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

Antimicrob Agents Chemother. 2021 May 18;65(6):e02263-20. doi: 10.1128/AAC.02263-20. Print 2021 May 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agar
  • Antibiotics, Antitubercular* / pharmacology
  • Diagnostic Tests, Routine
  • Drug Resistance, Bacterial / genetics
  • Eswatini
  • Humans
  • Microbial Sensitivity Tests
  • Mycobacterium tuberculosis* / genetics
  • Pharmaceutical Preparations*
  • Rifampin / pharmacology
  • Sensitivity and Specificity
  • Sputum
  • Tuberculosis, Multidrug-Resistant* / diagnosis
  • Tuberculosis, Multidrug-Resistant* / drug therapy
  • World Health Organization

Substances

  • Antibiotics, Antitubercular
  • Pharmaceutical Preparations
  • Agar
  • Rifampin