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During susceptibility testing of 743 isolates of Mycobacterium tuberculosis to pyrazinamide (PZA) using the Bactec 960 system, 57 (7.7%) isolates showed PZA resistance. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (4.4%) of these isolates as resistant, and 24 (3.2%) were susceptible. Erroneous results for resistance with the Bactec 960 were confirmed by testing the 24 discordant isolates for pyrazinamidase and mutations in the pncA gene.Pyrazinamide (PZA) is an important component of the multidrug regimen used to treat tuberculosis (TB). With increasing worldwide prevalence of drug-resistant TB, it is vital for laboratories to accurately detect resistance to first-line antimicrobials.The CLSI-recommended method for PZA testing (4) is the Bactec 460TB radiometric system (Becton Dickinson, Sparks, MD). Most laboratories have now replaced the 460TB system with the nonradiometric Bactec MGIT 960 (BT960) system (Becton Dickinson, Sparks, MD). Both methods utilize an acidified Middlebrook broth and a critical concentration of 100 g/ml.PZA is a prodrug which in Mycobacterium tuberculosis is converted to its active form, pyrazinoic acid (POA), by the enzyme pyrazinamidase (PZase) (5, 7). The absence of a functional PZase enzyme in an M. tuberculosis strain therefore indicates resistance to PZA. The pncA gene coding for PZase in M. tuberculosis has been sequenced, and mutations in this gene have been shown to be responsible for resistance to PZA (5,8,12). Tests both for PZase activity and for the detection of mutations in pncA may be utilized as alternative methods for the detection of PZA resistance in M. tuberculosis.All new M. tuberculosis isolates are tested for susceptibility to first-line drugs, including PZA, at the Public Health Laboratory, Toronto. During the report period, any isolate demonstrating PZA resistance by the BT960 was retested using the 460TB. If the 460TB PZA result was discordant, these isolates were further tested for the presence of PZase activity and mutations in the pncA gene, and testing in the BT960 was repeated.PZA susceptibility testing in the BT960 system was performed according to the manufacturer's instructions (2). Briefly, isolates of M. tuberculosis in Mycobacteria Growth Indicator Tubes (MGITs) were used as the test inocula. A drug-free control tube was inoculated with a 1:10 dilution of the inoculum, and the PZA test tube was inoculated with 0.5 ml of the inoculum and 0.1 ml of PZA. The tubes were monitored with the BT960 instrument until the growth control tube flagged positive. At that time, the instrument read the PZA test tube as either resistant (growth unit [GU] Ն 100) or susceptible (GU Ͻ 100). A blood agar purity plate from the inoculum was incubated for 3 days.PZA testing in the BT 460TB system was performed according to the manufacturer's instructions (13). Briefly, a drug-free control vial and the PZA test vial were each inoculated with 0.1 ml of inoculum from an actively growing culture. The vials were incubated and were read on...

BACKGROUND: The reported prevalence of pulmonary nontuberculous mycobacteria (NTM) infections is increasing.OBJECTIVE: To determine the ‘isolation prevalence’ of NTM in 2007 and compare it with previously published research that examined the increasing rates of isolation of NTM from clinical pulmonary specimens between 1997 and 2003.METHODS: Isolation prevalence was investigated retrospectively by reviewing a cohort of all positive pulmonary NTM culture results from the Tuberculosis and Mycobacteriology Laboratory, Public Health Laboratory (Toronto, Ontario) in 2007, which identifies at least 95% of NTM isolates in Ontario. Isolation prevalence was calculated as the number of persons with a pulmonary isolate in a calendar year divided by the contemporary population and expressed per 100,000 population. Changes in isolation prevalence from previous years were assessed for statistical significance using generalized linear models with a negative binomial distribution.RESULTS: In 2007, 4160 pulmonary isolates of NTM were collected from 2463 patients. The isolation prevalence of all species (excludingMycobacterium gordonae) was 19 per 100,000 population in 2007 – an increase from previous observations reported for Ontario – corresponding to an average annual increase of 8.5% from 1997 to 2007 (P<0.0001). Average annual increases in isolation prevalence ofMycobacterium aviumcomplex (8.8%, P<0.0001) andMycobacterium xenopi(7.3%, P=0.0005) were largely responsible for the overall increase, while prevalence rates of rapidly growing mycobacteria remained relatively stable.CONCLUSION: The isolation prevalence of pulmonary NTM continues to increase significantly in Ontario, supporting the belief that pulmonary NTM disease is increasingly common.

As the NTM prevalence rate in children with CF is within the range previously reported in adults and there are no reliable clinical predictors for isolation, annual sputum screening is needed to identify NTM in children. Further research is needed to determine the best sputum decontamination method for NTM culture in pediatric patients.

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