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.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium.

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, Ribeirão Preto, Santo André, Santos, São José do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30% and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9% were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8%) followed by Mycobacterium tuberculosis (28.0%). Mycobacterium xenopi was isolated in one case (0.8%) and in the remaining 17.4% the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirOo Preto, Santo AndrU, Santos, SOo JosU do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30 and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9 were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8) followed by Mycobacterium tuberculosis (28.0). Mycobacterium xenopi was isolated in one case (0.8) and in the remaining 17.4 the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.(AU)

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirÒo Preto, Santo AndrÚ, Santos, SÒo JosÚ do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30 and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9 were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8) followed by Mycobacterium tuberculosis (28.0). Mycobacterium xenopi was isolated in one case (0.8) and in the remaining 17.4 the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium.

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirÒo Preto, Santo André, Santos, SÒo José do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30

and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9

were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8

) followed by Mycobacterium tuberculosis (28.0

). Mycobacterium xenopi was isolated in one case (0.8

) and in the remaining 17.4

the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.