Needle in a haystack: Looking for tuberculosis in a low-incidence setting.

Background: Canada is a low-incidence country for tuberculosis (TB). The BC Public Health Laboratory diagnostic algorithm for pulmonary TB includes acid fast bacilli (AFB) smear and mycobacterial culture of all submitted sputa. TB nucleic acid amplification testing (NAT) is routinely performed on AFB-smear-positive (AFB+) sputa only. We assessed the laboratory-associated costs of implementing the international recommendations for TB NAT on AFB-smear-negative (AFB-) sputa. Methods: Two data sets were obtained: (1) all AFB- samples for a 3-year period (October 1, 2014-September 30, 2017) and (2) all AFB-, TB-culture-positive samples for the same period. One AFB- sample/patient from each defined diagnostic set of sputa was deemed eligible for TB NAT. To stratify patients by ordering location, a 1-year subset of data (October 1, 2016-September 30, 2017) was examined. Results: In the 3-year period, 0.7% of all diagnostic sets were AFB- and culture-positive. In the 1-year period, the provincial TB Services clinics submitted 26% of all AFB- samples received, but these constituted 78% of AFB-, culture-positive samples. Conclusions: The annual cost of TB NAT on one AFB- sputum sample from each eligible diagnostic set would total approximately $247,000. Targeting only TB Services clinic patients would reduce this cost to approximately $64,000/year while capturing more than 75% of AFB-, culture-positive patients. On the basis of our provincial positivity rate, it would cost approximately $6,000 to provide an early TB diagnosis for an AFB-, culture-positive patient. The cost-effectiveness to public health of this approach in a TB low-incidence setting needs to be carefully evaluated.

Historique: Le Canada est un pays à faible incidence de tuberculose. L'algorithme diagnostique de tuberculose pulmonaire du BC Public Health Laboratory inclut la recherche des bacilles acido-alcoolo-résistants (BAAR) par frottis et la culture mycobactérienne de toutes les expectorations soumises. Le test d'amplification de l'acide nucléique (TAN) pour dépister la tuberculose (TAN TB) est effectué systématiquement, mais seulement sur les expectorations positives au BAAR par frottis (BAAR+). Les chercheurs ont évalué quelle somme le laboratoire devrait investir pour mettre en œuvre les recommandations internationales visant l'utilisation du TAN TB sur des expectorations négatives BAAR par frottis (BAAR-). Méthodologie: Les chercheurs ont obtenu deux groupes de données : 1) tous les échantillons BAAR­ sur une période de trois ans (du 1er octobre 2014 au 30 septembre 2017) et 2) tous les échantillons BAAR­ dont la culture était positive à la tuberculose pendant la même période. Un échantillon de BAAR­ par patient de chaque groupe diagnostique d'expectoration était considéré comme admissible au TAN TB. Pour stratifier les patients selon la provenance du test, les chercheurs ont examiné un sous-groupe de données sur un an (du 1er octobre 2016 au 30 septembre 2017). Résultats: Pendant la période de trois ans, 0,7 % de tous les groupes diagnostiques étaient BAAR­ et positifs à la culture. Pendant la période d'un an, les cliniques provinciales de services pour la tuberculose ont soumis 26 % de tous les échantillons BAAR­ reçus, mais ils représentaient 78 % des échantillons BAAR­ positifs aux cultures. Conclusions: Le coût annuel du TAN TB sur un échantillon d'expectoration BAAR­ de chaque groupe diagnostique admissible totaliserait environ 247 000 $. Si on ciblait seulement les patients ayant consulté des services pour la tuberculose, ce coût fléchirait à environ 64 000 $ par année, mais saisirait plus de 75 % des patients BAAR­ dont la culture est positive. D'après le taux de positivité provinciale, un diagnostic précoce de tuberculose chez un patient BAAR­ positif à la culture coûterait environ 6 000 $. Le rapport coût-efficacité de cette approche pour la santé publique dans les milieux à faible incidence de tuberculose devra faire l'objet d'une évaluation attentive.

