The effect of M. tuberculosis lineage on clinical phenotype.

Six lineages of Mycobacterium tuberculosis sensu stricto (which excludes M. africanum) are described. Single-country or small observational data suggest differences in clinical phenotype between lineages. We present strain lineage and clinical phenotype data from 12,246 patients from 3 low-incidence and 5 high-incidence countries. We used multivariable logistic regression to explore the effect of lineage on site of disease and on cavities on chest radiography, given pulmonary TB; multivariable multinomial logistic regression to investigate types of extra-pulmonary TB, given lineage; and accelerated failure time and Cox proportional-hazards models to explore the effect of lineage on time to smear and culture-conversion. Mediation analyses quantified the direct effects of lineage on outcomes. Pulmonary disease was more likely among patients with lineage(L) 2, L3 or L4, than L1 (adjusted odds ratio (aOR) 1.79, (95% confidence interval 1.49-2.15), p<0.001; aOR = 1.40(1.09-1.79), p = 0.007; aOR = 2.04(1.65-2.53), p<0.001, respectively). Among patients with pulmonary TB, those with L1 had greater risk of cavities on chest radiography versus those with L2 (aOR = 0.69(0.57-0.83), p<0.001) and L4 strains (aOR = 0.73(0.59-0.90), p = 0.002). L1 strains were more likely to cause osteomyelitis among patients with extra-pulmonary TB, versus L2-4 (p = 0.033, p = 0.008 and p = 0.049 respectively). Patients with L1 strains showed shorter time-to-sputum smear conversion than for L2. Causal mediation analysis showed the effect of lineage in each case was largely direct. The pattern of clinical phenotypes seen with L1 strains differed from modern lineages (L2-4). This has implications for clinical management and could influence clinical trial selection strategies.

The effect of M. tuberculosis lineage on clinical phenotype.

Eight lineages of Mycobacterium tuberculosis sensu stricto are described. Single-country or small observational data suggest differences in clinical phenotype between lineages. We present strain lineage and clinical phenotype data from 12,246 patients from 3 low-incidence and 5 high-incidence countries. We used multivariable logistic regression to explore the effect of lineage on site of disease and on cavities on chest radiography, given pulmonary TB; multivariable multinomial logistic regression to investigate types of extra-pulmonary TB, given lineage; and accelerated failure time and Cox proportional-hazards models to explore the effect of lineage on time to smear and culture-conversion. Mediation analyses quantified the direct effects of lineage on outcomes. Pulmonary disease was more likely among patients with lineage(L) 2, L3 or L4, than L1 (adjusted odds ratio (aOR) 1.79, (95% confidence interval 1.49-2.15), p<0.001; aOR=1.40(1.09-1.79), p=0.007; aOR=2.04(1.65-2.53), p<0.001, respectively). Among patients with pulmonary TB, those with L1 had greater risk of cavities on chest radiography versus those with L2 (aOR=0.69(0.57-0.83), p<0.001) and L4 strains (aOR=0.73(0.59-0.90), p=0.002). L1 strains were more likely to cause osteomyelitis among patients with extra-pulmonary TB, versus L2-4 (p=0.033, p=0.008 and p=0.049 respectively). Patients with L1 strains showed shorter time-to-sputum smear conversion than for L2. Causal mediation analysis showed the effect of lineage in each case was largely direct. The pattern of clinical phenotypes seen with L1 strains differed from modern lineages (L2-4). This has implications for clinical management and could influence clinical trial selection strategies.

M. tuberculosis microvariation is common and is associated with transmission: Analysis of three years prospective universal sequencing in England.

BACKGROUND: The prevalence, association with disease status, and public health impact of infection with mixtures of M. tuberculosis strains is unclear, in part due to limitations of existing methods for detecting mixed infections. METHODS: We developed an algorithm to identify mixtures of M. tuberculosis strains using next generation sequencing data, assessing performance using simulated sequences. We identified mixed M. tuberculosis strains when there was at least one mixed nucleotide position, and where both the mixture's components were present in similar isolates from other individuals, compatible with transmission of the component strains. We determined risk factors for mixed infection among isolations of M. tuberculosis in England using logistic regression. We used survival analyses to assess the association between mixed infection and putative transmission. FINDINGS: 6,560 isolations of TB were successfully sequenced in England 2016-2018. Of 3,691 (56%) specimens for which similar sequences had been isolated from at least two other individuals, 341 (9.2%) were mixed. Mixed infection was more common in lineages other than Lineage 4. Among the 1,823 individuals with pulmonary infection with Lineage 4 M. tuberculosis, mixed infection was associated with significantly increased risk of subsequent isolation of closely related organisms from a different individual (HR 1.43, 95% CI 1.05,1.94), indicative of transmission. INTERPRETATION: Mixtures of transmissible strains occur in at least 5% of tuberculosis infections in England; when present in pulmonary disease, such mixtures are associated with an increased risk of tuberculosis transmission. FUNDING: Public Health England; NIHR Health Protection Research Units; European Union.

