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.

Outbreak of surgical infection caused by non-tuberculous mycobacteria in breast implants in Brazil.

SUMMARY: We investigated an outbreak caused by non-tuberculous mycobacteria (NTM) related to breast implant surgery in the city of Campinas, Brazil, by means of a retrospective cohort and molecular epidemiological study. A total of 492 records of individuals having breast surgery in 12 hospitals were evaluated. Twelve isolates were analysed using four different molecular typing methods. There were 14 confirmed cases, 14 possible cases and one probable case. One probable, nine possible and 12 confirmed cases were included in a cohort study; all occurred in eight of the hospitals and the confirmed cases in five. Univariate analysis showed that patients who had had breast reconstruction surgery in hospitals A and B were more likely to have NTM infections. No risk factor was independently associated with NTM infection in the multivariate model. The isolates obtained from patients at each hospital showed different molecular patterns, excluding isolates from hospital C that repeatedly showed the same genotype for approximately one year. In conclusion, this outbreak was caused by polyclonal strains at different institutions, and in one hospital a unique genotype caused most cases. No specific risk factors were found.

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.

Application of four molecular typing methods for analysis of Mycobacterium fortuitum group strains causing post-mammaplasty infections.

A cluster of cases of post-augmentation mammaplasty surgical site infections occurred between 2002 and 2004 in Campinas, in the southern region of Brazil. Rapidly growing mycobacteria were isolated from samples from 12 patients. Eleven isolates were identified as Mycobacterium fortuitum and one as Mycobacterium porcinum by PCR-restriction digestion of the hsp65 gene. These 12 isolates, plus six additional M. fortuitum isolates from non-related patients, were typed by pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: 16S-23S rRNA internal transcribed spacer (ITS) genotyping; randomly amplified polymorphic DNA (RAPD) PCR; and enterobacterial repetitive intergenic consensus (ERIC) PCR. Four novel M. fortuitum allelic variants were identified by restriction analysis of the ITS fragment. One major cluster, comprising six M. fortuitum isolates, and a second cluster of two isolates, were identified by the four methods. RAPD-PCR and ITS genotyping were less discriminative than ERIC-PCR. ERIC-PCR was comparable to PFGE as a valuable complementary tool for investigation of this type of outbreak.

Evaluation of mycobacteria growth indicator tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens.

The new BBL mycobacteria growth indicator tube (MGIT) was evaluated for its ability to detect mycobacteria directly from patient specimens and to determine the drug susceptibility of Mycobacterium tuberculosis isolates. A total of 85 respiratory specimens were tested. Specimens were digested, concentrated, examined microscopically for acid-fast bacilli, and inoculated into MGITs and onto Lowenstein-Jensen slants by standard procedures. The tubes were incubated at 37 degrees C and were examined daily for fluorescence to 365-nm UV light. All 25 specimens smear positive for acid-fast bacilli were tested for drug susceptibility in MGITs containing 1.0 mu g of rifampin per ml, 0.1 mu g of isoniazid per ml, 2.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. These results were compared with those obtained by testing the same M. tuberculosis isolates by the indirect proportion method at drug concentrations of 4.0 mu g of rifampin per ml, 0.2 mu g of isoniazid per ml, 2.0 mu g of ethambutol per ml. 4.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. No significant difference in the sensitivity of detection of M. tuberculosis isolates was found between the two methods. However, the time to detection was significantly shorter in MGITs. Drug susceptibility test results for M. tuberculosis isolates by the two methods demonstrated an excellent correlation. The mean time to reporting of drug susceptibility results was 5 days for MGITs versus 16 days for Lowenstein-Jensen slants. The results of this preliminary study indicate that the MGIT system appears to have potential for routine use in mycobacteriology for both the detection and the drug susceptibility testing of M. tuberculosis isolates. However, it is important to emphasize that simple nonautomated equipment should be developed to improve the accuracy of fluorescence detection.