Universal genotyping reveals province-level differences in the molecular epidemiology of tuberculosis.

OBJECTIVES: Compare the molecular epidemiology of tuberculosis (TB) between two large Canadian provinces-Ontario and British Columbia (BC)-to identify genotypic clusters within and across both provinces, allowing for an improved understanding of genotype data and providing context to more accurately identify clusters representing local transmission. DESIGN: We compared 24-locus Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) genotyping for 3,314 Ontario and 1,602 BC clinical Mycobacterium tuberculosis isolates collected from 2008 through 2014. Laboratory data for each isolate was linked to case-level records to obtain clinical and demographic data. RESULTS: The demographic characteristics of persons with TB varied between provinces, most notably in the proportion of persons born outside Canada, which was reflected in the large number of unique genotypes (n = 3,461). The proportion of clustered isolates was significantly higher in BC. Substantial clustering amongst non-Lineage 4 TB strains was observed within and across the provinces. Only two large clusters (≥10 cases/cluster) representing within province transmission had interprovincial genotype matches. CONCLUSION: We recommend expanding analysis of shared genotypes to include neighbouring jurisdictions, and implementing whole genome sequencing to improve identification of TB transmission, recognize outbreaks, and monitor changing trends in TB epidemiology.

Genotyping and Whole-Genome Sequencing to Identify Tuberculosis Transmission to Pediatric Patients in British Columbia, Canada, 2005-2014.

Background: Tuberculosis (TB) in children is often an indicator of recent transmission. Genotyping and whole-genome sequencing (WGS) can enhance pediatric TB investigations by confirming or refuting transmission events. Methods: Mycobacterium tuberculosis isolates from all pediatric patients <18 years with culture-confirmed TB in British Columbia (BC) from 2005 to 2014 (n = 49) were genotyped by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and compared with adult isolates. Genotypically clustered cases underwent WGS. Clinical, demographic, and contact data were reviewed for each case. Results: Twenty-three children were Canadian-born, 7 to Canadian-born parents (CBP) and 16 to foreign-born parents (FBP). Of the 26 foreign-born children, all were born in Asia (81%) or Africa (19%). Using molecular and epidemiological data, we determined that 15 children had acquired their infection within BC, and household transmission explained all 7 Canadian-born (FBP) children that acquired TB locally. In contrast, 6 of 7 Canadian-born (CBP) children were exposed via a non-household community source. Eight Canadian-born (FBP) children acquired their infections through travel to their parents' place of birth. All but 1 of the foreign-born children acquired their infection outside of BC. Conclusions: Genotyping and genomic data reveal that drivers of pediatric transmission vary according to a child's age, birthplace, and their parents' place of birth.

Universal Genotyping for Tuberculosis Prevention Programs: a 5-Year Comparison with On-Request Genotyping.

Prospective universal genotyping of tuberculosis (TB) isolates is used by many laboratories to detect clusters of cases and inform contact investigations. Prior to universal genotyping, most TB prevention programs genotyped isolates on request only, relying on requests from public health professionals whose knowledge of a patient's clinical, demographic, and epidemiological characteristics suggested potential transmission. To justify the switch from on-request to universal genotyping-particularly in the public health domain, with its limited resources and competing priorities-it is important to demonstrate the additional benefit provided by a universal genotyping program. We compared the clustering patterns revealed by retrospective 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat genotyping of all culture-positive isolates over a 5-year period to the patterns previously established by our genotyping-on-request program in the low-incidence setting of British Columbia, Canada. We found that 23.8% of isolates were requested during the study period, and while requested isolates had increased odds of belonging to a genotype cluster (adjusted odds ratio, 2.3; 95% confidence interval, 1.5 to 3.3), only 54.6% clustered with the requested comparator strain. Universal genotyping revealed 94 clusters ranging in size from 2 to 53 isolates (mean = 5) and involving 432 individuals. On-request genotyping missed 54 (57.4%) of these clusters and 130 (30.1%) clustered individuals. Our results underscore that TB patient networks are complex, with unrecognized linkages between patients, and a prospective province-wide universal genotyping program provides an informative, bias-free tool to explore transmission to a degree not possible with on-request genotyping.

