Abstract
We compared the ability of commercial and non-commercial, phenotypic and genotypic rapid drug susceptibility tests (DSTs) to detect rifampicin resistance (RR)-conferring ‘disputed’ mutations frequently missed by Mycobacterium Growth Indicator Tube (MGIT), namely L430P, D435Y, L452P, and I491F. Strains with mutation S450L served as positive control while wild-types were used as negative control. Of the 38 mutant strains, 5.7% were classified as RR by MGIT, 16.2% by Trek Sensititre MYCOTB MIC plate, 19.4% by resazurin microtiter plate assay (REMA), 50.0% by nitrate reductase assay (NRA), and 62.2% by microscopic observation direct susceptibility testing (MODS). Reducing MGIT rifampicin concentration to 0.5 µg/ml, and/or increasing incubation time, enhanced detection of disputed mutations from 5.7% to at least 65.7%, particularly for mutation I491F (from 0.0 to 75.0%). Compared with MGIT at standard pre-set time with 0.25 µg/ml ECOFF as breakpoint, we found a statistically significant increase in the ability of MGIT to resolve disputed mutants and WT strains at extended incubation period of 15 and 21 days, with 0.5 µg/ml and 1 µg/ml ECOFF respectively. MODS detected 75.0% of the I491F strains and NRA 62.5%, while it was predictably missed by all molecular assays. Xpert MTB/RIF, Xpert Ultra, and GenoscholarTB-NTM + MDRTB detected all mutations within the 81 bp RR determining region. Only GenoType MTBDRplus version 2 missed mutation L430P in 2 of 11 strains. Phenotypic and genotypic DSTs varied greatly in detecting occult rifampicin resistance. None of these methods detected all disputed mutations without misclassifying wild-type strains.
Highlights
RIF Ultra were endorsed by the WHO6,7
MGIT uses pre-set standard conditions of rifampicin concentration based on the WHO critical concentration (CC) of 1 μg/ml[22], inhibiting the growth of 99% of phenotypically wild-type (WT) strains, based on the proportion method that detects 1% of minority resistant strains, and pre-set incubation time, both of which were validated in several studies[23,24,25]
We explored whether extended incubation time in MGIT could reduce this overlap in minimum inhibitory concentration (MIC) values between WT and mutant strains and compared the ability of pheno- and genotypic rapid tests, both commercial and non-commercial, to detect occult rifampicin resistance due to ‘disputed’ mutations
Summary
RIF Ultra (hereinafter referred to as Ultra) were endorsed by the WHO6,7. In contrast with the short conventional probes of classic Xpert, Ultra was designed with four long sloppy molecular beacon probes. Discordances between phenotypic (in particular rapid liquid based, such as MGIT) and genotypic DSTs were largely attributable to occult resistance conferred by specific, uncommon rpoB mutations, referred to as ‘disputed’ mutations[10,11,12] These strains were not rare among retreatment cases from two low-income settings in Africa and Asia[13]. MGIT uses pre-set standard conditions of rifampicin concentration based on the WHO critical concentration (CC) of 1 μg/ml[22], inhibiting the growth of 99% of phenotypically wild-type (WT) strains, based on the proportion method that detects 1% of minority resistant strains, and pre-set incubation time, both of which were validated in several studies[23,24,25]. We explored whether extended incubation time in MGIT could reduce this overlap in MIC values between WT and mutant strains and compared the ability of pheno- and genotypic rapid tests, both commercial and non-commercial, to detect occult rifampicin resistance due to ‘disputed’ mutations
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