Louise Thibert

Major Mycobacterium tuberculosis Lineages Associate with Patient Country of Origin

ABSTRACT 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.

Extraction of Mycobacterium tuberculosis DNA: a question of containment.

ABSTRACT 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.

Sensitivities and Specificities of Spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing Methods for Studying Molecular Epidemiology of Tuberculosis

ABSTRACT The development of PCR-based genotyping modalities (spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat [MIRU-VNTR] typing) offers promise for real-time molecular epidemiological studies of tuberculosis (TB). However, the utility of these methods depends on their capacity to appropriately classify isolates. To determine the operating parameters of spoligotyping and MIRU-VNTR typing, we have compared results generated by these newer tests to the standard typing method, IS6110 restriction fragment length polymorphism, in analyses restricted to high-copy-number IS6110 isolates. Sensitivities of the newer tests were estimated as the percentages of isolates with identical IS6110 fingerprints that had identical spoligotypes and MIRU-VNTR types. The specificities of these tests were estimated as the percentages of isolates with unique IS6110 fingerprints that had unique spoligotypes and MIRU-VNTR types. The sensitivity of MIRU-VNTR typing was 52% (95% confidence interval [CI], 31 to 72%), and the sensitivity of spoligotyping was 83% (95% CI, 63 to 95%). The specificity of MIRU-VNTR typing was 56% (95% CI, 51 to 62%), and the specificity of spoligotyping was 40% (95% CI, 35 to 46%). The proportion of isolates estimated to be due to recent transmission was 4% by identical IS6110 patterns, 19% by near-identical IS6110 patterns, 33% by MIRU-VNTR typing, and 53% by spoligotyping. The low calculated specificities of spoligotyping and MIRU-VNTR typing led to misclassification of cases, inflated estimates of TB transmission, and low positive predictive values, suggesting that these techniques have unsuitable operating parameters for population-based molecular epidemiology studies.

Genomic Characterization of an Endemic Mycobacterium tuberculosis Strain: Evolutionary and Epidemiologic Implications

ABSTRACT In a study of 302 Mycobacterium tuberculosis clinical isolates from the low-incidence Canadian-born population of Quebec, we characterized a large endemic strain family by using genomic deletions. The DS6Quebec deleted region (11.4 kb) defined a strain family of 143 isolates encompassing two subgroups: one characterized by pyrazinamide (PZA) susceptibility and the other marked by a PZA-monoresistant phenotype. A second deletion (8 bp) in the pncA gene was shared by all 76 isolates with the PZA resistance phenotype, whereas a third DRv0961 deletion (970 bp) defined a further subset of 15 isolates. From their deletion profiles, we derived a most parsimonious evolutionary scenario and compared multiple standard genotyping modalities (using IS6110 restriction fragment length polymorphism [RFLP], spoligotyping, and mycobacterial interspersed repetitive units [MIRU]) across the deletion-based subgroups. The use of a single genotyping modality yielded an unexpectedly high proportion of clustered isolates for a high IS6110 copy strain (27% by IS6110 RFLP, 61% by MIRU, and 77% by spoligotyping). By combining all three modalities, only 14% were genotypically clustered overall, a result more congruent with the epidemiologic profile of reactivation tuberculosis, as suggested by the older age (mean age, 60 years), rural setting, and low proportion of epidemiologic links. These results provide insight into the evolution of genotypes in endemic strains and the potential for false clustering in molecular epidemiologic studies.

Widespread Pyrazinamide-Resistant Mycobacterium tuberculosis Family in a Low-Incidence Setting

ABSTRACT An unusually high prevalence of pyrazinamide (PZA) monoresistance in Mycobacterium tuberculosis has been observed in Quebec. In the absence of a recognized outbreak, we hypothesized that these isolates most likely represented reactivation of an old endemic strain in this low-incidence area. A case-control study of 77 PZA-resistant isolates with a specific Quebec mutation and 253 PZA-susceptible control M. tuberculosis isolates was undertaken. By molecular analysis, all 77 case isolates shared a unique mutation profile in the pncA gene which was not present in control isolates. While control isolates manifested diverse IS6110 restriction fragment length polymorphism (RFLP) patterns, spoligotypes, and major genetic groups, case isolates had similar but nonidentical IS6110 RFLP patterns, had common spoligotypes, and were confined to one major genetic group, suggesting a common clonal ancestor. By epidemiologic and geographic analyses, however, there were no significant differences between the cases and the controls. We conclude that a clonally related family of PZA-monoresistant M. tuberculosis isolates in Quebec represents historic rather than recent transmission.

Contact investigation and genotyping to identify tuberculosis transmission to children.

