Pyrazinamide (PZA) is an important first-line tuberculosis drug that is part of the currently used shortcourse tuberculosis chemotherapy. PZA is a prodrug that has to be converted to the active form pyrazinoic acid by pyrazinamidase (PZase) activity, encoded by the pncA gene of Mycobacterium tuberculosis, and loss of PZase activity is associated with PZA resistance. To further define the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we sequenced the pncA gene from a panel of 59 PZA-resistant clinical isolates from Canada, the United States, and Korea. Two strains that did not contain pncA mutations and had positive PZase turned out to be falsely resistant. Three PZase-negative strains (MIC, >900 g of PZA per ml) and one PZase-positive strain (strain 9739) (MIC, >300 g of PZA per ml) did not have pncA mutations. The remaining 53 of the 57 PZA-resistant isolates had pncA mutations, confirming that pncA mutation is the major mechanism of PZA resistance. Various new and diverse mutations were found in the pncA gene. Interestingly, 20 PZA-monoresistant strains and 1 multidrug-resistant isolate from Quebec, Canada, all had the same pncA mutation profile, consisting of an 8-nucleotide deletion and an amino acid substitution of Arg1403Ser. Strain typing indicated that these strains are highly related and share almost identical IS6110 patterns. These data strongly suggest the spread of a PZA-monoresistant strain, which has not previously been described.

Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that "clustered" tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with >6 IS6110 copies) or RFLP in combination with spoligotyping (for isolates with <6 IS6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS6110-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.Infection with Mycobacterium tuberculosis, the bacillus responsible for tuberculosis (TB), still results in almost 2 million global deaths each year despite the availability of effective chemotherapy and a partially effective vaccine (bacillus Calmette-Guérin [Mycobacterium bovis BCG]) for well over half a century (43). Even though there have been real advances in our level of understanding of M. tuberculosis metabolic pathways and in the identification of potential virulenceassociated molecules since the advent of the "postgenomic era" (8), significant improvements with respect to treatment, diagnosis, and prevention of TB still remain elusive. Recent descriptions of variably expressed virulence factors and disease outcomes that are associated with particular lineages of M. tuberculosis further emphasize the layers of complexity that need to be overcome in our efforts to combat this devastating disease (6,25,31,32,38).While the fact that TB phenotypic diversity exists is no longer in dispute, there is heightened interest in epidemiological circles in establishing the relevance of this diversity to human disease. Of particular note are the recent publications suggesting that strains belonging to the M. tuberculosis W/...

DNA fingerprinting of Mycobacterium tuberculosis by IS6110 restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism, M. tuberculosis remained viable during this process. These data suggest that the extraction of M. tuberculosis DNA should be performed within containment until complete.The standard method employed for DNA fingerprinting of Mycobacterium tuberculosis is IS6110 restriction fragment length polymorphism. This modality requires a large quantity (Ն2 g) of high-molecular-weight DNA (5-7). The DNA extraction protocols currently used for this purpose are based on chemical and enzymatic lysis of the bacterial cells followed by a chloroform-isoamyl alcohol-based DNA extraction, a somewhat lengthy process that raises methodological and biosafety issues. The cultivation of M. tuberculosis requires a biosafety level 3 containment facility and up to 4 weeks of culture time. The extraction necessitates a 2-to 3-day protocol harsh enough to lyse the bacteria yet sufficiently gentle to prevent DNA shearing.Due to the cumbersome nature of extraction protocols, there has been interest in determining at which point M. tuberculosis preparations can be considered inactivated and thus be safely removed from containment. Heating of the culture is widely used, and at least one report has deemed heating at 80°C as sufficient for inactivation (3). However, other reports have raised concerns as to the efficacy of heating (8) and even of further treatment with a combination of lysozyme and proteinase K (2). As such, the question of where and when these preparations may be safely manipulated remains unanswered. It is vital to address this issue, as improper inactivation and premature transfer of extraction mixtures from containment could result in the unnecessary exposure of laboratory personnel to M. tuberculosis.Here, the DNA from clinical M. tuberculosis isolates was extracted using standard protocols (all reagents were from Sigma-Aldrich unless noted). In brief, isolates were inoculated onto Lowenstein-Jensen (L-J) slants, and when luxuriant growth was apparent, all visible colonies were collected into 500 l Tris-EDTA buffer, pH 8.0, and heated for 20 min at 80°C. Lysozyme was then added to each tube (final concentration, 1 mg/ml), followed by incubation at 37°C for 2 h. Ten percent sodium dodecyl sulfate (final concentration, 1.1%) and proteinase K (final concentration, 0.2 mg/ml; Promega Inc.) were then added, and the tubes were vortexed gently and incubated for 20 min at 65°C. A mixture of N-acetyl-N,N,Ntrimethyl ammonium bromide (CTAB; final concentration, 40 mM) and NaCl (final concentration, 0.1 M) was added, followed immediately by the addition of NaCl alone (final concentration, 0.6 M). The tubes were then vortexed until the suspension turned milky and were incubated for 10 min at 65°C. Seven hundred fifty microliters of chloroform-isoamyl alcohol (24:1) was added to each tube, and ...

