We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.The emergence and spread of drug-resistant strains of Mycobacterium tuberculosis, especially multidrug-resistant (MDR) strains, are serious threats to the control of tuberculosis and comprise an increasing public health problem (40). Patients infected with MDR strains, which are defined as strains resistant to both rifampin (RIF) and isoniazid (INH), are difficult to cure and are more likely to remain sources of infection for a longer period of time than are patients with drug-susceptible strains (40).It is essential that rapid drug susceptibility tests be developed to prevent the spread of MDR M. tuberculosis. The time necessary for culture of specimens was reduced by the radiometric BACTEC 460TB system (BD Biosciences, Sparks, MD), the nonradiometric ESP II system (Trek Diagnostics, Westlake, OH), and other rapid broth methods, such as BACTEC MGIT 960 SIRE (BD Biosciences) (20). These drug susceptibility tests, however, still require 1 to 2 weeks for final determination and reporting to the clinician (23). Additional reductions in the detection period are needed.Drug resistance in M. tuberculosis is caused by mutations in relatively restricted regions of the genome (17, 39). Mutations associated with drug resistance occur in rpoB for RIF, katG and the promote...

This test is considered a promising diagnostic tool for TB, even for peripheral laboratories with limited equipment, such as those in developing countries.

BackgroundTransmission of tuberculosis (TB) to health care workers (HCWs) is a global issue. Although effective infection control measures are expected to reduce nosocomial TB, HCWs' infection has not been assessed enough in TB high burden countries. We conducted a cross-sectional study to determine the prevalence of TB infection and its risk factors among HCWs in Hanoi, Viet Nam.Methodology/Principal FindingsA total of 300 HCWs including all staff members in a municipal TB referral hospital received an interferon-gamma release assay (IGRA), QuantiFERON-TB Gold In-TubeTM, followed by one- and two-step tuberculin skin test (TST) and a questionnaire-based interview. Agreement between the tests was evaluated by kappa statistics. Risk factors for TB infection were analyzed using a logistic regression model. Among the participants aged from 20 to 58 years (median = 40), prevalence of TB infection estimated by IGRA, one- and two-step TST was 47.3%, 61.1% and 66.3% respectively. Although the levels of overall agreement between IGRA and TST were moderate, the degree of agreement was low in the group with BCG history (kappa = 0.29). Working in TB hospital was associated with twofold increase in odds of TB infection estimated by IGRA. Increased age, low educational level and the high body mass index also demonstrated high odds ratios of IGRA positivity.Conclusions/SignificancePrevalence of TB infection estimated by either IGRA or TST is high among HCWs in the hospital environment for TB care in Viet Nam and an infection control program should be reinforced. In communities with heterogeneous history of BCG vaccination, IGRA seems to estimate TB infection more accurately than any other criteria using TST.

Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome (36). Mutations in katG, the upstream region of the fabG1-inhA operon (P fabG1-inhA ), and inhA are responsible for INH resistance (36). The katG gene encodes the bifunctional catalase-peroxidase enzyme that converts INH to an active form (35).Previously, we developed a DNA sequencing-based method to detect mutations in regions associated with INH resistance in M. tuberculosis, including katG and P fabG1-inhA (28). Consequently, five novel mutations in katG associated with INH resistance were found (28). In the present study, we cloned 21 katG mutants, including 15 novel mutants, and compared their INH oxidase activities. Certain katG mutations were shown to cause high-level INH resistance, which suggests the possibility of determining the degree of INH resistance, such as high-or low-level resistance, by detecting these katG mutations. Furthermore, to detect these mutations in ordinary-scale clinical laboratories without sequencing, we developed a new hybridization-based line probe assay (LiPA) for INH resistance in M. tuberculosis isolates, which can be applied easily in clinical use. MATERIALS AND METHODSBacterial strains and plasmids. One hundred eight Inh r M. tuberculosis isolates were obtained from single patients at

We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n ‫؍‬ 316), Mycobacterium avium (n ‫؍‬ 71), Mycobacterium intracellulare (n ‫؍‬ 51), Mycobacterium kansasii (n ‫؍‬ 54), and other Mycobacterium species (n ‫؍‬ 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.