Multicentre study of nitrate reductase assay for rapid detection of rifampicin-resistant M. tuberculosis.

SETTING: Four regional laboratories belonging to the Mycobacteria Reference Laboratory of São Paulo State, Brazil. OBJECTIVE: To evaluate the nitrate reductase assay (NRA) for rifampicin (RMP) susceptibility testing of Mycobacterium tuberculosis directly from clinical sputum samples of patients with pulmonary tuberculosis (TB). DESIGN: Performance of the NRA for detection of M.tuberculosis susceptibility to RMP was evaluated with 210 clinical sputum samples received by the participating laboratories during 2005 and 2006 and compared with the results of the direct proportion method. RESULTS: Susceptibility tests performed using the NRA and the direct proportion method showed 204 susceptible isolates and six isolates resistant to RMP by both methods. NRA sensitivity and specificity for RMP was 100%. The NRA results of susceptibility tests against RMP were available in 15 days for 87% of the samples. The results showed that NRA may yield a rapid answer in determining resistance for the majority of sputum samples with smear results reported as 3+ and 2+. CONCLUSION: The results demonstrate the feasibility of NRA for screening resistant strains in sputum samples from patients with pulmonary TB. NRA represents a rapid and low-cost alternative method that might be used in microbiological laboratories where resources are scarce.

Multicentre evaluation of an automated BACTEC 960 system for susceptibility testing of Mycobacterium tuberculosis.

SETTING: Three mycobacteria reference laboratories in the south-eastern part of Brazil. OBJECTIVE: To evaluate the automated Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis. DESIGN: Performance of the automated BACTEC MGIT 960 (M960) system for testing M. tuberculosis susceptibility to streptomycin (SM), isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) was evaluated with 95 clinical isolates and compared to the results of the radiometric BACTEC 460TB (B460) system, the proportion method (PM), and the resistance ratio method (RRM). Judicial susceptibility profiles of 88 isolates were defined based on two or more concordant results among B460, PM and RRM, and used as a reference for comparison with M960 results. RESULTS: Agreement rates between M960 and conventional methods were 95.2% with B460, 96.6% with the PM and 93.4% with the RRM. The lowest agreement rates were obtained for SM with the RRM and for EMB with B460. When comparing M960 with judicial susceptibility profiles, the agreement rate was 97.9%. The agreement rates obtained for INH and RMP were 99.2% and for SM and EMB they were 96.2% and 96.9%, respectively. The mean time to reporting the M960 results was 6.9 days. CONCLUSION: M960 offers great improvements when compared to the proportion and resistance ratio methods and would benefit patient treatment.

Differentiation of Mycobacterium tuberculosis from other mycobacteria with rho-nitrobenzoic acid using MGIT960.

SETTING: Mycobacteria growth in media with the addition of inhibitory substances has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. OBJECTIVE: To develop a rapid PNB test using the automated BACTEC MGIT960 system and to evaluate its usefulness in the screening of mycobacterial isolates. DESIGN: PNB tests were performed in 93 MTC strains and 61 NTM strains from the Instituto Adolfo Lutz Culture Collection. PNB was added to Löwenstein-Jensen (LJ) medium and to BACTEC MGIT960 medium. RESULTS: The MTC strains were all PNB-susceptible, confirming the original identification. Among 10 NTM species, all were found to be resistant to PNB, except for one strain of M. kansasii and another of M. marinum. The median time to obtain presumptive identification of MTC by inhibition test in the BACTEC MGIT960 system was 6.3 days and for NTM it was 2.5 days. The presumptive identification of MTC in LJ was mostly obtained after day 20. CONCLUSION: The key finding of this analysis was the possibility of combining the traditionally accepted method proposed by Tsukamura and Tsukamura in 1964 with the modern, safe and rapid BACTEC MGIT960 methodology.

A population-based study of drug resistance and transmission of tuberculosis in an urban community.

