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Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

ABSTRACT: Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG?TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10?? to 2.49×10??) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10??, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10??). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.

SUBMITTER: Bergval I

PROVIDER: S-EPMC3250395 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

Bergval Indra I Kwok Brian B Schuitema Anja A Kremer Kristin K van Soolingen Dick D Klatser Paul P Klatser Paul P Anthony Richard R

PloS one 20120103 1

Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis geno ...[more]

PMID: 22235262

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Project description:BACKGROUND: Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential. OBJECTIVE: To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan. MATERIALS AND METHODS: Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009-2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin. RESULTS: Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains. CONCLUSIONS: Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.

Project description:BACKGROUND:Accurate diagnosis of tuberculosis, especially by using rapid molecular assays, can reduce transmission of drug resistant tuberculosis in communities. However, the frequency of resistance conferring mutations varies with geographic location of Mycobacterium tuberculosis, and this affects the efficiency of rapid molecular assays in detecting resistance. This has created need for characterizing drug resistant isolates from different settings to investigate frequencies of resistance conferring mutations. Here, we describe the prevalence and patterns of rifampicin- and isoniazid- resistance conferring mutations in isolates from Uganda, which could be useful in the management of MDR-TB patients in Uganda and other countries in sub-Saharan Africa. RESULTS:Ninety seven M. tuberculosis isolates were characterized, of which 38 were MDR, seven rifampicin-resistant, 12 isoniazid-mono-resistant, and 40 susceptible to rifampicin and isoniazid. Sequence analysis of the rpoB rifampicin-resistance determining region (rpoB/RRDR) revealed mutations in six codons: 588, 531, 526, 516, 513, and 511, of which Ser531Leu was the most frequent (40%, 18/45). Overall, the three mutations (Ser531Leu, His526Tyr, Asp516Tyr) frequently associated with rifampicin-resistance occurred in 76% of the rifampicin resistant isolates while 18% (8/45) of the rifampicin-resistant isolates lacked mutations in rpoB/RRDR. Furthermore, sequence analysis of katG and inhA gene promoter revealed mainly the Ser315Thr (76%, 38/50) and C(-15)T (8%, 4/50) mutations, respectively. These two mutations combined, which are frequently associated with isoniazid-resistance, occurred in 88% of the isoniazid resistant isolates. However, 20% (10/50) of the isoniazid-resistant isolates lacked mutations both in katG and inhA gene promoter. The sensitivity of sequence analysis of rpoB/RRDR for rifampicin-resistance via detection of high confidence mutations (Ser531Leu, His526Tyr, Asp516Tyr) was 81%, while it was 77% for analysis of katG and inhA gene promoter to detect isoniazid-resistance via detection of high confidence mutations (Ser315Thr, C(-15)T, T(-8)C). Furthermore, considering the circulating TB genotypes in Uganda, the isoniazid-resistance conferring mutations were more frequent in M. tuberculosis lineage 4/sub-lineage Uganda, perhaps explaining why this genotype is weakly associated with MDR-TB. CONCLUSION:Sequence analysis of rpoB/RRDR, katG and inhA gene promoter is useful in detecting rifampicin/isoniazid resistant M. tuberculosis isolates in Uganda however, about ≤20% of the resistant isolates lack known resistance-conferring mutations hence rapid molecular assays may not detect them as resistant.

Project description:BackgroundWhile rifampicin (RFP) and isoniazid (INH) are the most commonly used first-line antituberculosis drugs, multidrug resistance in Mycobacterium tuberculosis poses a threat to the success of tuberculosis (TB) control programs. Clinical practice guidelines and expert consensuses recommend drug susceptibility testing (DST) before the initiation of antituberculosis treatment. However, traditional DST is time-consuming and has high requirements for laboratory conditions. The recently developed molecular diagnostic techniques, such as DNA microarray, offer new options. We thus investigated the diagnostic value of DNA microarray in detecting RFP + INH-resistant TB, with an attempt to identify simple, efficient, and accurate drug-resistant TB testing methods.MethodsThe clinical features and DST results of patients diagnosed with pulmonary tuberculosis by Bactec MGIT 960 liquid culture system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) who received DNA microarray analysis in our center from July 2019 to July 2020 were retrospectively analyzed. Level of agreement between liquid culture and DNA microarray technology was assessed by using the Cohen kappa coefficient. With the results of liquid culture as the gold standard, the sensitivity and specificity of the DNA microarray were calculated, and the receiver operating characteristic (ROC) curves were used to assess the diagnostic values of the DNA microarray in detecting RFP + INH-resistant TB.ResultsA total of 825 patients were enrolled. The sensitivity and specificity of DNA microarray were 0.84 and 0.94, respectively, in the detection of RFP resistance, with an area under the curve (AUC) of 0.89 [95% confidence interval (CI): 0.87-0.91)] and a Cohen kappa coefficient of 0.78 (95% CI: 0.72-0.83). For INH resistance, the sensitivity and specificity of the DNA microarray were 0.73 and 0.97, respectively, with an AUC of 0.85 (95% CI: 0.82-0.87) and a Cohen kappa coefficient of 0.75 (95% CI: 0.70-0.80).ConclusionsThe DNA microarray had high specificity and sensitivity in detecting RFP + INH-resistant TB. As a rapid, accurate, and practical technique, it can be routinely performed in clinical laboratories.

Dataset Information

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

Publications

Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

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