Pamela Chedore

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”.

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.

Non-tuberculous mycobacteria in children with cystic fibrosis: Isolation, prevalence, and predictors†

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.

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.

Routine use of the Gen-Probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory.

The new version of the Amplified Mycobacterium Tuberculosis Direct Test, MTD2 (Gen-Probe Inc., San Diego, CA) has been implemented as part of the regular testing algorithm for detecting Mycobacterium tuberculosis (MTB) in selected respiratory and non-respiratory specimens in our laboratory. At the Central Public Health Laboratory, Etobicoke, Ontario, we receive specimens for the detection of mycobacteria from all areas of the Province of Ontario. The laboratory processes approximately 25,000 specimens per year, and receives approximately 2000 reference cultures for identification. There are 600 to 700 new cases of tuberculosis detected yearly. Over the 1-year period (1997-98), 823 specimens were tested by MTD2 and the results were compared with radiometric culture (Bactec, Becton Dickinson, Sparks, MD) and clinical diagnosis, giving an overall sensitivity, specificity, positive predictive value and negative predictive values of 100%, 99.6%, 97.4% and 100%, respectively. Two hundred and two cases of respiratory TB and 56 cases of extrapulmonary TB were detected by MTD2 within 0-4 days of specimen arrival in the laboratory. By appropriate selection of specimens for testing, the MTD2 can provide a fast, accurate, and cost-effective method for the detection of MTB in clinical specimens.

Phenotypic and Molecular Characterization of Clinical Isolates of Mycobacterium elephantis from Human Specimens

ABSTRACT 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 44368T 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.

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.

Method for Inactivating and Fixing Unstained Smear Preparations of Mycobacterium tuberculosis for Improved Laboratory Safety

ABSTRACT 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.

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)).

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.