Mycobacterium tuberculosis transmission in Birmingham, UK, 2009-19: An observational study.

Background: Over 10-years of whole-genome sequencing (WGS) of Mycobacterium tuberculosis in Birmingham presents an opportunity to explore epidemiological trends and risk factors for transmission in new detail. Methods: Between 1st January 2009 and 15th June 2019, we obtained the first WGS isolate from every patient resident in a postcode district covered by Birmingham's centralised tuberculosis service. Data on patients' sex, country of birth, social risk-factors, anatomical locus of disease, and strain lineage were collected. Poisson harmonic regression was used to assess seasonal variation in case load and a mixed-effects multivariable Cox proportionate hazards model was used to assess risk factors for a future case arising in clusters defined by a 5 single nucleotide polymorphism (SNP) threshold, and by 12 SNPs in a sensitivity analysis. Findings: 511/1653 (31%) patients were genomically clustered with another. A seasonal variation in diagnoses was observed, peaking in spring, but only among clustered cases. Risk-factors for a future clustered case included UK-birth (aHR=2·03 (95%CI 1·35-3·04), p < 0·001), infectious (pulmonary/laryngeal/miliary) tuberculosis (aHR=3·08 (95%CI 1·98-4·78), p < 0·001), and M. tuberculosis lineage 3 (aHR=1·91 (95%CI 1·03-3·56), p = 0·041) and 4 (aHR=2·27 (95%CI 1·21-4·26), p = 0·011), vs. lineage 1. Similar results pertained to 12 SNP clusters, for which social risk-factors were also significant (aHR 1·72 (95%CI 1·02-2·93), p = 0·044). There was marked heterogeneity in transmission patterns between postcode districts. Interpretation: There is seasonal variation in the diagnosis of genomically clustered, but not non-clustered, cases. Risk factors for clustering include UK-birth, infectious forms of tuberculosis, and infection with lineage 3 or 4. Funding: Wellcome Trust, MRC, UKHSA.

Effectiveness of nationwide programmatic testing and treatment for latent tuberculosis infection in migrants in England: a retrospective, population-based cohort study.

BACKGROUND: In low-incidence countries, tuberculosis mainly affects migrants, mostly resulting from reactivation of latent tuberculosis infection (LTBI) acquired in high-incidence countries before migration. A nationwide primary care-based LTBI testing and treatment programme for migrants from high-incidence countries was therefore established in high tuberculosis incidence areas in England. We aimed to assess the effectiveness of this programme. METHODS: We did a retrospective, population-based cohort study of migrants who registered in primary care between Jan 1, 2011, and Dec 31, 2018, in 55 high-burden areas with programmatic LTBI testing and treatment. Eligible individuals were aged 16-35 years, born in a high-incidence country, and had entered England in the past 5 years. Individuals who tested interferon-γ release assay (IGRA)-negative were advised about symptoms of tuberculosis, whereas those who tested IGRA-positive were clinically assessed to rule out active tuberculosis and offered preventive therapy. The primary outcome was incident tuberculosis notified to the national Enhanced Tuberculosis Surveillance system. FINDINGS: Our cohort comprised 368 097 eligible individuals who had registered in primary care, of whom 37 268 (10·1%) were tested by the programme. 1446 incident cases of tuberculosis were identified: 166 cases in individuals who had IGRA testing (incidence 204 cases [95% CI 176-238] per 100 000 person-years) and 1280 in individuals without IGRA testing (82 cases [77-86] per 100 000 person-years). Overall, in our primary analysis including all diagnosed tuberculosis cases, a time-varying association was identified between LTBI testing and treatment and lower risk of incident tuberculosis (hazard ratio [HR] 0·76 [95% CI 0·63-0·91]) when compared with no testing. In stratified analysis by follow-up period, the intervention was associated with higher risk of tuberculosis diagnosis during the first 6 months of follow-up (9·93 [7·63-12·9) and a lower risk after 6 months (0·57 [0·41-0·79]). IGRA-positive individuals had higher risk of tuberculosis diagnosis than IGRA-negative individuals (31·9 [20·4-49·8]). Of 37 268 migrants who were tested, 6640 (17·8%) were IGRA-positive, of whom 1740 (26·2%) started preventive treatment. LTBI treatment lowered the risk of tuberculosis: of 135 incident cases in the IGRA-positive cohort, seven cases were diagnosed in the treated group (1·87 cases [95% CI 0·89-3·93] per 1000 person-years) and 128 cases were diagnosed in the untreated group (10·9 cases [9·16-12·9] per 1000 person-years; HR 0·14 [95% CI 0·06-0·32]). INTERPRETATION: A low proportion of eligible migrants were tested by the programme and a small proportion of those testing positive started treatment. Despite this, programmatic LTBI testing and treatment of individuals migrating to a low-incidence region is effective at diagnosing active tuberculosis earlier and lowers the long-term risk of progression to tuberculosis. Increasing programme participation and treatment rates for those testing positive could substantially impact national tuberculosis incidence. FUNDING: National Institute for Health Research Health Protection Research Unit in Respiratory Infections.

