Extensive Homoplasy but No Evidence of Convergent Evolution of Repeat Numbers at MIRU Loci in Modern Mycobacterium tuberculosis Lineages.

More human deaths have been attributable to Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.

Can colony-forming unit testing be used to extend the shelf life of BCG vaccines?

A recent shortage in supply of Bacille Calmette-Guérin (BCG), the live attenuated vaccine given to protect against tuberculosis (TB) caused major disruption to global vaccination programs. In this study, we assessed whether quantification of viable bacteria, could be used to inform the use of the BCG vaccine beyond its manufacturer-assigned expiration date. The viability of a single batch of BCG-Denmark was tested in three independent laboratories. There was high inter-vial and inter-laboratory variability in viability counts, however all three laboratories detected a decrease in BCG viability over time. Despite this, there was no difference in the rate of BCG scar formation in infants who were vaccinated with this batch of BCG before and after its manufacturer-assigned expiration date. This study demonstrates the potential for using BCG viability counts to guide the use of BCG vaccine beyond the manufacturer-assigned expiration date.

Mutations associated with in vitro resistance to bedaquiline in Mycobacterium tuberculosis isolates in Australia.

Genome-wide analysis of 517 Mycobacterium tuberculosis isolates from New South Wales, Australia, have identified previously reported and 8 new mutations in Rv0678 and atpE genes linked to in vitro resistance to bedaquiline, a new class of antimycobacterial drugs. These mutations were present in 2.9% of prospectively sequenced genomes but in 10.6% of strains that were phenotypically multidrug-resistant. However only 14% of isolates with these mutations demonstrated elevated minimum inhibitory concentrations to bedaquiline.

16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

BACKGROUND: Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways. METHODS: We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants. RESULTS: Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade. CONCLUSION: Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

Genotype heterogeneity of Mycobacterium tuberculosis within geospatial hotspots suggests foci of imported infection in Sydney, Australia.

In recent years the State of New South Wales (NSW), Australia, has maintained a low tuberculosis incidence rate with little evidence of local transmission. Nearly 90% of notified tuberculosis cases occurred in people born in tuberculosis-endemic countries. We analyzed geographic, epidemiological and genotypic data of all culture-confirmed tuberculosis cases to identify the bacterial and demographic determinants of tuberculosis hotspot areas in NSW. Standard 24-loci mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-24) typing was performed on all isolates recovered between 2009 and 2013. In total 1692/1841 (91.9%) cases with confirmed Mycobacterium tuberculosis infection had complete MIRU-24 and demographic data and were included in the study. Despite some year-to-year variability, spatio-temporal analysis identified four tuberculosis hotspots. The incidence rate and the relative risk of tuberculosis in these hotspots were 2- to 10-fold and 4- to 8-fold higher than the state average, respectively. MIRU-24 profiles of M. tuberculosis isolates associated with these hotspots revealed high levels of heterogeneity. This suggests that these spatio-temporal hotspots, within this low incidence setting, can represent areas of predominantly imported infection rather than clusters of cases due to local transmission. These findings provide important epidemiological insight and demonstrate the value of combining tuberculosis genotyping and spatiotemporal data to guide better-targeted public health interventions.

Antibiotic susceptibility of diverse Mycobacterium abscessus complex strains in New South Wales, Australia.

Members of the Mycobacterium abscessus complex are emerging pathogens of increasing importance, causing both respiratory and soft tissue infections, but precise speciation is problematic. This study was performed to examine the subspecies and antibiotic susceptibility of M. abscessus complex isolates collected during 2013 at the statewide New South Wales Mycobacterium Reference Laboratory (NSW MRL), Australia. Mycobacterium abscessus subsp. abscessus accounted for more than half of all M. abscessus isolates (n = 24, 57.1%), and M. abscessus subsp. massiliense comprised the remainder of the isolates (n = 18, 42.9%). There were no M. abscessus subsp. bolletii isolates. The prevalence of antibiotic resistance to all antibiotics, apart from amikacin was high, with 26.3% of isolates being reliably susceptible to only amikacin. Most M. abscessus subsp. abscessus isolates (80%) demonstrated inducible clarithromycin resistance whereas the majority of M. abscessus subsp. massiliense isolates (94.4%) remained susceptible to clarithromycin. There was a good correlation between the erm(41) genotype and clarithromycin susceptibility results after 14 days of incubation for most isolates with only three exceptions. Further studies correlating in vitro susceptibility profiles with clinical outcomes of M. abscessus infections treated with combination antimicrobial therapy are warranted.

Epidemiology of Peripheral Lymph Node Tuberculosis and Genotyping of M. tuberculosis Strains: A Case-Control Study.

