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

E. Ardizzoni, E. Ariza, D. Mulengwa, Q. Mpala, R. de la Tour, G. Maphalala, F. Varaine, B. Kerschberger, P. Graulus, A. L. Page, S. Niemann, Andries Dreyer, A. Van Deun, T. Decroo, L. Rigouts, B. C. de Jong

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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.

Original languageEnglish
Article numbere02263-20
JournalAntimicrobial Agents and Chemotherapy
Issue number6
Number of pages11
Publication statusPublished - 2021


  • 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
  • World Health Organization

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