SETTING: A low-income neighborhood of Sao Paulo, Brazil. OBJECTIVE: To determine the incidence, risk factors and transmission patterns of multidrug-resistant tuberculosis (MDR-TB). DESIGN: Prospective longitudinal study of patients with pulmonary TB (PTB). METHODS: Sputum culture-confirmed patients with PTB were recruited between March 2000 and May 2002. Bivariate and multivariate logistic regression analyses were performed to identify factors associated with MDR-TB. Mycobacterium tuberculosis isolates were tested for drug susceptibility and typed by IS6110-RFLP analysis. RESULTS: Of 420 patients, respectively 71% and 27% were new and previously treated; 15.5% of the patients' M. tuberculosis isolates were resistant to at least one drug; of these, 11% and 27% were found among new and previously treated cases, respectively. Respectively 1% and 16.7% of the new and previously treated cases were MDR-TB. RFLP analysis showed that new transmission of MDR strains was uncommon. By multivariate logistic regression analysis, previous TB and hospitalization in the 24 months before TB diagnosis were identified as independent predictors of MDR-TB. CONCLUSIONS: The results showed an intermediate level of MDR-TB incidence in a neighborhood of Sao Paulo and identified predictors that can be targeted for intervention by national and local TB control programs.

Evaluation of a rapid differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with rho-nitrobenzoic acid and thiophene-2-carboxylic acid hydrazide.

SETTING: Mycobacterial growth in media to which inhibitory substances are added has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. Thiophene-2-carboxylic acid hydrazide (TCH) is useful in the differentiation of MTC when performed together with other tests. OBJECTIVE: To develop a test using PNB or TCH added to culture medium, and to evaluate its usefulness in the screening of mycobacteria isolates. DESIGN: In 2001, PNB testing was performed in 109 M. tuberculosis strains identified by Instituto Adolfo Lutz (IAL) and 52 NTM strains from the institute's culture collection. The drugs were added to Löwenstein-Jensen (LJ) medium and to BBL-MGIT. RESULTS: Species differentiation of MTC with the MGIT/TCH method was similar to that observed using the conventional LJ/TCH method. The accuracy of the MGIT/PNB method to differentiate NTM and MTC strains was 99.4%. The BBL-MGIT system allowed presumptive identification in 3-11 days, compared to > or =12 days with LJ medium. CONCLUSION: A simple, low-cost test using growth inhibitors may be incorporated into a modern, safe and quick methodology enabling differentiation of MTC and NTM.

Prevalence of katG Ser315 substitution and rpoB mutations in isoniazid-resistant Mycobacterium tuberculosis isolates from Brazil.

SETTING: Four hundred and sixty-eight isoniazid (INH) resistant Mycobacterium tuberculosis isolates recovered from a selected Brazilian population. OBJECTIVE: To check for susceptibility to other chemotherapeutic drugs used in TB treatment, and to ascertain mutations involved in INH and rifampicin (RMP) resistance. DESIGN: Antimicrobial susceptibility to RMP, streptomycin and ethambutol (EMB) was evaluated by the resistance ratio method and pyrazinamide (PZA) by activity assay. Single strand conformation polymorphism (SSCP) and sequence analysis were performed in samples from this panel to confirm mutations in codon 315 of the katG and in a 69-bp region of the rpoB gene. RESULTS: Combined resistance to INH+RMP, INH+ PZA, INH+EMB, and INH+RMP+PZA was shown in respectively 272 (58.1%), 126 (26.9%), 47 (10%), 116 (24.8%) isolates. No katG mutation was found in 19 (39.6%) of 48 strains tested. Ser315Thr substitution was found in 29 (60.4%). All RMP-resistant strains tested (n = 25) showed rpoB mutations. S531L substitution was found in 15 (60%). CONCLUSION: INH-resistant strains isolated from selected Brazilian populations frequently show resistance to other first-line anti-tuberculosis drugs. rpoB mutation was responsible for RMP resistance in all strains. Among INHr strains, katG mutations were shown in only 60.4%. Genetic approaches targeting the rpoB gene but not the katG gene have a high sensitivity to detect resistance among Brazilian M. tuberculosis strains.

Stability and expression of enteropathogenic Escherichia coli adherence plasmid pMS49 in the presence of human colostrum

Adhesion of enteropathogenic Escherichia coli (EPEC) to HeLa cells is inhibited by human colostrum. In the present study we investigated the effect of colostrum on the stability of pMS49, an EPEC adherence plasmid coding for localized adhesion and ampicillin (Ap) resistance. The plasmid was highly stable after serial passage of bacterial cultures in Tryptic Soy Broth containing 67%, 50%, 10% (v/v) or no human colostrum. A few variants (0.4%) with a low adherence were observed regardless of the treatment given. Human colostrum did not enhance their emergence. No bactericidal or bacteriostatic effect of colostrum was observed under the experimental conditions used. A specific process regulating plasmid expression is supposed to occur in EPRC strains, giving rise to variants with a lower concentration of the outher-membrane adherence-related protein and consequently lower adherence ability. This process seems to also occur for Ap-resistance genes coded in the same plasmid