Canadian multicenter laboratory study for standardized second-line antimicrobial susceptibility testing of Mycobacterium tuberculosis.

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.

Potential for erroneous results indicating resistance when using the Bactec MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide.

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.

Nontuberculous mycobacterial lung infections in Ontario, Canada: clinical and microbiological characteristics.

STUDY OBJECTIVES: The aim of this study was to determine gender and clinical phenotype frequencies in pulmonary nontuberculous mycobacterial (NTM) infection and the frequency of disease in NTM isolates. DESIGN: The study is a retrospective observational cohort study of two overlapping cohorts: population cohort and clinical cohort. Setting The study was conducted at the University Health Network and Ontario Mycobacteriology Laboratory in Toronto, Ontario, Canada. PATIENTS OR PARTICIPANTS: The population cohort consisted of all patients with one or more pulmonary NTM isolates in Ontario in 2003. The clinical cohort consisted of all patients with one or more pulmonary NTM isolates at our hospital in 2002-2003. Interventions The study entailed the review of laboratory records and demographics (both cohorts) and detailed clinical records (clinical cohort). MEASUREMENTS AND RESULTS: In the population cohort (N = 1651), females comprised 48% overall and 51% with microbiological disease criteria. In the clinical cohort (N = 552), females comprised 48% overall and 55% with NTM disease. In the population cohort, 45% fulfilled microbiological disease criteria, and in the clinical cohort 46% of patients had disease. Patients with MAC isolates fulfilled microbiological disease criteria in 51% of population cohort cases and all disease criteria in 52% of clinical cohort cases. Women more commonly fulfilled microbiological disease criteria in the population cohort (51 vs. 45%, P = 0.02) and all disease criteria in the clinical cohort (53 vs. 40%, P = 0.03). Among clinical cohort patients, 26% (13 women, 44 men) had fibrocavitation, while 62% (101 women, 37 men) had nodular bronchiectasis. CONCLUSIONS: Women comprised a small majority with disease. Nodular bronchiectasis in women was most common, but significant proportions of each gender with each radiographic type were observed. NTM isolation, particularly MAC, was frequently associated with disease.

Mycobacterium tuberculosis in Ontario, Canada: Insights from IS6110 restriction fragment length polymorphism and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping.

A collection of 1,308 clinical Mycobacterium tuberculosis isolates from Ontario, Canada, was genotyped by IS6110 restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. RFLP or >12 MIRU-VNTR loci were necessary for resolution of Indo-Oceanic strains. The low clustering rate and high strain diversity indicate that, in Ontario, most tuberculosis results from reactivation of latent infections.

Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates from Ontario, Canada.

OBJECTIVES: Ontario bears the greatest burden of tuberculosis in Canada, with 40% of all cases and 60% of multidrug-resistant cases. The purpose of this study was to genotypically characterize isoniazid- and rifampicin-resistant isolates and compare these results with phenotypic drug susceptibility testing data. This is the first Canadian study to examine gene mutations that contribute to multidrug-resistant tuberculosis. METHODS: A total of 751 tuberculosis isolates were tested for drug resistance using phenotypic antimicrobial susceptibility testing methods. Isolates were then characterized using molecular methods. Following DNA extraction, PCR amplification and sequence analysis were performed on the rifampicin resistance region of rpoB, as well as the region surrounding katG315 and the inhA promoter region associated with isoniazid resistance. RESULTS: Eighteen different mutation types were found in the rpoB region of rifampicin-resistant isolates. Isolates with mutations at residues rpoB531 (64.1%), rpoB526 (15.2%) and rpoB516 (8.7%) were the most common. In addition, an insertion was found at residue 514. Three phenotypically rifampicin-resistant isolates (3.3%) were genotypically wild-type. In isoniazid-resistant strains, mutations were found most commonly at katG315 (45.4%) as well as at the inhA promoter region (28.6%). Thirty-nine isolates (25.3%) were phenotypically isoniazid-resistant but genotypically wild-type. The katG315 mutation was statistically associated with multidrug-resistant isolates. CONCLUSIONS: This study expands the knowledge of mutations that potentially contribute to drug resistance in tuberculosis and lays the foundation for developing molecular-based tests to determine drug resistance in clinical tuberculosis isolates.

