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Setting The prairie provinces of Canada. Objective To characterize tuberculosis (TB) transmission among the Indigenous and non-Indigenous Canadian-born peoples of the prairie provinces of Canada. Design A prospective epidemiologic study of consecutively diagnosed adult (age ≥ 14 years) Canadian-born culture-positive pulmonary TB cases on the prairies, hereafter termed "potential transmitters," and the transmission events generated by them. "Transmission events" included new positive tuberculin skin tests (TSTs), TST conversions, and secondary cases among contacts. Results In the years 2007 and 2008, 222 potential transmitters were diagnosed on the prairies. Of these, the vast majority (198; 89.2%) were Indigenous peoples who resided in either an Indigenous community (135; 68.2%) or a major metropolitan area (44; 22.2%). Over the 4.5-year period between July 1st, 2006 and December 31st 2010, 1085 transmission events occurred in connection with these potential transmitters. Most of these transmission events were attributable to potential transmitters who identified as Indigenous (94.5%). With a few notable exceptions most transmitters and their infected contacts resided in the same community type. In multivariate models positive smear status and a higher number of close contacts were associated with increased transmission; adjusted odds ratios (ORs) and 95% confidence intervals (CIs), 4.30 [1.88, 9.84] and 2.88 [1.31, 6.34], respectively. Among infected contacts, being Indigenous was associated with disease progression; OR and 95% CI, 3.59 [1.27, 10.14] and 6.89 [2.04, 23.25] depending upon Indigenous group, while being an infected casual contact was less likely than being a close contact to be associated with disease progression, 0.66 [0.44, 1.00]. Conclusion In the prairie provinces of Canada and among Canadian-born persons, Indigenous peoples account for the vast majority of cases with the potential to transmit as well as the vast majority of infected contacts. Active case finding and preventative therapy measures need to focus on high-incidence Indigenous communities.

The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.

The purpose of this study was to establish a standardized protocol for second-line antimicrobial susceptibility testing of Mycobacterium tuberculosis using the Bactec MGIT 960 system in Canadian laboratories. Four Canadian public health laboratories compared the susceptibility testing results of 9 second-line antimicrobials between the Bactec 460 and Bactec MGIT 960 systems. Based on the data generated, we have established that the Bactec MGIT 960 system provides results comparable to those obtained with the previous Bactec 460 method. The critical concentrations established for the testing of the antimicrobials used are as follows: amikacin, 1 μg/ml; capreomycin, 2.5 μg/ml; ethionamide, 5 μg/ml; kanamycin, 2.5 μg/ml; linezolid, 1 μg/ml; moxifloxacin, 0.25 μg/ml; ofloxacin, 2 μg/ml; p -aminosalicylic acid, 4 μg/ml; rifabutin, 0.5 μg/ml.

Sequencing of the 16S ribosomal DNA (rDNA )for identification of nontuberculous mycobacteria (NTM) has contributed to the establishment of more than 35 new species during the last decade. Increasingly, NTM are accepted as potential or proven pathogens. We identified, by 16S rDNA sequence analysis, slowly growing NTM isolates negative by AccuProbe (GenProbe, San Diego, CA) that previously were identified by using conventional biochemical techniques, to determine the accuracy of reporting AccuProbenegative NTM prior to sequence-based identification. Of 82 strains, 30 were deemed novel. An attempt was made to determine the clinical importance of previously misidentified novel species. Clinical cases are described for a number of strains previously identified as Mycobacterium terrae complex, Mycobacterium scrofulaceum, and Mycobacterium avium complex. As sequence-based identification methods become more commonplace in clinical microbiology laboratories, there is a need to understand the significance of previously undescribed species, which often mimic and subsequently are identified as well-established species. Many nontuberculous mycobacteria (NTM) are ubiquitous in the environment and may colonize and occasionally cause infection in humans. Mycobacterium avium complex, Mycobacterium kansasii, and members of the Mycobacterium fortuitum complex are causative agents for most NTM infections and currently have a well-understood environmental epidemiology. 1 Other NTM species rarely cause infection

Rural Underpinnings for Resiliency and Linkages (RURAL) is an example of a Safe Schools/Healthy Students (SS/HS) project. RURAL provides services to children and families in a rural Kansas county. The county's rates of juvenile alcohol and illicit drug use are higher than state averages. The RURAL project was designed to enhance existing partnerships among three school districts, the local community mental health center, law enforcement, the regional prevention center, and Fort Hays State University. A significant number of evidence-based strategies were implemented in the schools and community in a short period of time. Using a public health approach to increase school safety and promote healthy behaviors, RURAL introduced strategies designed to provide universal prevention for the school population, early intervention for at-risk children and families, and intensive services for those with the greatest needs. Services were provided to students (preschool–young adults) and their families. School psychologists held key roles in the development, implementation, and management of the project. Preliminary evaluation findings are presented, and the importance of local process and outcome evaluation is discussed. © 2003 Wiley Periodicals, Inc. Psychol Schs 40: 531–547, 2003.