Molecular Epidemiology of Tuberculosis in British Columbia, Canada: A 10-Year Retrospective Study.

Background: Understanding regional molecular epidemiology allows for the development of more efficient tuberculosis prevention strategies in low-incidence settings. Methods: We analyzed 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) genotyping for 2290 Mycobacterium tuberculosis clinical isolates collected in the province of British Columbia (BC), Canada, in 2005-2014. Laboratory data for each isolate were linked to case-level clinical and demographic data. These data were used to describe the molecular epidemiology of tuberculosis across the province. Results: We detected >1500 distinct genotypes across the 4 major M. tuberculosis lineages, reflecting BC's diverse population. Disease site and clustering rates varied across lineages, and MIRU-VNTR was used to group the 2290 isolates into 189 clusters (2-70 isolates per cluster), with an overall clustering rate of 42.4% and an estimated local transmission rate of 34.1%. Risk factors for clustering varied between Canadian-born and foreign-born individuals; the former had increased odds (odds ratio, 7.8; 95% confidence interval [CI], 6.2-9.6) of belonging to a genotypic cluster, although nearly one-quarter of clusters included both Canadian- and foreign-born persons. Large clusters (≥10 cases) occurred more frequently within the M. tuberculosis Euro-American lineage, and individual-level risk factors associated with belonging to a large cluster included being Canadian born (adjusted odds ratio, 3.3; 95% CI, 2.3-4.8), residing in a rural area (2.3; 1.2-4.5), and illicit drug use (2.0; 1.2-3.4). Conclusions: Although tuberculosis in BC largely arises through reactivation of latent tuberculosis in foreign-born persons, locally transmitted infections occur in discrete populations with distinct disease and risk factor profiles, representing groups for targeted interventions.

Complete Genome Sequence of Mycobacterium chimaera SJ42, a Nonoutbreak Strain from an Immunocompromised Patient with Pulmonary Disease.

Mycobacterium chimaera, a nontuberculous mycobacterium (NTM) belonging to the Mycobacterium avium complex (MAC), is an opportunistic pathogen that can cause respiratory and disseminated disease. We report the complete genome sequence of a strain, SJ42, isolated from an immunocompromised male presenting with MAC pneumonia, assembled from Illumina and Oxford Nanopore data.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Nontuberculous mycobacterial breast implant infections.

BACKGROUND: For reasons that are unclear, the incidence of nontuberculous mycobacterial disease is increasing worldwide. Periprosthetic nontuberculous mycobacterial infections following augmentation mammaplasty and breast reconstruction have been reported previously in the form of case reports. METHODS: This retrospective case series examines periprosthetic nontuberculous mycobacterial infections in two western Canadian cities (Edmonton, Alberta, and Vancouver, British Columbia) over a 10-year time period. RESULTS: Ten patients were identified, four of whom had bilateral infections. The most common isolate was Mycobacterium fortuitum. Clinical features were similar to nonmycobacterial periprosthetic infections. The median time to onset of symptoms was 4.5 weeks and the median time to culture an organism was 5.4 weeks. The median duration of antibiotic therapy was 22 weeks. Patients required a mean of three additional operations after diagnosis. Nine patients underwent explantation of the involved implant(s). Reimplantation was performed in six patients a median of 11.5 months after explantation. All cases of reimplantation were successful. CONCLUSIONS: Experience with this postoperative complication is limited, as nontuberculous mycobacteria represent a minority of the pathogens responsible for periprosthetic infections. In the absence of specific features with which to identify patients at risk, the surgeon must be aware of the possibility of this infection. To achieve earlier diagnosis, the clinician should have a high index of suspicion in a patient with delayed onset of symptoms, negative preliminary cultures, and a periprosthetic infection that fails to resolve following a course of conventional antimicrobial treatment. With appropriate treatment, nontuberculous mycobacterial periprosthetic infections can be managed successfully.