Epidemiology of Mycobacterium abscessus in England: an observational study.

BACKGROUND: Mycobacterium abscessus has emerged as a significant clinical concern following reports that it is readily transmissible in health-care settings between patients with cystic fibrosis. We linked routinely collected whole-genome sequencing and health-care usage data with the aim of investigating the extent to which such transmission explains acquisition in patients with and without cystic fibrosis in England. METHODS: In this retrospective observational study, we analysed consecutive M abscessus whole-genome sequencing data from England (beginning of February, 2015, to Nov 14, 2019) to identify genomically similar isolates. Linkage to a national health-care usage database was used to investigate possible contacts between patients. Multivariable regression analysis was done to investigate factors associated with acquisition of a genomically clustered strain (genomic distance <25 single nucleotide polymorphisms [SNPs]). FINDINGS: 2297 isolates from 906 patients underwent whole-genome sequencing as part of the routine Public Health England diagnostic service. Of 14 genomic clusters containing isolates from ten or more patients, all but one contained patients with cystic fibrosis and patients without cystic fibrosis. Patients with cystic fibrosis were equally likely to have clustered isolates (258 [60%] of 431 patients) as those without cystic fibrosis (322 [63%] of 513 patients; p=0·38). High-density phylogenetic clusters were randomly distributed over a wide geographical area. Most isolates with a closest genetic neighbour consistent with potential transmission had no identifiable relevant epidemiological contacts. Having a clustered isolate was independently associated with increasing age (adjusted odds ratio 1·14 per 10 years, 95% CI 1·04-1·26), but not time spent as an hospital inpatient or outpatient. We identified two sibling pairs with cystic fibrosis with genetically highly divergent isolates and one pair with closely related isolates, and 25 uninfected presumed household contacts with cystic fibrosis. INTERPRETATION: Previously identified widely disseminated dominant clones of M abscessus are not restricted to patients with cystic fibrosis and occur in other chronic respiratory diseases. Although our analysis showed a small number of cases where person-to-person transmission could not be excluded, it did not support this being a major mechanism for M abscessus dissemination at a national level in England. Overall, these data should reassure patients and clinicians that the risk of acquisition from other patients in health-care settings is relatively low and motivate future research efforts to focus on identifying routes of acquisition outside of the cystic fibrosis health-care-associated niche. FUNDING: The National Institute for Health Research, Health Data Research UK, The Wellcome Trust, The Medical Research Council, and Public Health England.

Prosthetic hip joint infection by Bacillus Calmette-Guerin therapy following intravesical instillation for bladder cancer identified using whole-genome sequencing: a case report.