BACKGROUND: This study examined potential risk factors of lymph node tuberculosis (LNTB), including phylogenetic lineages of Mycobacterium tuberculosis (MTB), in comparison to pulmonary tuberculosis (PTB) in a setting with an ethnically diverse population. METHODS: We conducted a case-control study at a major tuberculosis clinic in Sydney, Australia, which included all patients with peripheral LNTB seen at the clinic between 2000 and 2012. Controls were randomly selected patients with PTB seen at the same clinic during the study period. Epidemiological data were extracted from the hospital electronic database and medical records. Associations between LNTB and age, sex, ethnicity, comorbidities and phylogenetic lineages of MTB in comparison to PTB were examined using logistic regression in univariate and multivariate analyses. RESULTS: There were 212 cases with LNTB and 424 randomly selected controls with PTB. Among patients with LNTB, 74% were female and the mean age (standard deviation, SD) was 42 (16) years. Among patients with PTB, 43% were female and the mean age was 44 (22) years. Females, 45 to 64-year-olds and Southern Asians had an increased risk for LNTB (OR 3.13, 95% CI 2.10-4.67; OR 2.50, 95% CI 1.29-4.84; OR 3.95, 95% CI 1.54-10.12 respectively). Patients with diabetes were at a higher risk of PTB (OR 0.40, 95% CI 0.19 - 0.83 for LNTB). A subset analysis showed that patients infected with the East African Indian strain of MTB were more likely to develop LNTB (OR 10.07, 95% CI 2.37-42.77). CONCLUSIONS: An increased risk for LNTB (but still lower rates than for PTB) was found among females, people aged 45 to 64 years and people born in Southern Asia. An increased risk for PTB was found among patients with diabetes. The East African Indian strain of MTB was significantly associated with a higher likelihood of LNTB compared to other MTB strains.

Genome sequencing reveals novel deletions associated with secondary resistance to pyrazinamide in MDR Mycobacterium tuberculosis.

OBJECTIVES: Detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates presents significant challenges in settings with no dominant clonal lineages, such as Australia. We assessed the utility of WGS versus standard PCR amplification assays for the characterization of pyrazinamide resistance in MDR-TB isolates identified in New South Wales, Australia, over an 8 year period. METHODS: PCR amplicon sequencing was used to identify molecular markers associated with antibiotic resistance in pyrazinamide-resistant MDR-TB isolates recovered by the New South Wales Mycobacterium Reference Laboratory between 2007 and 2014. WGS was subsequently performed on two isolates for which pncA amplification failed. RESULTS: WGS identified two novel genomic deletions associated with in vitro resistance to pyrazinamide in MDR-TB. One isolate also carried a second deletion involving the genes dfrA and thyA associated with resistance to para-aminosalicylic acid. CONCLUSIONS: Steadily decreasing sequencing costs are increasing the appeal of WGS as an alternative approach for detecting complex patterns of pyrazinamide resistance in MDR-TB.

Added value of whole-genome sequencing for management of highly drug-resistant TB.

OBJECTIVES: Phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis takes several weeks to complete and second-line DST is often poorly reproducible, potentially leading to compromised clinical decisions. Following a fatal case of XDR TB, we investigated the potential benefit of using whole-genome sequencing to generate an in silico drug susceptibility profile. METHODS: The clinical course of the patient was reviewed, assessing the times at which phenotypic DST data became available and changes made to the therapeutic regimen. Whole-genome sequencing was performed on the earliest available isolate and variants associated with drug resistance were identified. RESULTS: The final DST report, including second-line drugs, was issued 10 weeks after patient presentation and 8 weeks after initial growth of M. tuberculosis. In the interim, the patient may have received a compromised regimen that had the potential to select for further drug resistance. The in silico susceptibility profile, extrapolated from evolving evidence in the literature, provided comparable or superior data to the DST results for second-line drugs and could be generated in a much shorter timeframe. CONCLUSIONS: We propose routine whole-genome sequencing of all MDR M. tuberculosis isolates in adequately resourced settings. This will improve individual patient care, monitor for transmission events and advance our understanding of resistance-associated mutations.

Fluoroquinolone resistance in non-multidrug-resistant tuberculosis-a surveillance study in New South Wales, Australia, and a review of global resistance rates.

