Anna Engström

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.

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.

Detection of rifampicin resistance in Mycobacterium tuberculosis by padlock probes and magnetic nanobead-based readout.

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.

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.

Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors

Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the chromosome. Multi-drug-resistant tuberculosis (MDR-TB) is an increasing public health threat, and prompt detection of such strains is of critical importance. As rolling circle amplification of padlock probes can be used to robustly distinguish single-nucleotide variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to develop a rapid molecular diagnostic test for MDR-TB. A proof-of-concept test was established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315 (AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The molecular diagnostic test produces visual signals corresponding to the respective genotypes on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid information about the drug-susceptibility pattern can assist clinicians to choose suitable treatment regimens and take appropriate infection control actions rather than prescribing empirical treatment, thereby helping to control the spread of MDR-TB in the community.

Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

ABSTRACT The ability of pyrosequencing to detect a resistant minority population of a heteroresistant Mycobacterium tuberculosis strain was investigated by performing a titration study. A mutant signal was noted only when the proportion of mutant DNA in the DNA target was 35 to 50%, showing that the sensitivity is significantly lower than that of phenotypic drug susceptibility test methods.