title: Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.
abstract: The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.
authors.affiliation: DST/NRF Centre of Excellence for Biomedical Tuberculosis Research/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics,Faculty of Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: E M
authors.initials: EM
authors.lastname: Streicher
authors.forename: I
authors.initials: I
authors.lastname: Bergval
authors.forename: K
authors.initials: K
authors.lastname: Dheda
authors.forename: E C
authors.initials: EC
authors.lastname: Böttger
authors.forename: N C
authors.initials: NC
authors.lastname: Gey van Pittius
authors.forename: M
authors.initials: M
authors.lastname: Bosman
authors.forename: G
authors.initials: G
authors.lastname: Coetzee
authors.forename: R M
authors.initials: RM
authors.lastname: Anthony
authors.forename: P D
authors.initials: PD
authors.lastname: van Helden
authors.forename: T C
authors.initials: TC
authors.lastname: Victor
authors.forename: R M
authors.initials: RM
authors.lastname: Warren
mesh_terms: D000583:Amikacin / Q000008:administration & dosage / Q000627:therapeutic use*; D000995:Antitubercular Agents / Q000008:administration & dosage / Q000627:therapeutic use*; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D027081:DNA Gyrase / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D015242:Ofloxacin / Q000008:administration & dosage / Q000627:therapeutic use*; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D000583:Amikacin; D015242:Ofloxacin; D027081:DNA Gyrase