Comparison of in-house and commercial 16S rRNA sequencing with high-performance liquid chromatography and genotype AS and CM for identification of nontuberculous mycobacteria.

Sequencing of the 16S gene or other targets and line probe assay are in wide use for the identification of nontuberculous mycobacteria. We compared in-house and commercial sequencing with 3 sequence databases against high-performance liquid chromatography (HPLC) and line probe assay (HAIN Genotype AS and CM) for the identification of 84 reference, clinical, and unique strains representing 41 species. Consensus of methods was used as reference standard. Sequencing identification was more specific and flexible than HPLC, but it was limited by database content and quality as well as fragment length. No one database satisfied all requirements. In-house sequencing was lower in cost than commercial sequencing or line probe assay.

Hot tub lung mimicking classic acute and chronic hypersensitivity pneumonitis: two case reports.

Pulmonary disease in otherwise healthy patients can occur by secondary exposure to nontuberculous mycobacteria from hot tubs. The pathology of hot tub lung may be related to an infection, a hypersensitivity reaction or both. Previous reports of hot tub lung have highlighted distinct pathological features that have distinguished this entity from classic hypersensitivity pneumonitis. Two cases of hot tub lung in Ontario, which presented at very different time points in their disease course, are reported; one patient presented more fulminantly with a clinical picture resembling subacute hypersensitivity pneumonitis, and the other presented with chronic disease. Both cases exhibited clinical, radiological and pathological findings closely mimicking classic subacute and chronic hypersensitivity pneumonitis.

Aging, COPD, and other risk factors do not explain the increased prevalence of pulmonary Mycobacterium avium complex in Ontario.

BACKGROUND: The cause of observed increases in pulmonary Mycobacterium avium complex (pMAC) isolation and disease is unexplained. To explore possible causes of the increase in pMAC isolation and disease prevalence in Ontario, Canada, we studied age and other population-level risk factors. METHODS: We determined age and sex of patients with pMAC disease between 2003 and 2008. We then estimated whether the potential effect of population aging and changes in prevalence of HIV infection, solid organ transplant, COPD, and tumor necrosis factor-α (TNF-α) inhibition have contributed to the observed increase in pMAC disease. RESULTS: During 2003 to 2008, pMAC isolation and disease prevalence (per 100,000) both increased (8.44 to 12.62 and 4.35 to 6.81, respectively). The total number of cases of disease increased by 348 (2.46 per 100,000). Based on actual contemporary population changes, aging could explain 70 additional cases (increase of 0.57 per 100,000). The increase in self-reported COPD prevalence could potentially explain 11 (95% CI, 0-42) additional cases (increase of 0.09 per 100,000 [95% CI, 0-0.34 per 100,000]). HIV infection, solid organ transplant, and TNF-α inhibition combined could potentially explain no more than 73 additional cases (increase of 0.60 per 100,000). CONCLUSIONS: Although population aging appears to be a major risk factor, the increase in pMAC disease in Ontario could be only partly explained by aging, increases in COPD, HIV, solid organ transplantation, and TNF-α inhibition therapy. The increase in pMAC is likely multifactorial and may be affected by environmental or pathogen factors not addressed in this study.

Isolation prevalence of pulmonary nontuberculous mycobacteria in Ontario in 2007.

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 (excluding Mycobacterium 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 of Mycobacterium avium complex (8.8%, P<0.0001) and Mycobacterium 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.

Non-tuberculous mycobacteria in children with cystic fibrosis: isolation, prevalence, and predictors.

