Thin-Layer-Agar-Based Direct Phenotypic Drug Susceptibility Testing on Sputum in Eswatini Rapidly Detects Mycobacterium tuberculosis Growth and Rifampicin Resistance Otherwise Missed by WHO-Endorsed Diagnostic Tests

Abstract

Xpert MTB/RIF rapidly detects resistance to rifampicin (RR); however, this test misses I491F-RR conferring rpoB mutation, common in southern Africa. In addition, Xpert MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis (MTB). We aimed to investigate the ability of thin-layer agar (TLA) direct drug-susceptibility testing (DST) to detect MTB and its drug-resistance profiles in field conditions in Eswatini. Consecutive samples were tested in parallel with Xpert MTB/RIF and TLA for rifampicin (1.0 μg/ml) and ofloxacin (2.0 μg/ml). TLA results were compared at the Reference Laboratory in Antwerp with indirect-DST on Löwenstein-Jensen or 7H11 solid media and additional phenotypic and genotypic testing to resolve discordance. TLA showed a positivity rate for MTB detection of 7.1% versus 10.0% for Xpert MTB/RIF. Of a total of 4,547 samples included in the study, 200 isolates were available for comparison to the composite reference. Within a median of 18.4 days, TLA detected RR with 93.0% sensitivity (95% confidence interval [CI], 77.4 to 98.0) and 99.4% specificity (95% CI, 96.7 to 99.9) versus 62.5% (95% CI, 42.7 to 78.8) and 99.3% (95% CI, 96.2 to 99.9) for Xpert MTB/RIF. Eight isolates, 28.6% of all RR-confirmed isolates, carried the I491F mutation, all detected by TLA. TLA also correctly identified 183 of the 184 ofloxacin-susceptible isolates (99.5% specificity; 95% CI, 97.0 to 99.9). In field conditions, TLA rapidly detects RR, and in this specific setting, it contributed to detection of additional RR patients over Xpert MTB/RIF, mainly but not exclusively due to I491F. TLA also accurately excluded fluoroquinolone resistance.

Keywords: MDR; TLA; XDR; Xpert MTB/RIF; resistance detection; rpoB I491F mutant; tuberculosis.

FIG 1

Phenotypic rifampicin resistance testing results from the site (direct, TLA) and the reference laboratory (indirect, LJ). Xpt, Xpert MTB/RIF; INC, result inconclusive (error, invalid, no result); RMP, rifampicin; GC, growth control; NTM, nontuberculous mycobacteria; IND, indeterminate; NA, not available; (n), administrative errors excluded; for details refer to Table S1. In the gray area, results for 172 isolates with indirect-DST, Xpert, and TLA results available, including administrative errors; *, 100 isolates selected for WGS, in addition to all RR detected by any method.

FIG 2

Number of positive plates and cumulative percentage per day of reading. Sm⁺, sputum smear microscopy positive; Sm⁻, sputum smear microscopy negative. Indication of median positivity and drug susceptibility results for rifampicin-susceptible (S) isolates (black line with dashes and dots), rifampicin-resistant isolates with mutations different from I491F (solid black line), and rifampicin-resistant isolates with I491F mutation only (dashed black line).

FIG 3

Sample collection sites in the Shiselweni region (Google map, modified). Distance and time of driving from the site of collection to the closest of the three TB laboratories (shown by red squares).