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.

Potent antimycobacterial activity of the pyridoxal isonicotinoyl hydrazone analog 2-pyridylcarboxaldehyde isonicotinoyl hydrazone: a lipophilic transport vehicle for isonicotinic acid hydrazide.

The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

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.

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.

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.

Phenotypically occult multidrug-resistant Mycobacterium tuberculosis: dilemmas in diagnosis and treatment.

OBJECTIVES: The clinical significance of the emergence of Mycobacterium tuberculosis (MTB) isolates that contain rpoB mutations (genotypic resistance), but are phenotypically susceptible to rifampicin (RIF G(R) P(S)), remains uncertain. The aim of this study was to determine the prevalence of MTB cases that demonstrate this discordant rifampicin resistance pattern and to establish whether these patients have poorer treatment outcomes with rifampicin-based regimens. METHODS: rpoB sequencing was performed on all MTB isolates demonstrating phenotypic resistance to one or more first-line antituberculosis agents (excluding rifampicin). Rifampicin MICs were determined for rpoB mutation-positive isolates and clinical case notes were reviewed to identify treatment outcomes in these patients. RESULTS: Of the 214 phenotypically drug (excluding rifampicin)-resistant isolates tested, 5 contained rpoB mutations (4 isoniazid resistant and 1 pyrazinamide resistant). These isolates demonstrated elevated rifampicin MICs (low-level resistance), despite testing susceptible using phenotypic broth-based methods. One patient experienced a relapse of tuberculosis (TB) 2 years after completion of a rifampicin-containing regimen. These findings are consistent with a recent study that reported treatment failure with rifampicin-based regimens in patients with isoniazid-resistant MTB and genotypic rifampicin resistance. CONCLUSIONS: While MTB RIF G(R) P(S) strains remain relatively uncommon, they can be associated with low-level rifampicin resistance and poorer treatment outcomes with rifampicin-based regimens. This recently recognized form of multidrug-resistant TB should be adequately detected and managed.

Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 2010. 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 2010, a total of 1,051 cases were identified by bacteriology; an annual reporting rate of 4.7 cases per 100,000 population. Twelve children aged less than 10 years had bacteriologically-confirmed tuberculosis. Results of in vitro drug susceptibility testing were available for 1,050 isolates for isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PYZ). A total of 126 (12%) isolates of M. tuberculosis were resistant to at least one of these anti-tuberculosis agents. Resistance to at least INH and RIF (defined as multi-drug resistance, MDR) was detected in 37 (3.5%) isolates, including three Australians with extensive travel in high burden TB countries; 33 were from the respiratory tract (sputum n=28, bronchoscopy n=5). Nineteen (65.5%) of the MDR-TB-positive sputum specimens were smear-positive, as were single samples from bronchoscopy and urine. Sixteen patients with MDR-TB were from the Torres Strait Protected Zone. If these Papa New Guinea nationals are excluded from the analysis, the underlying MDR-TB rate in Australian isolates was 2.0%. One case of extensively drug-resistant TB (defined as MDR-TB with additional resistance to a fluoroquinolone and an injectable agent) was detected in 2010.

Editor's choice: Implications of isoniazid resistance in Mycobacterium bovis Bacillus Calmette-Guérin used for immunotherapy in bladder cancer.

We report a case of sepsis from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) with low-level isoniazid resistance following intravesical treatment for bladder cancer. Isoniazid resistance in BCG has therapeutic implications when it causes infections after intravesical instillation. For these circumstances, we propose some modifications to existing treatment guidelines for BCG infection.

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.

Rolling circle amplification and multiplex allele-specific PCR for rapid detection of katG and inhA gene mutations in Mycobacterium tuberculosis.

The aim of the study was to compare a novel, rolling circle amplification (RCA) assay for detection of common isoniazid (INH) resistance mutations in Mycobacterium tuberculosis with a multiplex allele-specific PCR (MAS-PCR) and sequencing of katG and the fabG1-inhA promoter region. One or more mutations were identified by RCA, MAS-PCR, and sequencing in 21 (68%), 22 (71%), and 23 (74%), respectively, of 31 epidemiologically unrelated INH-resistant isolates, and in none of 8 INH-susceptible isolates. The RCA assay is a rapid, inexpensive, and practical screening method for INH resistance in M. tuberculosis in countries with high prevalence of INH resistance.

Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing.

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference 'gold standard'. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is the commonest pathogen causing food-borne disease among humans and animals in Australia. A multiplex PCR-based reverse line blot (mPCR/RLB) system was developed to rapidly identify S. Typhimurium phage types and strains within them. The system comprised 32 biotin-labelled primer sets and 38 amino-labelled probes, based on sequences that were either phage-type-related or derived from temperate phages ST64B, P22, Gifsy-1 or Gifsy-2. The system was developed and evaluated using 168 S. Typhimurium isolates, representing 46 phage types. RLB patterns, based on a combination of positive hybridization and grading of signal intensities, validated by sequencing, differentiated S. Typhimurium isolates into 102 types. Some clusters contained isolates belonging to a single phage type while others contained isolates belonging to more than one. Most phage types exhibited at least two RLB profiles. The feasibility of this system was evaluated during investigations of three outbreaks, due to two different phage types. Within each outbreak, isolates showed identical RLB patterns, whereas sporadic isolates of corresponding phage types showed various patterns. The mPCR/RLB system was compared with multilocus variable-number tandem-repeat analysis (MLVA). The two methods demonstrated similar discriminatory abilities. Based on these preliminary results, the mPCR/RLB system is a promising tool for molecular identification of most common S. Typhimurium phage types. It could be used as an alternative to, or in conjunction with, MLVA for rapid strain typing during outbreaks.

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.

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.

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.