Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Çoban, Ahmet Yılmaz; Uzun, Meltem

doi:10.1590/0074-0276130697

Cited by 7 publications

(5 citation statements)

References 17 publications

(21 reference statements)

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“… Farnia et al (2008) reported that both INH and RIF sensitivities, specificities and agreements were 100%. In another study, Coban and Uzun (2013) reported that the sensitivity, specificity, PPV, NPV and agreement of the MGDA for INH and RIF were 100-95.6%, 97.6-100%, 96.8-100%, 100-98% and 98.6-98.6%, respectively.…”

Section: Discussionmentioning

confidence: 97%

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

Çoban

Akbal

Uzun

et al. 2015

Mem. Inst. Oswaldo Cruz

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The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosis isolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

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Section: Discussionmentioning

confidence: 97%

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

Çoban

Akbal

Uzun

et al. 2015

Mem. Inst. Oswaldo Cruz

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“…Such practices may drastically reduce the proportion of (or eradicate) F420H2-deficient mutants present in the original sample. In addition, efforts to develop MG decolorization assays for detection of drug-resistant TB are expected to be fruitless for these mutants (51)(52)(53)(54). We did not determine the basis for the greater recovery of F420H2-deficient mutants on 7H11 vs. 7H10 media despite 4x higher total MG concentrations in the former.…”

Section: Discussionmentioning

confidence: 99%

Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Rifat

Ioerger

et al. 2018

Preprint

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SIGNIFICANCENitroimidazole pro-drugs represent a promising new class of anti-tuberculosis drugs. Reliable methods to assure nitroimidazole susceptibility are critical to assure their optimal use. Yet, the spectrum of nitroimidazole resistance mutations remains incompletely characterized. Using 161 pretomanid-resistant Mycobacterium tuberculosis isolates selected in pretomanid-treated mice, we discovered a novel resistance determinant, Rv2983, required for cofactor F420 biosynthesis and characterized the remarkable diversity of mutations in this and 5 other genes involved in nitroimidazole activation. We show that F420H2-deficient nitroimidazole-resistant mutants are hypersusceptible to the selective decontaminant malachite green used in solid media to isolate mycobacteria and may evade detection on such media. These results have important implications for development and clinical use of genotypic and phenotypic methods for nitroimidazole susceptibility testing. ABSTRACTDelamanid represents one of two novel antimicrobial classes approved to treat tuberculosis in over 40 years. Pretomanid is another promising nitroimidazole pro-drug in clinical development.Characterization of the full spectrum of mutations conferring resistance to nitroimidazoles and their related phenotypes in Mycobacterium tuberculosis will inform development of suitable genotypic and phenotypic drug susceptibility tests. Here, we used a range of pretomanid doses to select pretomanid-resistant mutants in two pathologically distinct murine TB models. The frequency of spontaneous pretomanid resistance mutations was approximately 10 -5 CFU.Pretomanid demonstrated dose-dependent bactericidal activity and selective amplification of resistant mutants. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 90% of which were found in 1 of 5 genes previously associated with nitroimidazole activation and resistance. The remaining 10% harbored isolated mutations in Rv2983. Complementing an Rv2983 mutant with a wild-type copy of Rv2983 restored wild-type susceptibility to pretomanid and delamanid, confirming that loss of Rv2983 function causes nitroimidazole resistance. By quantifying F420 and its precursor Fo in Mycobacterium smegmatis overexpressing Rv2983 and an M. tuberculosis Rv2983 mutant, we provide evidence that Rv2983 is necessary for F420 biosynthesis and nitroimidazole activation, perhaps as the guanylyltransferase CofC. F420H2-deficient mutants displayed hypersusceptibility to malachite green (MG), a selective decontaminant present in solid media used to isolate and propagate mycobacteria from clinical samples. The wide diversity of mutations causing high-level pretomanid resistance and MG hypersusceptibility of most mutants poses significant challenges to clinical detection of nitroimidazole resistance using either genotypic or phenotypic methods.

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“…Such practices may drastically reduce the proportion of (or eradicate) F 420 H 2 -deficient mutants present in the original sample. In addition, efforts to develop MG decolorization assays for detection of drug-resistant TB are expected to be fruitless for these mutants (47)(48)(49)(50). We did not determine the basis for the greater recovery of F 420 H 2 -deficient mutants on 7H11 vs. 7H10 media despite 4x higher total MG concentrations in the former.…”

Section: Bashiri Et Al Recently Revised the F 420 Biosynthetic Pathway Based On Biochemical Evidence Thatmentioning

confidence: 92%

Mutations in fbiD ( Rv2983 ) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Rifat

Ioerger

et al. 2020

Antimicrob Agents Chemother

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The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Cited by 7 publications

References 17 publications

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Mutations in fbiD ( Rv2983 ) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

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