[This corrects the article DOI: 10.1371/journal.pgph.0001788.].

BACKGROUND Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS We examined whether APS-1 could be classified as a disease mediated by interferon-gamma. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-gamma using Aire(-/-)Ifng(-/-) mice and Aire(-/-) mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS Patients with APS-1 had enhanced interferon-gamma responses in blood and in all examined autoimmunity-affected tissues. Aire(-/-) mice had selectively increased interferon-gamma production by T cells and enhanced interferon-gamma, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire(-/-) mice normalized interferon-gamma responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-gamma, normalized interferon-gamma and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjogren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-gamma-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

AbstractEight lineages ofMycobacterium tuberculosis sensu strictoare 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.

BACKGROUND:Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE:We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS:We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS:Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS:Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

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. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd.

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 ITBI 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 ITBI 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-gamma 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, ofwhom 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 ITBI 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. ITBI 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 ITBI 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. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd.

The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a data compendium of 12,289 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole-genome sequencing and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay. It is the largest matched phenotypic and genotypic dataset for M. tuberculosis to date. Here, we provide a summary detailing the breadth of data collected, along with a description of how the isolates were selected, collected, and uniformly processed in CRyPTIC partner laboratories across 23 countries. The compendium contains 6,814 isolates resistant to at least 1 drug, including 2,129 samples that fully satisfy the clinical definitions of rifampicin resistant (RR), multidrug resistant (MDR), pre-extensively drug resistant (pre-XDR), or extensively drug resistant (XDR). The data are enriched for rare resistance-associated variants, and the current limits of genotypic prediction of resistance status (sensitive/resistant) are presented by using a genetic mutation catalogue, along with the presence of suspected resistance-conferring mutations for isolates resistant to the newly introduced drugs bedaquiline, clofazimine, delamanid, and linezolid. Finally, a case study of rifampicin monoresistance demonstrates how this compendium could be used to advance our genetic understanding of rare resistance phenotypes. The data compendium is fully open source and it is hoped that it will facilitate and inspire future research for years to come.

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. 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. Infection with lineages other than Lineage 4 were associated with mixed infection. 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 Unit Oxford; European Union. Research in Context Evidence Before This Study We searched Pubmed using the search terms ‘tuberculosis’ and ‘mixed’ or ‘mixture’ for English Language articles published up to 1 April 2019. Studies, most performed without the benefit of genomic sequencing, report mixed TB infection from a range of medium and high prevalence areas and show it to be associated with delayed treatment response. Modelling suggests detection and treatment of mixed TB infection is an important goal for TB eradication campaigns. Although routine DNA sequencing of M. tuberculosis isolates is becoming widespread, efficient methods for detecting mixed infection from such data are underdeveloped, and the true prevalence of mixed infection and its association with transmission is unclear. Added Value of This Study This study investigated a large series of TB isolations obtained as part of a routine Mycobacterial sequencing program by two reference laboratories, in a low incidence area, England. We developed an efficient generalisable approach to identify transmitted mixed M. tuberculosis infection; our approach is capable of sensitive and specific detection of a single mixed nucleotide position. We identified mixed infection of similar strains (‘microvariation’) in about 9.2% of the M. tuberculosis samples which we were able to assess, and found evidence of increased transmission from individuals with mixed infection. Implications of All the Available Evidence TB microvariation is a risk factor for TB transmission, even in the low incidence area studied. Although an efficient and highly specific technique identifying microvariation exists, it relies on comparison with similar sequences isolated from other patients. Sharing of sequence data from the many TB sequencing programs being deployed globally will increase the sensitivity of microvariation detection, and may assist targeted public health interventions.

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y(12) represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

IMPORTANCE Chilblain-like lesions have been one of the most frequently described cutaneous manifestations during the COVID-19 pandemic. Their etiopathogenesis, including the role of SARSCoV-2, remains elusive. OBJECTIVE To examine the association of chilblain-like lesions with SARS-CoV-2 infection. DESIGN, SETTING, AND PARTICIPANTS This prospective case series enrolled 17 adolescents who presented with chilblain-like lesions from April 1 to June 30, 2020, at a tertiary referral academic hospital in Italy. MAIN OUTCOMES AND MEASURES Macroscopic (clinical and dermoscopic) and microscopic (histopathologic) analysis contributed to a thorough understanding of the lesions. Nasopharyngeal swab, serologic testing, and in situ hybridization of the skin biopsy specimenswere performed to test for SARS-CoV-2 infection. Laboratory tests explored signs of systemic inflammation or thrombophilia. Structural changes in peripheral microcirculation were investigated by capillaroscopy. RESULTS Of the 17 adolescents (9 [52.9%] male; median [interquartile range] age, 13.2 [12.5-14.3] years) enrolled during the first wave of the COVID-19 pandemic, 16 (94.1%) had bilaterally localized distal erythematous or cyanotic lesions. A triad of red dots (16 [100%]), white rosettes (11 [68.8%]), and white streaks (10 [62.5%]) characterized the dermoscopic picture. Histologic analysis revealed a remodeling of the dermal blood vessels with a lobular arrangement, wall thickening, and a mild perivascular lymphocytic infiltrate. SARS-CoV-2 infection was excluded by molecular and serologic testing. In situ hybridization did not highlight the viral genome in the lesions. CONCLUSIONS AND RELEVANCE This study delineated the clinical, histologic, and laboratory features of chilblain-like lesions that emerged during the COVID-19 pandemic, and its findings do not support their association with SARS-CoV-2 infection. The lesions occurred in otherwise healthy adolescents, had a long but benign course to self-resolution, and were characterized by a microvascular remodeling with perivascular lymphocytic infiltrate but no other signs of vasculitis. These results suggest that chilblain-like lesions do not imply a concomitant SARS-CoV-2 infection. Ongoing studies will help clarify the etiopathogenic mechanisms.