The critical concentration (CC) for ethambutol testing on the Bactec MGIT 960 M. tuberculosis susceptibility testing has been questioned in recent publications. In this study, we correlate susceptibility results from the Bactec 460, MGIT 960 and embB gene sequencing to determine if the Bactec MGIT 960 adequately detects ethambutol resistance. We discovered discrepancies between the methods that highlight a need to re-evaluate ethambutol susceptibility testing recommendations, namely by considering lowering currently recommended CC on the MGIT 960. Further studies on the clinical significance of low-level ethambutol resistance are also required.

Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.

Mycobacterium branderi , a potential human pathogen first characterized in 1995, has been isolated from respiratory tract specimens. We report here a case in which M. branderi was the only organism isolated upon culture from a hand infection. This isolate, along with a second isolate from a bronchial specimen, was subjected to conventional identification tests for mycobacterial species. Further analysis by high-performance liquid chromatography (HPLC) of mycolic acids and 16S rRNA gene sequencing was performed, and the antibiotic susceptibility profile was determined for both strains. Biochemical tests and the HPLC pattern were consistent with that of M. branderi and M. celatum , which are very similar. The 16S rRNA gene sequence of both strains corresponded to that of M. branderi and enabled us to confidently differentiate this organism from other closely related species such as M. celatum . This contributes to a further understanding of the status of this species as a potential human pathogen as well as illustrating the need for molecular diagnostics as a complementary method for the identification of rare mycobacterial species.

16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium . Previous studies have shown that Mycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii . In the present study, we have used the recA gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis , M. smegmatis , and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between M. gastri and M. kansasii to 75.7% between M. aurum and M. leprae . Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae . We propose that recA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the Mycobacterium species.

Killer immunoglobulin-like receptors (KIR) on natural killer (NK) cells interact with other immune cells to monitor the immune system and combat infectious diseases, such as tuberculosis (TB). The balance of activating and inhibiting KIR interactions helps determine the NK cell response. In order to examine the enrichment or depletion of KIRs as well as to explore the association between TB status and inhibitory/stimulatory KIR haplotypes, we performed KIR genotyping on samples from 93 Canadian First Nations (Dene, Cree, and Ojibwa) individuals from Manitoba with active, latent, or no TB infection, and 75 uninfected Caucasian controls. There were significant differences in KIR genes between Caucasians and First Nations samples and also between the First Nations ethnocultural groups (Dene, Cree, and Ojibwa). When analyzing ethnicity and tuberculosis status in the study population, it appears that the KIR profile and centromeric haplotype are more predictive than the presence or absence of individual genes. Specifically, the decreased presence of haplotype B centromeric genes and increased presence of centromeric-AA haplotypes in First Nations may contribute to an inhibitory immune profile, explaining the high rates of TB in this population.

Mutations in the erm(41) gene of M.abscessus group organisms are associated with differences in inducible macrolide resistance, with current recommendations being to hold rapidly growing isolates for up to 14 days in order to ensure that resistance which develops more slowly can be detected. This study aimed to determine the ideal incubation time for accurate identification of inducible macrolide resistance as well as to determine if there was an association between the time taken to detect inducible resistance in M.abscessus group organisms and their erm(41) sequevar. We amplified and sequenced the erm(41) genes of a total of 104 M.abscessus group isolates and determined their sequevars. The isolates were tested for phenotypic clarithromycin resistance at days 7, 10, 14 and 21, using Trek Diagnostics Sensititre RAPMYCO microbroth dilution plates. Associations between erm(41) gene sequevars and time to detection of resistance were evaluated using Fisher’s exact test in R. The samples included in this study fell into 14 sequevars, with the majority of samples falling into Sequevar02 (16), Sequevar06 (15), Sequevar08 (7) and Sequvar 15 (31), and several isolates that were in small clusters, or unique. The majority (82.7%) of samples exhibiting inducible macrolide resistance were interpreted as resistant by day 7. Two isolates in Sequevar02, which has a T28C mutation that is associated with sensitivity, showed intermediate resistance at day 14, though the majority (13) were sensitive at day 14. The majority of isolates with inducible macrolide resistance fell into Sequevars 06,08 and 15, none of which contain the T28C mutation. These sequevars were analyzed to determine if there was any correlation between sequevar and time to detection of resistance. None was found. Based on these findings, we recommend the addition of a day 7 read to the CLSI guidelines to improve turn-around-times for these isolates. It is also recommended that erm(41) gene sequencing be added to routine phenotypic testing for the resolution of cases with difficult-to-interpret phenotypic results.

Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit – variable number of tandem repeat (MIRU–VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU–VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.

The current gold standard for Mycobacterium tuberculosis complex (MTBC) genotyping is insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) as it provides the highest discriminatory power of all available MTBC genotyping methods. However, RFLP is labour intensive and the interpretation of data from this method can be susceptible to errors. In 2001 a rapid, reproducible variable number of tandem repeat (VNTR) based typing method using 12 mycobacterial interspersed repetitive units (MIRU) was developed. Despite this advancement, this method lacked the discriminatory power of IS6110-RFLP. More recently a set of 24 MIRU-VNTR loci was reported to have greater discriminatory power than the original 12 locus system and may exceed that of RFLP when combined with spoligotyping. We compared the 24 locus method to the 12 locus method in order to improve surveillance of tuberculosis in Canada. A random sample of 650 MTBC isolates from British Columbia, Saskatchewan, Manitoba and Quebec Canada was genotyped using the 24 MIRU loci. Comparison of the data for the 12 and 24 MIRU loci showed an increase of the Hunter-Gaston discriminatory index (HGDI) from 0.895 (12 loci) to 0.920 (24 loci). The implementation of the 24 locus MIRU-VNTR methods offers improvement in discriminatory power over the traditional 12 locus method. For long-term surveillance of MTBC within Canada, the use of 24 MIRU-VNTR loci will provide rapid, highly discriminatory molecular epidemiology information.

Rifampin is a key component of standard short-course first-line therapy against Mycobacterium tuberculosis (MTB). Rifampin monoresistant MTB, previously a rare phenomenon, is now being reported at increasing rates worldwide. We report a mutation in the rpoB region leading to low level rifampin monoresistance in a cluster of HIV-positive patients. All rifampin monoresistant isolates identified from 2004 to 2006 underwent susceptibility confirmation, sequencing of rpoB and genotyping. Three patients were found to have a previously undocumented 3 base pair insertion at codon 525 in the rpoB region. The earliest initial case was infected with fully susceptible MTB. Disease relapse occurred 7 months later with a genotypically identical MTB isolate, showing acquired rifampin monoresistance. MTB isolates from 2 subsequent patients showed primary rifampin monoresistance with an identical genotype to the index case. Patients with rifampin monoresistant MTB tend to have poorer outcomes than those with fully susceptible strains. Risk factors for the development of rifampin monoresistance include co-morbid HIV infection and previously treated tuberculosis. HIV infection has been associated with malabsorption of anti-tuberculous medications leading to sub-therapeutic levels of administered drugs. These factors may have played a role in the development of this previously undocumented mutation.

A case of presumed bacillus Calmette-Guérin (BCG) cystitis in an elderly female patient following direct intravesical BCG instillation treatment for papillary transitional cell carcinoma is reported. The organism cultured from urine samples was eventually identified as a rifampin-resistantMycobacterium bovisBCG isolate. Because the patient had received rifampin monotherapy during the course of treatment for presumed BCG disease, the clinical picture favoured acquired rifampin resistance. Sequencing of the target gene for rifampin (rpoB) confirmed a known mutation responsible for conferring high levels of resistance to both rifampin and rifabutin (Ser531Tyr). To the authors' knowledge, this is the first reported case ofM bovisBCG disease in a non-HIV patient where the organism had acquired drug resistance to rifampin, and the second reported case ofM bovisBCG that had acquired drug resistance. The present case demonstrates the necessity to re-evaluate appropriate guidelines for the effective treatment of BCG disease.

Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being d -mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of ≤1 μg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of “farcy.” By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense . Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing ≥98.4% identity with each other and ≥98.6% identity with the sequence of five bovine strains of M. senegalense . There was ≤97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense . This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.