BACKGROUND: Fluoroquinolones (FQs) are used for drug-susceptible tuberculosis (TB) in patients unable to tolerate first-line agents. Current trials are also investigating these drugs in empiric first-line TB therapy, to improve outcomes and allow for shortened treatment regimens. Widespread FQ use in the community has resulted in FQ resistance in many microorganisms, including Mycobacterium tuberculosis. Despite this, FQ drug susceptibility testing (DST) is rarely performed in non-multidrug-resistant TB (non-MDR-TB). METHODS: We conducted a 1-year surveillance study of FQ resistance on all MTB isolates from New South Wales (NSW), Australia. In addition, we performed a literature review of previous studies assessing FQ resistance in non-MDR-TB to summarize the global extent of this resistance pattern. RESULTS: Two (0.6%) out of 357 MTB isolates from NSW were found to be FQ-resistant. One isolate was an MDR strain (11% of all MDR-TB). The other was isoniazid-monoresistant (0.3% of all non-MDR-TB). Eleven studies from 10 countries had performed FQ resistance surveillance on non-MDR-TB. In the majority of these studies, FQ resistance was found to be low (mean 1%; 95% confidence interval 0.2-2%). CONCLUSIONS: FQ resistance in non-MDR-TB is uncommon in NSW, Australia. The existing global evidence suggests that FQ resistance remains largely confined to MDR-TB strains. In the majority of TB endemic regions, however, FQ resistance in non-MDR-TB has not been assessed. Knowledge of the prevalence of FQ resistance in MTB is essential to guide the rational use of these drugs, including their feasibility as first-line agents.

Temporal dynamics of Mycobacterium tuberculosis genotypes in New South Wales, Australia.

BACKGROUND: Molecular epidemiology of Mycobacterium tuberculosis, its transmission dynamics and population structure have become important determinants of targeted tuberculosis control programs. Here we describe recent changes in the distribution of M. tuberculosis genotypes in New South Wales (NSW), Australia and compared strain types with drug resistance, site of disease and demographic data. METHODS: We evaluated all culture-confirmed newly identified tuberculosis cases in NSW, Australia, from 2010-2012. M. tuberculosis population structure and clustering rates were assessed using 24-loci Mycobacterial interspersed repetitive unit (MIRU) analysis and compared to MIRU data from 2006-2008. RESULTS: Of 1177 tuberculosis cases, 1128 (95.8%) were successfully typed. Beijing and East African Indian (EAI) lineage strains were most common (27.6% and 28.5%, respectively) with EAI strains increasing in relative abundance from 11.8% in 2006-2008 to 28.5% in 2010-2012. Few cases of multi-drug resistant tuberculosis were identified (18; 1.7%). Compared to 12-loci, 24-loci MIRU provided improved cluster resolution with 695 (61.6%) and 227 (20.1%) clustered cases identified, respectively. Detailed analysis of the largest cluster identified (an 11 member Beijing cluster) revealed wide geographic diversity in the absence of documented social contact. CONCLUSIONS: EAI strains of M. tuberculosis recently overtook Beijing family as a prevalent cause of tuberculosis in NSW, Australia. This lineage appeared to be less commonly related to multi-drug resistant tuberculosis as compared to Beijing strain lineage. The resolution provided by 24-loci MIRU typing was insufficient for reliable assessment of transmissions, especially of Beijing family strains.

Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis.

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S-23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.

Rolling circle amplification for direct detection of rpoB gene mutations in Mycobacterium tuberculosis isolates from clinical specimens.

Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.

Tuberculosis in Australia: bacteriologically-confirmed cases and drug resistance, 2011. A report of the Australian Mycobacterium Reference Laboratory Network.

The Australian Mycobacterium Reference Laboratory Network collects and analyses laboratory data on new cases of disease caused by the Mycobacterium tuberculosis complex. In 2011, a total of 1,057 cases were identified bacteriologically; an annual reporting rate of 4.6 cases per 100,000 population. Eighteen children aged less than 15 years plus an additional 11 children from the Torres Strait Protected Zone had bacteriologically-confirmed tuberculosis. Results of in vitro drug susceptibility testing were available for 1,056 isolates for isoniazid, rifampicin, ethambutol, and pyrazinamide. A total of 107 (10.0%) isolates of M. tuberculosis were resistant to at least one of these anti-tuberculosis agents. Resistance to at least isoniazid and rifampicin (defined as multi-drug resistance, MDR) was detected in 25 (2.4%) isolates; 18 were from the respiratory tract (sputum n=14, bronchoscopy n=3, tissue n=1). Ten (55.6%) of the MDR-TB-positive sputum specimens were smear-positive, as was a single sample from a lymph node. Ten patients with MDR-TB were Papua New Guinea (PNG) nationals in the Torres Strait Protected Zone. If these PNG nationals are excluded from the analysis, the underlying MDR-TB rate in Australia was 1.4%. No cases of extensively drug-resistant TB (defined as MDR-TB with additional resistance to a fluoroquinolone and an injectable agent) were detected in 2011.