BACKGROUND: Joint replacement is an effective intervention and prosthetic joint infection (PJI) is one of the most serious complications of such surgery. Diagnosis of PJI is often complex and requires multiple modalities of investigation. We describe a rare cause of PJI which highlights these challenges and the role of whole-genome sequencing to achieve a rapid microbiological diagnosis to facilitate prompt and appropriate management. CASE PRESENTATION: A 79-year-old man developed chronic hip pain associated with a soft-tissue mass, fluid collection and sinus adjacent to his eight-year-old hip prosthesis. His symptoms started after intravesical Bacillus Calmette-Guerin (BCG) therapy for bladder cancer. Synovasure™ and 16S polymerase chain reaction (PCR) tests were negative, but culture of the periarticular mass and genome sequencing diagnosed BCG infection. He underwent a two-stage joint revision and a prolonged duration of antibiotic therapy which was curative. CONCLUSIONS: BCG PJI after therapeutic exposure can have serious consequences, and awareness of this potential complication, identified from patient history, is essential. In addition, requesting appropriate testing is required, together with recognition that traditional diagnostics may be negative in non-pyogenic PJI. Advanced molecular techniques have a role to enhance the timely management of these infections.

Whole-Genome Sequencing for Predicting Clarithromycin Resistance in Mycobacterium abscessus.

Mycobacterium abscessus is emerging as an important pathogen in chronic lung diseases, with concern regarding patient-to-patient transmission. The recent introduction of routine whole-genome sequencing (WGS) as a replacement for existing reference techniques in England provides an opportunity to characterize the genetic determinants of resistance. We conducted a systematic review to catalogue all known resistance-determining mutations. This knowledge was used to construct a predictive algorithm based on mutations in the erm(41) and rrl genes which was tested on a collection of 203 sequentially acquired clinical isolates for which there were paired genotype/phenotype data. A search for novel resistance-determining mutations was conducted using a heuristic algorithm. The sensitivity of existing knowledge for predicting resistance in clarithromycin was 95% (95% confidence interval [CI], 89 to 98%), and the specificity was 66% (95% CI, 54 to 76%). The subspecies alone was a poor predictor of resistance to clarithromycin. Eight potential new resistance-conferring single nucleotide polymorphisms (SNPs) were identified. WGS demonstrated probable resistance-determining SNPs in regions that the NTM-DR line probe cannot detect. These mutations are potentially clinically important, as they all occurred in samples that were predicted to be inducibly resistant and for which a macrolide would therefore currently be indicated. We were unable to explain all resistance, raising the possibility of the involvement of other as yet unidentified genes.

Identifying Mixed Mycobacterium tuberculosis Infection and Laboratory Cross-Contamination during Mycobacterial Sequencing Programs.

The detection of laboratory cross-contamination and mixed tuberculosis infections is an important goal of clinical mycobacteriology laboratories. The objective of this study was to develop a method to detect mixtures of different Mycobacterium tuberculosis lineages in laboratories performing mycobacterial next-generation sequencing (NGS). The setting was the Public Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive mycobacterial growth indicator tubes. We analyzed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, which were obtained during development and production use of a diagnostic process using NGS. The counts of the most common (major) variant and all other variants (nonmajor variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development. For each sample, we determined the nonmajor variant proportions at 55 sets of lineage-defining positions. The nonmajor variant proportion in the two most mixed lineage-defining sets (F2 metric) was compared with that of the 47 least-mixed lineage-defining sets (F47 metric). The following three patterns were observed: (i) not mixed by either metric; (ii) high F47 metric, suggesting mixtures of multiple lineages; and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevations relative to F47. Pattern ii was observed in batches, with similar patterns in the M. tuberculosis H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in the patterns were 97%, 2.8%, and 0.001%, respectively. The F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis genomes.

Control of Artifactual Variation in Reported Intersample Relatedness during Clinical Use of a Mycobacterium tuberculosis Sequencing Pipeline.

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artifactual variation between Mycobacterium tuberculosis isolates during routine next-generation sequencing of Mycobacterium spp., we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted and sequenced, reads were mapped, and consensus sequences were determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of nonmycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of nonmycobacterial bacterial DNA, we found significant increases in minor variant frequencies, of more than 1.5-fold, in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high-variation regions strongly influenced by the amount of nonmycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we demonstrated an approach identifying critical genomic regions contributing to clinically relevant artifactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multistep laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

Machine learning for classifying tuberculosis drug-resistance from DNA sequencing data.