BACKGROUND: Screening for non-tuberculous mycobacteria (NTM) is recommended for adults with cystic fibrosis (CF). The relevance of this organism in North American pediatric CF patients is unclear as there is limited NTM prevalence data for children. We aimed to determine the prevalence of NTM in children with CF from a single expectorated sputum and identify clinical predictors of NTM isolation. Additionally, we compared two different sputum decontamination methods before mycobacterial culture. METHODS: From March to November 2004, all sputum-producing patients aged 6-18 years attending the CF clinic at the Hospital for Sick Children in Toronto, Canada, were screened for NTM. Sputum samples were processed by both a single (N-acetyl-l-cysteine + NaOH) and double (N-acetyl-l-cysteine + NaOH + oxalic Acid) decontamination method. Using our CF clinic database and patient charts we analyzed differences in FEV(1), age, sex, pancreatic sufficiency, body mass index, bacterial colonization, and antibiotic use between NTM positive and negative patients. RESULTS: Of 98 study patients, 6 (6.1%) were positive for NTM, 2 with Mycobacterium abscessus, and 4 with Mycobacterium avium complex. One patient with M. abscessus had clinically significant lung disease requiring treatment. We found no predictors of NTM isolation. The double decontamination method allowed detection of only half (3/6) of the positive NTM cultures. CONCLUSIONS: 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.

Isolation prevalence of pulmonary non-tuberculous mycobacteria in Ontario, 1997 2003.

BACKGROUND: The prevalence of pulmonary non-tuberculous mycobacteria (NTM) infection is reportedly increasing. A study was undertaken of the "isolation prevalence" of pulmonary NTM in Ontario, Canada between 1997 and 2003 and the frequency of pulmonary NTM "disease". METHODS: In a retrospective cohort, the "isolation prevalence" was studied by reviewing all positive NTM culture results from the Tuberculosis and Mycobacteriology Laboratory, Ministry of Health and Long-Term Care in Toronto from 1997 to 2003. This laboratory identifies at least 90% of NTM isolates in Ontario, Canada. Prevalence was compared between years using a negative binomial model. To study the frequency of "disease" (defined by American Thoracic Society criteria), the records of patients who had an isolate in 2003 and had been assessed at our hospital were reviewed. RESULTS: 22,247 pulmonary isolates were obtained from 10,231 patients. The "isolation prevalence" of all species (excluding Mycobacterium gordonae) was 9.1/100,000 in 1997, rising to 14.1/100,000 by 2003 (p<0.0001) with a mean annual increase of 8.4%. Similar increases were observed for individual species. 200 patients assessed at our institution were studied using American Thoracic Society criteria for "disease". Microbiological criteria were fulfilled by 37%. Of patients with adequate data, 74% fulfilled clinical criteria, 77% fulfilled radiological criteria and 33% fulfilled all criteria. CONCLUSIONS: The "isolation prevalence" of pulmonary NTM has significantly and rapidly increased in Ontario; a sizeable proportion of patients are likely to have "disease".

Outbreak of acupuncture-associated cutaneous Mycobacterium abscessus infections.

BACKGROUND: Cutaneous atypical mycobacterial infections have been increasingly described in association with cosmetic and alternative procedures. OBJECTIVE: We report an outbreak of acupuncture-associated mycobacteriosis. Between April and December 2002, 32 patients developed cutaneous mycobacteriosis after visiting an acupuncture practice in Toronto, Canada. RESULTS: Of 23 patients whose lesions were biopsied, 6 (26.1%) had culture-confirmed infection with Mycobacterium abscessus. These isolates were genetically indistinguishable by amplified fragment length polymorphism. The median incubation period was 1 month. Of 24 patients for whom clinical information was available, 23 (95.8%) had resolution of their infection. All patients developed residual scarring or hyperpigmentation. CONCLUSION: Nontuberculous mycobacteria should be recognized as an emerging, but preventable, cause of acupuncture-associated infections.

Mycobacterium lacus sp. nov., a novel slowly growing, non-chromogenic clinical isolate.

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)).

Phenotypic and molecular characterization of clinical isolates of Mycobacterium elephantis from human specimens.

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.

Method for inactivating and fixing unstained smear preparations of mycobacterium tuberculosis for improved laboratory safety.

The inactivation of smears that contain Mycobacterium tuberculosis for microscopy before removal of the material from a biosafety cabinet is an important safety factor in preventing the potential transmission of tuberculosis to laboratory workers. The fixing and inactivating properties of heat flaming, 70% ethanol, and 1, 3, and 5% phenol in ethanol for smears containing M. tuberculosis were investigated. Heat flaming failed to inactivate the smear material, whereas 5% phenol in ethanol successfully fixed and inactivated all smears containing M. tuberculosis both from concentrated sputum samples and from culture material.