title: Molecular detection of early appearance of drug resistance during Mycobacterium tuberculosis infection.
abstract: During the early development of drug resistance in Mycobacterium tuberculosis (M. tuberculosis) infection only a small proportion of resistant bacteria are present within a milieu of sensitive bacteria. This complicates the use of molecular methods to predict the presence of a resistant phenotype and has been largely ignored in many of the newly developed molecular methods. In this study, mixtures of DNA from M. tuberculosis strains with known wild-type and mutant sequences were used to evaluate the sensitivity of three different molecular methods for detection of drug resistance. The dot-blot and amplification refractory mutation system (ARMS) methods showed sensitivities that approach those of routine phenotypic methods and are able to detect the presence of mutant sequences at a ratio of 1 in 50 (corresponding to 2% mutant sequences). This is 10-fold more sensitive than the commercial kit. The ARMS method was also used to investigate the use of molecular methods to identify mixed infections, and both drug-resistant and susceptible strain populations were identified in a single clinical isolate. These findings highlight the applicability of molecular methods to the rapid detection of drug resistance in tuberculosis patients, particularly in those who are non-compliant and in contacts of known drug-resistant tuberculosis patients, and assistance in limiting the spread of drug-resistant strains.
authors.affiliation: MRC Center for Molecular and Cellular Biology, Department of Medical Biochemistry, University of Stellenbosch, Tygerberg, South Africa. TV@sun.ac.za
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Victor
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Lee
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.forename: Annemarie M
authors.initials: AM
authors.lastname: Jordaan
authors.forename: Gian
authors.initials: G
authors.lastname: van der Spuy
authors.forename: Paul D
authors.initials: PD
authors.lastname: van Helden
authors.forename: Robin
authors.initials: R
authors.lastname: Warren
mesh_terms: D004252:DNA Mutational Analysis / Q000145:classification; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic / Q000382:microbiology; D012680:Sensitivity and Specificity; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D011933:Reagent Kits, Diagnostic