Motivation: Correct and rapid determination of Mycobacterium tuberculosis (MTB) resistance against available tuberculosis (TB) drugs is essential for the control and management of TB. Conventional molecular diagnostic test assumes that the presence of any well-studied single nucleotide polymorphisms is sufficient to cause resistance, which yields low sensitivity for resistance classification. Summary: Given the availability of DNA sequencing data from MTB, we developed machine learning models for a cohort of 1839 UK bacterial isolates to classify MTB resistance against eight anti-TB drugs (isoniazid, rifampicin, ethambutol, pyrazinamide, ciprofloxacin, moxifloxacin, ofloxacin, streptomycin) and to classify multi-drug resistance. Results: Compared to previous rules-based approach, the sensitivities from the best-performing models increased by 2-4% for isoniazid, rifampicin and ethambutol to 97% (P < 0.01), respectively; for ciprofloxacin and multi-drug resistant TB, they increased to 96%. For moxifloxacin and ofloxacin, sensitivities increased by 12 and 15% from 83 and 81% based on existing known resistance alleles to 95% and 96% (P < 0.01), respectively. Particularly, our models improved sensitivities compared to the previous rules-based approach by 15 and 24% to 84 and 87% for pyrazinamide and streptomycin (P < 0.01), respectively. The best-performing models increase the area-under-the-ROC curve by 10% for pyrazinamide and streptomycin (P < 0.01), and 4-8% for other drugs (P < 0.01). Availability and implementation: The details of source code are provided at http://www.robots.ox.ac.uk/~davidc/code.php. Contact: david.clifton@eng.ox.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

Evaluation of Whole-Genome Sequencing for Mycobacterial Species Identification and Drug Susceptibility Testing in a Clinical Setting: a Large-Scale Prospective Assessment of Performance against Line Probe Assays and Phenotyping.

Use of whole-genome sequencing (WGS) for routine mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-line Mycobacterium tuberculosis resistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728 M. tuberculosis complex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777 M. tuberculosis complex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), respectively (P = 0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy, but work is needed to increase the proportion of predictions made.

Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study.

BACKGROUND: Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater-cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. METHODS: We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. FINDINGS: Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. INTERPRETATION: HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. FUNDING: Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDRsl Assays.

In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

Insidious Risk of Severe Mycobacterium chimaera Infection in Cardiac Surgery Patients.

BACKGROUND: An urgent UK investigation was launched to assess risk of invasive Mycobacterium chimaera infection in cardiothoracic surgery and a possible association with cardiopulmonary bypass heater-cooler units following alerts in Switzerland and The Netherlands. METHODS: Parallel investigations were pursued: (1) identification of cardiopulmonary bypass-associated M. chimaera infection through national laboratory and hospital admissions data linkage; (2) cohort study to assess patient risk; (3) microbiological and aerobiological investigations of heater-coolers in situ and under controlled laboratory conditions; and (4) whole-genome sequencing of clinical and environmental isolates. RESULTS: Eighteen probable cases of cardiopulmonary bypass-associated M. chimaera infection were identified; all except one occurred in adults. Patients had undergone valve replacement in 11 hospitals between 2007 and 2015, a median of 19 months prior to onset (range, 3 months to 5 years). Risk to patients increased after 2010 from <0.2 to 1.65 per 10000 person-years in 2013, a 9-fold rise for infections within 2 years of surgery (rate ratio, 9.08 [95% CI, 1.81-87.76]). Endocarditis was the most common presentation (n = 11). To date, 9 patients have died. Investigations identified aerosol release through breaches in heater-cooler tanks. Mycobacterium chimaera and other pathogens were recovered from water and air samples. Phylogenetic analysis found close clustering of strains from probable cases. CONCLUSIONS: We identified low but escalating risk of severe M. chimaera infection associated with heater-coolers with cases in a quarter of cardiothoracic centers. Our investigations strengthen etiological evidence for the role of heater-coolers in transmission and raise the possibility of an ongoing, international point-source outbreak. Active management of heater-coolers and heightened clinical awareness are imperative given the consequences of infection.

Identifying lineage effects when controlling for population structure improves power in bacterial association studies.

Bacteria pose unique challenges for genome-wide association studies because of strong structuring into distinct strains and substantial linkage disequilibrium across the genome(1,2). Although methods developed for human studies can correct for strain structure(3,4), this risks considerable loss-of-power because genetic differences between strains often contribute substantial phenotypic variability(5). Here, we propose a new method that captures lineage-level associations even when locus-specific associations cannot be fine-mapped. We demonstrate its ability to detect genes and genetic variants underlying resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection, recombination and penetrance confer high power to recover known antimicrobial resistance mechanisms and reveal a candidate association between the outer membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method pinpoints locus-specific effects where possible and boosts power by detecting lineage-level differences when fine-mapping is intractable.