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.

Tuberculosis and homelessness in Montreal: a retrospective cohort study.

BACKGROUND: Montreal is Canada's 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. METHODS: We 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. RESULTS: There 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. CONCLUSIONS: TB cases among homeless persons in Montreal raise concerns about delayed diagnosis and ongoing local transmission.

Major Mycobacterium tuberculosis lineages associate with patient country of origin.

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 >or=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.

Mycobacterium haemophilum epididymal abscess in a renal transplant patient.

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.

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.

Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada.

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.

Extraction of Mycobacterium tuberculosis DNA: a question of containment.

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.

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.

Tuberculosis in the Inuit community of Quebec, Canada.

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.

Genomic characterization of an endemic Mycobacterium tuberculosis strain: evolutionary and epidemiologic implications.

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 DS6(Quebec) 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.

Diversity of Mycobacterium tuberculosis isolates in an immigrant population: evidence against a founder effect.

Population-based studies have used DNA typing of Mycobacterium tuberculosis organisms to estimate the extent of ongoing tuberculosis transmission in various communities and to characterize associated risk factors. The finding of matched DNA "fingerprints" among isolates from an immigrant subgroup may reflect transmission in the adopted country but could also reflect limited diversity among M. tuberculosis organisms within that immigrant community. The authors sought to determine which hypothesis is more likely to explain the high frequency of matched isolates among Haitian-born tuberculosis patients in Montreal, Quebec, Canada. The authors determined the number of different bacterial genotypes in this community as compared with other foreign-born tuberculosis patients and applied a recently described measure of genetic similarity between M. tuberculosis organisms ("genetic distance"). Among 76 Haitian-born tuberculosis patients diagnosed during 1996-1998, the authors identified 47 distinct genotypes on the basis of standard IS6110 DNA typing and categorical analysis. In genetic distance analysis, these 47 genotypes showed as great a genetic diversity as that observed among the 191 distinct genotypes identified in 216 other foreign-born tuberculosis patients. A mycobacterial "founder effect" is unlikely to account for the high proportion of shared isolates among Haitian-born Montrealers. Recent transmission remains the most likely explanation.

Widespread pyrazinamide-resistant Mycobacterium tuberculosis family in a low-incidence setting.

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.