Macrolides are an important tool in the treatment of Mycobacterium avium complex infections. Here, we evaluate the use of 23S rRNA gene sequencing for the rapid detection of macrolide resistance. Routine sequencing of the 23S rRNA gene is highly specific for macrolide resistance but lacks in sensitivity.

A group of pigmented, slowly growing mycobacteria identified by 16S rRNA gene sequencing as ‘MCRO 33’ (GenBank accession no. AF152559) have been isolated from several clinical specimens in various laboratories across Canada. Genotypically, the organism is most closely related to Mycobacterium simiae. However, it presents with a similar phenotypic profile to Mycobacterium scrofulaceum. Several reference strains obtained from ATCC and TMC culture collections, previously identified as M. scrofulaceum or M. simiae, have also been found to possess the MCRO 33 16S rRNA gene sequence. Biochemical testing, susceptibility testing, HPLC, hsp65 gene and 16S–23S spacer (ITS1) sequencing were performed on clinical and reference strains to characterize further this unique species. Of the clinical strains, one was isolated from a cervix biopsy whereas all other clinical isolates were obtained from respiratory samples. In one patient, symptoms, imaging and repeat clinical specimens positive on culture for this organism were suggestive of active clinical disease. The description of this species, for which the name Mycobacterium parascrofulaceum sp. nov. is proposed, follows the present trend of a large number of novel Mycobacterium species identified due in great part to sequence-based methods. The type strain is HSC68T (=ATCC BAA-614T=DSM 44648T).

Setting: The Canadian province of Manitoba. Objective: To confirm the putative hypervirulence observed in Mycobacterium tuberculosis Type1 strain and further characterize the progression and manifestation of pulmonary tuberculosis caused by this strain in comparison to other common clinical isolates from Manitoba. Design : C3H and BALB/c mice were exposed to aerosols either of Type1, Type2, Type5, Type72 or H37Rv strain to study their respective survival profiles. Additionally, bacillary loads and lung histology were examined at 15 days post-exposure. Results: In both mouse models, Type-1 infected mice succumbed to disease significantly earlier than other strains ( p ⩽0.0002). Differences between average log 10 CFU values between clinical isolates were less than 1log10 difference. In C3H mice, the amount of granulomatous inflammation was highest in Type1 infected mice but not significantly different than all clinical strains. In contrast, BALB/c mice infected with Type1 induced the lowest amount of granulomatous inflammation compared to other clinical isolates. Conclusion: Our results indicate that although mice infected with the Type1 strain died significantly earlier than mice infected with other clinical strains in both C3H and BALB/c mice, the hypervirulence of the Type1 strain is not attributed to the growth rate of the organism, as differences in growth between clinical M. tuberculosis isolates were insignificant.

Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies. Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems. In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry. The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs's method, and the Yamane procedure. Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet. All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M. tuberculosis H37Ra. Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis. Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations. The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M. tuberculosis H37Ra. While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M. tuberculosis.

Molecular techniques are playing an important role in the diagnosis of nontuberculous mycobacterial infections. This case report describes a chronic soft tissue infection in an immunocompetent patient caused by a previously undescribed pigmented, rapidly growing Mycobacterium species, emphasizing the importance of clinical suspicion and effective laboratory techniques in the diagnosis and treatment of infection.

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.

A strain of a novel non-chromogenic mycobacterium was isolated from synovial tissue from a 68-year-old female with bursitis of her right elbow. The slowly growing strain had a unique PCR-restriction enzyme analysis (PRA) profile of the hsp65 gene and 16S rRNA gene sequence in comparison with other mycobacterium species. The most closely related species, as determined by 16S rRNA gene sequence analysis, are Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium ulcerans and members of the Mycobacterium tuberculosis complex. The HPLC and biochemical profiles resembled those of Mycobacterium gastri, although differences were noted in the peak-height ratio of the HPLC pattern and the nitrate and pyrazinamidase tests. On the basis of PRA, HPLC, biochemical and 16S rRNA gene sequence analyses, the name Mycobacterium lacus sp. nov. is proposed for this potential pathogen. The type strain is strain NRCM 00-255(T) (= ATCC BAA-323(T) = DSM 44577(T)).

