b) plates.

FIG 4

Diagrammatic representation of BDQ DST results for discordance isolates by method stratified by CRS susceptible (n = 14) (a) and resistant (n = 10) (b). AP, agar proportion; BMD, broth microdilution.