Direct identification of mycobacteria in primary liquid detection media by partial sequencing of the 65-kilodalton heat shock protein gene.

We investigated extending the use of direct partial hsp65 gene sequencing for the identification of mycobacteria to isolates in primary liquid detection media as an economical, feasible, and more rapid means of identification. During the course of the study, the hsp65 sequence-based identifications for isolates from 670 primary liquid detection media determined to be positive for acid-fast bacilli were compared to the identifications derived from Accuprobes, biochemical test panels, or 16S rRNA gene sequencing. Preliminary analysis indicated a 97.6% concordance, with a final agreement of 99.1% between the identification algorithms. hsp65 sequencing costs (32.84 US dollars) were greater than the cost of identification with Accuprobe (9 US dollars) but less than the cost of the biochemical test panel identification (average cost, 98.90 US dollars) and equivalent to the cost of 16S rRNA sequencing, although there was a referral cost (59.85 US dollars) for the shipping of isolates to another reference laboratory. Analysis indicated that our laboratory would have recognized a cost savings of approximately 12,000 US dollars by using hsp65 sequencing to identify isolates from specimens with a negative fluorescent- smear status and would have achieved further savings by using it as an alternative to biochemical panel testing for fluorescent-smear-positive specimens. The time to identification by hsp65 gene sequencing was slightly longer than that required by the Accuprobe assay (1 versus 2 days), shorter than that required by the biochemical test panels (2 days versus 26 days on average), and more rapid than referral for 16S rRNA gene sequencing.

Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources.

We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium-M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, and M. terrae. Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

Predictors of clustering of tuberculosis in Greater Vancouver: a molecular epidemiologic study.

BACKGROUND: The understanding of how tuberculosis is transmitted can be improved by combining DNA fingerprinting of Mycobacterium tuberculosis with conventional epidemiologic methods. We used such techniques to determine the predictors of clustering of identical isolates from tuberculosis patients in Vancouver. METHODS: We used the restriction fragment length polymorphism (RFLP) technique and, if necessary, spoligotyping to determine DNA patterns of M. tuberculosis isolates from all patients with newly diagnosed tuberculosis in Greater Vancouver reported to the Division of Tuberculosis Control from January 1995 to March 1999. Isolates were considered to be part of a cluster if they had an identical DNA pattern. We also collected demographic and epidemiologic data. Predictors associated with being in a cluster were analyzed in a multivariate logistic regression model. RESULTS: Isolates from 793 patients (430 men) were identified; 137 (17.3%) were considered to be in clusters. After adjustment for multiple potential predictors, we found that the following patients were more likely to be part of a cluster: Canadian-born Aboriginals (v. foreign-born patients) (adjusted odds ratio [OR] 6.0, 95% confidence interval [CI] 3.0-11.7), Canadian-born non-Aboriginals (v. foreign-born patients) (adjusted OR 3.6, 95% CI 2.1-6.3), and injection drug users (v. patients who did not inject drugs) (adjusted OR 3.9, 95% CI 1.9-8.1). Patients with a prior history of tuberculosis were less likely to be part of a cluster than were patients with no history of tuberculosis (adjusted OR 0.3, 95% CI 0.1-0.8). INTERPRETATION: Our findings indicate the need to target groups at high risk of tuberculosis more aggressively to prevent transmission and to treat latent infection. DNA fingerprinting may be a useful adjunct to conventional epidemiologic methods to monitor the transmission of tuberculosis in an inner-city setting.