Objective: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Platelets are key regulators of thrombosis, inflammation, immunity and are prime candidates for a role in the pathogenesis of COVID-19. The objective of this study was to analyze the platelet phenotype in COVID-19. Approach and Results: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in human megakaryocytes and platelets in COVID-19 patients and following incubation with the virus in vitro . We show a direct interaction between SARS-CoV-2 and megakaryocytes that alters the platelet transcriptome and platelet activity. COVID-19 platelets are hyperreactive and have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. We find transcriptomic changes mediated by SARS-CoV-2 do not occur following exposure of megakaryocytes with a coronavirus responsible for the common cold, CoV-OC43. In a cohort of 3,915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication use, and biomarkers of inflammation and thrombosis, platelet count, size, and immaturity are each associated with increased critical illness and all-cause mortality. Conclusions: Our findings demonstrate that SARS-CoV-2 virions invade megakaryocytes and platelets, inducing alterations to the platelet transcriptome and activation profile, which correlate with critical illness and mortality in hospitalized COVID-19 patients.

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.

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.

Background: Mycobacterium abscessus has emerged as a significant clinical concern following reports that it is readily transmissible in healthcare settings between patients with Cystic Fibrosis (CF). Methods: We analysed consecutive M. abscessus whole-genome sequencing data from England (February-2015 to November-2019) to identify genomically similar isolates. Linkage to a national healthcare usage database was used to investigate possible contacts between patients. Multivariable regression analysis was performed to investigate factors associated with acquisition of a genomically clustered strain (within ≤25 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 ≥10 patients, all but one contained patients with CF and patients without CF. Patients with CF were equally likely to have clustered isolates as those without CF. High-density phylogenetic clusters were randomly distributed over a wide geographical area. The majority of 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 but not time spent as an hospital inpatient/outpatient. We identified two CF sibling pairs with genetically highly divergent isolates and 1 pair with closely related isolates, as well as 25 uninfected presumed household contacts with CF. Interpretation: Previously identified widely disseminated dominant clones of M. abscessus are not restricted to CF patients and occur in other chronic respiratory diseases. Our analysis does not support person-to-person transmission as an important mechanism in M. abscessus dissemination at a national level in England. Funding Statement: The research was supported by the National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance (HPRU 2012-10041) at the University of Oxford in partnership with Public Health England (PHE) and by Oxford NIHR Biomedical Research Centre. T Peto, AS Walker and DW Crook are NIHR Senior Investigators. Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. The report presents independent research funded by NIHR. Declaration of Interests: SL is supported by a Medical Research Council Clinical Research Training Fellowship. DWE declares lecture fees from Gilead, outside the submitted work. TMW is a Wellcome Trust Clinical Career Development Fellow (214560/Z/18/Z). The remaining authors declare no conflicts of interest. Ethics Approval Statement: This work was carried out as a public health investigation with internal approval from Public Health England and therefore ethics committee approval was not required.

OBJECTIVES:Tuberculosis (TB) is a serious re-emergent public health problem in the UK. In response to rising case incidence a National TB Strain-Typing Service based on molecular strain-typing was established. This facilitates early detection and investigation of clusters, targeted public health action, and prevention of further transmission. We review the added public health value of investigating molecular TB strain-typed (ST) clusters.METHODS:A structured questionnaire for each ST cluster investigated in England between 1 January 2010 and 30 June 2013 was completed. Questions related to epidemiological links and public health action and the perceived benefits of ST cluster investigation.RESULTS:There were 278 ST cluster investigations (CIs) involving 1882 TB cases. Cluster size ranged from 2 to 92. CIs identified new epidemiological links in 36% of clusters; in 18% STs were discordant refuting transmission thought to have occurred. Additional public health action was taken following 23% of CI.CONCLUSIONS:We found positive benefits of TB molecular ST and CI, in identifying new epidemiological links between cases and taking public health action and in refuting transmission and saving resources. This needs to be translated to a decrease in transmission to provide evidence of public health value in this low prevalence high resource setting.

Wyllie, David H; Davidson, Jennifer A; Grace Smith, E; Rathod, Priti; Crook, Derrick W; Peto, Tim EA; Robinson, Esther; Walker, Tim; Campbell, Colin; (2018) A Quantitative Evaluation of MIRU-VNTR Typing Against Whole-Genome Sequencing for Identifying Mycobacterium tuberculosis Transmission: A Prospective Observational Cohort Study. EBioMedicine, 34. pp. 122-130. ISSN 2352-3964 DOI: https://doi.org/10.1016/j.ebiom.2018.07.019 Downloaded from: http://researchonline.lshtm.ac.uk/id/eprint/4655180/ DOI: https://doi.org/10.1016/j.ebiom.2018.07.019

The clinical phenotype of zoonotic tuberculosis and its contribution to the global burden of disease are poorly understood and probably underestimated. This shortcoming is partly because of the inability of currently available laboratory and in silico tools to accurately identify all subspecies of the Mycobacterium tuberculosis complex (MTBC). We present SNPs to Identify TB (SNP-IT), a single-nucleotide polymorphism-based tool to identify all members of MTBC, including animal clades. By applying SNP-IT to a collection of clinical genomes from a UK reference laboratory, we detected an unexpectedly high number of M. orygis isolates. M. orygis is seen at a similar rate to M. bovis, yet M. orygis cases have not been previously described in the United Kingdom. From an international perspective, it is possible that M. orygis is an underestimated zoonosis. Accurate identification will enable study of the clinical phenotype, host range, and transmission mechanisms of all subspecies of MTBC in greater detail.

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 resistancedetermining 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.