Nocardia is an uncommon pathogen, but immunosuppression, its main risk factor, is becoming more frequent. We aimed to evaluate changes in the annual incidence of nocardiosis and in the susceptibility profile of its aetiological agents. Demographic data were analysed for all isolates of Nocardia forwarded to the provincial public health laboratory of Quebec, Canada during the last two decades. Population incidence could be measured from 1997 onwards. Resistance patterns were analysed for those isolates selected for in vitro susceptibility testing. Throughout Quebec, 575 incident cases were identified between 1997 and 2008. The annual incidence of Nocardia infection/colonization increased from 0.33 (1997-1998) to 0.87 (2007-2008) per 100,000 inhabitants (p 0.001). In a small subset of patients for whom detailed clinical information was available, 59% of isolates corresponded to genuine infections. Nocardia farcinica predominated in specimens representing invasive infections (blood, brain, lung or pleural aspirates). Isolates were often non-susceptible to several antimicrobials, with the exception of amikacin and linezolid. Overall, 43% of 157 isolates were non-susceptible to trimethoprim-sulphamethoxazole. In conclusion, Nocardia infection/colonization remains rare. However, from 1997-1998, a progressive increase in incidence was noted in the province of Quebec. In regions such as ours, where a substantial proportion of invasive isolates are non-susceptible in vitro to trimethoprim-sulphamethoxazole, the latter may no longer be the empirical treatment of choice in immunosuppressed and severely ill patients with nocardiosis.

In low-incidence countries targeting tuberculosis (TB) elimination, TB remains a problem of a few high-risk groups. In Canada, Aboriginals, and particularly the Arctic Inuit communities, have witnessed dramatic decreases in TB during the 1960s to 1970s, but rates remain at least 10 to 20 times higher than the national average. We are describing the results of an integrated traditional and molecular epidemiology study of all culture-positive Mycobacterium tuberculosis cases in the Arctic Inuit communities of Quebec from 1990 until 2000. The demographic characteristics of the 46 TB cases included in the study were most notable for a bimodal age distribution (48% under 25 years). Genotyping analysis using multiple modalities (IS6110 restriction fragment length polymorphism, spoligotype, mycobacterial interspersed repetitive units-variable number tandem repeats) showed that 76% (35/46) of TB cases were clustered (six clusters, median size four cases) and estimated that at least 62.5% of TB cases were due to ongoing transmission. By integrating the epidemiologic and genotyping data, we observed that the genotyping clustering results were concordant with recognized epidemiologic links but most notably identified previously unrecognized intervillage transmission. This study demonstrates significant ongoing transmission in a geographically isolated, low-density population. In a resource-rich country such as Canada, these communities illustrate some of the persistent challenges of TB control and elimination.