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing ∼440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

Eleven strains of a rapidly growing mycobacterium were isolated from patient specimens originating from various regions of the province of Ontario, Canada, over a 2-year period. Unique high-performance liquid chromatography (HPLC) and PCR-restriction enzyme pattern analysis (PRA) profiles initially suggested a new Mycobacterium species, while sequencing of the 16S rRNA gene revealed a sequence match with Mycobacterium sp. strain MCRO 17 (GenBank accession no. X93028), an isolate determined to be unique which is to date uncharacterized, and also a close similarity to M. elephantis (GenBank accession no. AJ010747), with six base pair variations. A complete biochemical profile of these isolates revealed a species of mycobacteria with phenotypic characteristics similar to those of M. flavescens. HPLC, PRA, and 16S rRNA sequencing of strain M. elephantis DSM 44368 T and result comparisons with the clinical isolates revealed that these strains were in fact M. elephantis, a newly described species isolated from an elephant. All strains were isolated from human samples, 10 from sputum and 1 from an axillary lymph node. Since 1997 alone, 17 new species of mycobacteria have been established. With the exponential discovery of new species, phenotypic profiles determined by conventional testing are less reliable as a means of accurately identifying the 95 mycobacterial species currently established (J. P. Euzeby, http://www .bacterio.cict.fr/m/mycobacterium.html). The increased use of sequence-based identification techniques, as well as high-performance liquid chromatography (HPLC) analysis of mycolic acids, has become a necessity in identifying less common species of mycobacteria. In this study we examined 11 mycobacterial strains, which gave unique and identical profiles when analyzed by HPLC and PCR-restriction pattern enzyme analysis (PRA). Sequencing of the 16S rRNA gene revealed a perfect identity with an uncharacterized Mycobacterium strain from the GenBank database designated MCRO 17 (GenBank accession no. X93028) and a close relationship to M. elephantis DSM 44368 T (GenBank accession no. AJ010747). Ten strains were isolated from sputa of different patients, and the 11th strain was isolated from an axillary lymph node from a 27-year-old male. This patient presented with lymphadenopathy, and after surgical excision, histopathology of the node showed caseating granulomas. Subsequent mycobacterial culture yielded a lightly pigmented mycobacterium that corresponded to the above cluster. The patients resided in various regions of the province of Ontario, Canada. This paper describes a cluster of mycobacterial species isolated on 11 separate occasions from human specimens, subsequently identified as M. elephantis based on further character

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.

Setting Nationwide Canadian public health surveillance. Objective Description of demographic features and disease characteristics of drug-resistant tuberculosis (TB) in Canada over a 12 year period. Design Continuous surveillance of all cases of culture-confirmed TB in Canada. Demographic and microbiologic features were analyzed and comparisons between drug-susceptible, multidrug-resistant (MDR), and drug-resistant not-MDR were made. Cases of extensively drug resistant TB are described. Results 15,993 cases of culture-confirmed TB were reported during the study period. There were 5 cases of XDR-TB, 177 cases of MDR-TB, and 1,234 cases of first-line drug resistance not-MDR. The majority of drug-resistant cases were reported in foreign-born individuals, with drug-resistant cases diagnosed earlier post-arrival in Canada compared to drug-susceptible cases. In MDR-TB isolates, there was a high rate of drug-resistance to other first- and second-line drugs, making reliable empiric therapeutic recommendations for MDR-TB difficult. There was a statistically significant association between both MDR and drug-resistance not-MDR, and the risk of a negative treatment outcome (defined as treatment failure, absconded, or treatment ongoing >3 yrs). Conclusion Drug-resistance complicates TB management even in developed nations with well-established TB control programs. The predominantly international origin of drug-resistant cases highlights the need for global strategies to combat TB.

Background Outcome of Mycobacterium tuberculosis (Mtb) infection is affected by virulence of the infecting strain of Mtb, host environment, co-morbidities, and the genetic composition of the host, specifically the presence or absence of genes involved in immune responses/regulation. It is hypothesized that specific killer immunoglobulin-like receptor (KIR) genes may be associated with Mtb infection and clinical outcome. This cross-sectional study examined the KIR gene frequencies, profiles, and haplotypes of individuals with active tuberculosis, latent tuberculosis infection, compared to TB and HIV negative healthy controls. Results Analysis of KIR gene frequencies revealed differences among disease status groups, suggesting that enrichment or depletion of specific KIR genes may direct the disease outcome. Mtb infected individuals were more likely to have a centromeric-AA haplotype compared to controls. Conclusion The differences in KIR gene frequencies and haplotypes may result in differential cytokine expression, contributing to different disease outcomes, and suggest a genetic influence on Mtb susceptibility and pathogenesis.

Background In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms. Methods An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. Results Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. Conclusions This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.