DNA Mutational Analysis/classification ; DNA Primers/genetics ; DNA Probes/genetics ; Humans ; Mycobacteriumtuberculosis/drugeffects ; Mycobacteriumtuberculosis/genetics* ; Mycobacteriumtuberculosis/isolation & purification ; Nucleic Acid Hybridization/methods ; Polymerase Chain Reaction/methods ; Reagent Kits, Diagnostic/microbiology ; Sensitivity and Specificity ; Tuberculosis, Multidrug-Resistant/microbiology*
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.