Pontus Juréen

Fluoroquinolone Resistance in Mycobacterium tuberculosis and Mutations in gyrA and gyrB

ABSTRACT This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 → Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.

Pyrazinamide Resistance and pncA Gene Mutations in Mycobacterium tuberculosis

ABSTRACT Thirty-four pyrazinamide-resistant and 37 pyrazinamide-susceptible Mycobacterium tuberculosis complex strains were analyzed for pncA gene mutations. None of the sensitive strains had any mutations, apart from silent mutations, whereas all but one resistant strain showed pncA mutations. By using sequencing as a means of early resistance detection, the inconsistency of phenotypic pyrazinamide assays can be circumvented.

Discordant Resistance to Kanamycin and Amikacin in Drug-Resistant Mycobacterium tuberculosis

ABSTRACT It is generally thought that there is full cross-resistance in Mycobacterium tuberculosis between the aminoglycoside drugs kanamycin and amikacin. However, kanamycin resistance and amikacin susceptibility were seen in 43 of 79 (54%) multidrug-resistant Estonian isolates, indicating that there might be a need to test the resistance of M. tuberculosis isolates to both drugs.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis by Pyrosequencing Technology

ABSTRACT We developed an assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis based on Pyrosequencing technology, involving a technique for real-time sequencing. A 180-bp region of the rpoB gene was amplified in clinical isolates of both rifampin-resistant and -susceptible M. tuberculosis. The PCR products were subjected to Pyrosequencing analysis using four different sequencing primers in four overlapping reactions. These four sequencing reactions covered the 81-bp region where >96% of the mutations associated with rifampin resistance are located. The results were compared to those obtained with two other molecular methods, the line probe assay and cycle sequencing, and the phenotypic BACTEC method. The genotypic determination methods all detected the mutations that previously have been correlated with rifampin resistance. In addition, Pyrosequencing analysis and the two other molecular methods found additional mutations within the rpoB gene in phenotypically susceptible strains. We found that Pyrosequencing technology, in particular, offers high accuracy, short turnaround time, and a potentially high throughput in detection of rifampin resistance in M. tuberculosis.

Detection of First- and Second-Line Drug Resistance in Mycobacterium tuberculosis Clinical Isolates by Pyrosequencing

ABSTRACT Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.

Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis

OBJECTIVES The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. METHODS We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. RESULTS The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n = 4) for which the MICs were 4-8 mg/L. CONCLUSIONS Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.

Challenging a dogma: antimicrobial susceptibility testing breakpoints for Mycobacterium tuberculosis

The rise in multidrug-resistant tuberculosis makes it increasingly important that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce clinically meaningful and technically reproducible results. Unfortunately, this is not always the case because mycobacteriology specialists have not followed generally accepted modern principles for the establishment of susceptibility breakpoints for bacterial and fungal pathogens. These principles specifically call for a definition of the minimum inhibitory concentrations (MICs) applicable to organisms without resistance mechanisms (also known as wild-type MIC distributions), to be used in combination with data on clinical outcomes, pharmacokinetics and pharmacodynamics. In a series of papers the authors have defined tentative wild-type MIC distributions for M. tuberculosis and hope that other researchers will follow their example and provide confirmatory data. They suggest that some breakpoints are in need of revision because they either (i) bisect the wild-type distribution, which leads to poor reproducibility in antimicrobial susceptibility testing, or (ii) are substantially higher than the MICs of wild-type organisms without supporting clinical evidence, which may result in some strains being falsely reported as susceptible. The authors recommend, in short, that susceptibility breakpoints for antituberculosis agents be systematically reviewed and revised, if necessary, using the same modern tools now accepted for all other bacteria and fungi by the scientific community and by the European Medicines Agency and the European Centre for Disease Prevention and Control. For several agents this would greatly improve the accuracy and reproducibility of antimicrobial susceptibility testing of M. tuberculosis.

Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis

OBJECTIVES The aim of this study was to clarify the conflicting data regarding cross-resistance and drug-resistance mechanisms for the cyclic peptide capreomycin and the aminoglycosides amikacin and kanamycin by comparing genotypes and phenotypes of clinical isolates and in vitro selected mutants of Mycobacterium tuberculosis. METHODS The genes rrs and tlyA and the promoter region of eis of 152 M. tuberculosis clinical isolates (including 55 capreomycin resistant) and 44 in vitro selected capreomycin-, amikacin- and kanamycin-resistant mutants were sequenced. In addition, MICs of capreomycin, amikacin and kanamycin on Middlebrook 7H10 were determined. RESULTS The results clearly show major differences in genotypes and cross-resistance patterns to amikacin and kanamycin between the capreomycin-resistant clinical isolates and in vitro selected mutants. tlyA mutations were found almost exclusively among the in vitro selected capreomycin-resistant mutants, while only four were found among the clinical isolates, of which two were capreomycin susceptible. In contrast, 53 of the 55 capreomycin-resistant clinical isolates had a mutation at position 1401 in rrs and were resistant to capreomycin, amikacin and kanamycin. Low-level resistance to kanamycin was correlated to mutations in the promoter region of eis. CONCLUSIONS Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.

Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

ABSTRACT The distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.

Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis

OBJECTIVES To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. METHODS The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. RESULTS Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. CONCLUSIONS Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.