In this study the nucleotide sequence of the pncA gene from 59 Mycobacterium tuberculosis clinical isolates was analyzed. Mutations in the pncA gene were identified in 29 of 40 pyrazinamide-resistant isolates, and no pyrazinamidase activity was detected in 39 of them. Twelve mutations found in this work have not been described previously

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Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.The Mycobacterium avium complex (MAC) has been increasingly recognized as an important group of organisms causing severe opportunistic infections in patients in the final stages of AIDS (1,3,5,8). MAC has been responsible for 96% of these infections (12). Species identification of the isolated mycobacteria in AIDS patients and rapid and accurate diagnosis of the infection are important due to the existence of antimicrobial drugs that are able to suppress symptomatic infection (9). PCR has been the most rapid and sensitive method for identifying MAC infection (6) and has the potential to reduce the time taken for the identification and species determination of M. avium to a few days. DNA fragments specific for the genus Mycobacterium and for the species M. avium can be amplified by using primers derived from the 16S rRNA gene (10,13,14,16). In this work, we report the results of experiments using a technique in which DNA from blood samples of AIDS patients was purified and used for the detection of the genus Mycobacterium and the species M. avium.Blood specimens. Sixty-nine samples from 47 AIDS patients of the Hospital of the Universidade Federal do Rio de Janeiro, a national reference center for AIDS, and of the Public Hospitals of Rio Grande do Sul, Brazil, were analyzed. The samples were collected in the period between 1996 and 1999. Fourteen out of 47 patients had more than one aliquot (5 ml) of whole blood collected in a period of 24 h, the number of samples collected being dependent on the seriousness of disease. The samples were collected at time intervals of between 8 and 12 h. Of these patients, six had two samples collected and eight had three samples collected during this period. This was possible because the patients were hospitalized. All samples were prepared by the lysis centrifugation technique.Each aliquot was inoculated in Löwenstein-Jensen medium, and 500 l was used for DNA purification followed by PCR amplification. The "gold standard" used was based on the clinical diagnosis for mycobacteremia caused by M. avium. The clinical diagnosis was based on the symptoms presented by the patients and on the isolation of the organism by microbiological methods and according to therapeutic criteria (resolution of the process during the treatment).Isolation and DNA detection procedures. The DNA was purified with DNAzol (Gibco BRL and Life Technologies, Rockville, Md.). The DNAzol procedure developed by Chomezynski et al. (4) is based on the use of a guanidine detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from a cell lysate. DNA was amplified by PCR with the primers derived from the 16S rRNA sequence, mycgen-f (5Ј-AG A...