Background: Tuberculosis (TB) in young children is an indicator of ongoing community transmission. We examined contact investigations related to pediatric TB, yield for source case identifications and genotypes for relevant Mycobacterium tuberculosis isolates in a low-incidence setting. Methods: We reviewed public health data for all patients with TB aged <18 years reported to Montreal authorities during 1996 to 2000. M. tuberculosis isolates from patients of all ages were subjected to IS6110-based genotyping, supplemented by spoligotyping, to compare isolates from children and adults during the same years. Results: Sixty-six patients aged <18 years were diagnosed with active TB from 1996 to 2000. Mean age was 11.1 years (standard deviation 6.7 years). Twenty-five children (38%) were Canadian-born, all with at least one foreign-born parent. Nineteen children were diagnosed after contact investigations of known adult cases; 8 underwent no contact investigation. For the remaining 39 children, a total of 616 contacts were identified. The median number of contacts per child was 9 (interquartile range, 6–10). Four hundred eighty-one contacts (78%) underwent tuberculin testing; 188 (39%) were reactors and 186 (39%) began treatment of latent TB. Investigations uncovered 4 probable source cases, all involving parents or other relatives. M. tuberculosis genotyping for 38 children identified up to 14 additional possible source cases; in only one was a possible epidemiologic link evident from public health records. Conclusions: Among largely foreign-born children with active TB, contact investigations were extensive and often identified latent tuberculosis infection—but rarely source cases. However, genotyping suggested substantial, previously unrecognized transmission to children despite low overall incidence.

Mycobacterium haemophilum Epididymal Abscess in a Renal Transplant Patient

ABSTRACT Mycobacterium haemophilum is an established cause of cutaneous lesions in immunocompromised hosts. We report the first known case of epididymal abscess, which highlights the need to work up all specimens that are acid-fast bacillus-positive for M. haemophilum from immunocompromised hosts, regardless of body site.

Tuberculosis and homelessness in Montreal: a retrospective cohort study

BackgroundMontreal is Canadas second-largest city, where mean annual tuberculosis (TB) incidence from 1996 to 2007 was 8.9/100,000. The objectives of this study were to describe the epidemiology of TB among homeless persons in Montreal and assess patterns of transmission and sharing of key locations.MethodsWe reviewed demographic, clinical, and microbiologic data for all active TB cases reported in Montreal from 1996 to 2007 and identified persons who were homeless in the year prior to TB diagnosis. We genotyped all available Mycobacterium tuberculosis isolates by IS6110 restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping, and used a geographic information system to identify potential locations for transmission between persons with matching isolates.ResultsThere were 20 cases of TB in homeless persons, out of 1823 total reported from 1996-2007. 17/20 were Canadian-born, including 5 Aboriginals. Homeless persons were more likely than non-homeless persons to have pulmonary TB (20/20), smear-positive disease (17/20, odds ratio (OR) = 5.7, 95% confidence interval (CI): 1.7-20), HIV co-infection (12/20, OR = 14, 95%CI: 4.8-40), and a history of substance use. The median duration from symptom onset to diagnosis was 61 days for homeless persons vs. 28 days for non-homeless persons (P = 0.022). Eleven homeless persons with TB belonged to genotype-defined clusters (OR = 5.4, 95%CI: 2.2-13), and ten potential locations for transmission were identified, including health care facilities, homeless shelters/drop-in centres, and an Aboriginal community centre.ConclusionsTB cases among homeless persons in Montreal raise concerns about delayed diagnosis and ongoing local transmission.

Canadian Multicenter Laboratory Study for Standardized Second-Line Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis

ABSTRACT 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.

Mycobacterium tuberculosis transmission over an 11-year period in a low-incidence, urban setting.

SETTING Montreal, Canada, has a mean annual tuberculosis (TB) incidence of 9 per 100,000 population, 1996-2007. OBJECTIVE To characterise potential Mycobacterium tuberculosis transmission by patient subgroups defined by age, sex, birthplace, smear and human immunodeficiency virus status, and to estimate the proportion of cases that resulted from transmission between these patient subgroups. DESIGN Retrospective study using DNA fingerprinting techniques, with clinical and demographic information from the public health department. Among cases with matching fingerprints, a pulmonary index case was identified. The transmission index was defined as the average number of subsequent TB cases generated directly or indirectly from an index case, and was compared among subgroups, including Haitian immigrants. RESULTS Compared to non-Haitian foreign-born index cases, Canadian-born index cases were associated with 2.38 times as many (95%CI 1.24-4.58) subsequent cases, while Haitian-born index cases were associated with 3.58 times as many (95%CI 1.74-7.36). Smear-positive index cases were not independently associated with increased transmission. However, middle-aged Canadian-born index patients were associated with a disproportionate number of subsequent cases. CONCLUSION In Montreal, index patients from several high-risk groups are associated with subsequent transmission. This approach can be applied to other low-incidence settings to identify where targeted interventions could potentially further reduce transmission.