Summary Background Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect ...microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. Methods In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data. Findings We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis . The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0·0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters. Interpretation Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts. Funding Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.
Patients born outside the UK have contributed to a 20% rise in the UK's tuberculosis incidence since 2000, but their effect on domestic transmission is not known. Here we use whole-genome sequencing ...to investigate the epidemiology of tuberculosis transmission in an unselected population over 6 years.
We identified all residents with Oxfordshire postcodes with a Mycobacterium tuberculosis culture or a clinical diagnosis of tuberculosis between Jan 1, 2007, and Dec 31, 2012, using local databases and checking against the national Enhanced Tuberculosis Surveillance database. We used Illumina technology to sequence all available M tuberculosis cultures from identified cases. Sequences were clustered by genetic relatedness and compared retrospectively with contact investigations. The first patient diagnosed in each cluster was defined as the index case, with links to subsequent cases assigned first by use of any epidemiological linkage, then by genetic distance, and then by timing of diagnosis.
Although we identified 384 patients with a diagnosis of tuberculosis, country of birth was known for 380 and we sequenced isolates from 247 of 269 cases with culture-confirmed disease. 39 cases were genomically linked within 13 clusters, implying 26 local transmission events. Only 11 of 26 possible transmissions had been previously identified through contact tracing. Of seven genomically confirmed household clusters, five contained additional genomic links to epidemiologically unidentified non-household members. 255 (67%) patients were born in a country with high tuberculosis incidence, conferring a local incidence of 109 cases per 100,000 population per year in Oxfordshire, compared with 3·5 cases per 100,000 per year for those born in low-incidence countries. However, patients born in the low-incidence countries, predominantly UK, were more likely to have pulmonary disease (adjusted odds ratio 1·8 95% CI 1·2-2·9; p=0·009), social risk factors (4·4 2·0-9·4; p<0·0001), and be part of a local transmission cluster (4·8 1·6-14·8; p=0·006).
Although inward migration has contributed to the overall tuberculosis incidence, our findings suggest that most patients born in high-incidence countries reactivate latent infection acquired abroad and are not involved in local onward transmission. Systematic screening of new entrants could further improve tuberculosis control, but it is important that health care remains accessible to all individuals, especially high-risk groups, if tuberculosis control is not to be jeopardised.
UK Clinical Research Collaboration (Wellcome Trust, Medical Research Council, National Institute for Health Research NIHR), and NIHR Oxford Biomedical Research Centre.
Summary Background Health-care workers have been implicated in nosocomial outbreaks of Staphylococcus aureus , but the dearth of evidence from non-outbreak situations means that routine health-care ...worker screening and S aureus eradication are controversial. We aimed to determine how often S aureus is transmitted from health-care workers or the environment to patients in an intensive care unit (ICU) and a high-dependency unit (HDU) where standard infection control measures were in place. Methods In this longitudinal cohort study, we systematically sampled health-care workers, the environment, and patients over 14 months at the ICU and HDU of the Royal Sussex County Hospital, Brighton, England. Nasal swabs were taken from health-care workers every 4 weeks, bed spaces were sampled monthly, and screening swabs were obtained from patients at admission to the ICU or HDU, weekly thereafter, and at discharge. Isolates were cultured and their whole genome sequenced, and we used the threshold of 40 single-nucleotide variants (SNVs) or fewer to define subtypes and infer recent transmission. Findings Between Oct 31, 2011, and Dec 23, 2012, we sampled 198 health-care workers, 40 environmental locations, and 1854 patients; 1819 isolates were sequenced. Median nasal carriage rate of S aureus in health-care workers at 4-weekly timepoints was 36·9% (IQR 35·7–37·3), and 115 (58%) health-care workers had S aureus detected at least once during the study. S aureus was identified in 8–50% of environmental samples. 605 genetically distinct subtypes were identified (median SNV difference 273, IQR 162–399) at a rate of 38 (IQR 34–42) per 4-weekly cycle. Only 25 instances of transmission to patients (seven from health-care workers, two from the environment, and 16 from other patients) were detected. Interpretation In the presence of standard infection control measures, health-care workers were infrequently sources of transmission to patients. S aureus epidemiology in the ICU and HDU is characterised by continuous ingress of distinct subtypes rather than transmission of genetically related strains. Funding UK Medical Research Council, Wellcome Trust, Biotechnology and Biological Sciences Research Council, UK National Institute for Health Research, and Public Health England.
Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a ...rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows.
We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics.
Compared with routine results, WGS predicted species with 93% (95% CI 90–96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91–95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% 95% CI 10–26) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6–10), a median of 21 days (IQR 14–32) faster than final reference laboratory reports were produced (median of 31 days IQR 21–44), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows.
We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis.
National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.
Summary Background Strong immunosuppression, including chemotherapy and immune-depleting antibodies followed by autologous haemopoietic stem-cell transplantation (aHSCT), has been used to treat ...patients with multiple sclerosis, improving control of relapsing disease. We addressed whether near-complete immunoablation followed by immune cell depleted aHSCT would result in long-term control of multiple sclerosis. Methods We did this phase 2 single-arm trial at three hospitals in Canada. We enrolled patients with multiple sclerosis, aged 18–50 years with poor prognosis, ongoing disease activity, and an Expanded Disability Status Scale of 3·0–6·0. Autologous CD34 selected haemopoietic stem-cell grafts were collected after mobilisation with cyclophosphamide and filgrastim. Immunoablation with busulfan, cyclophosphamide, and rabbit anti-thymocyte globulin was followed by aHSCT. The primary outcome was multiple sclerosis activity-free survival (events were clinical relapse, appearance of a new or Gd-enhancing lesion on MRI, and sustained progression of Expanded Disability Status Scale score). This study was registered at ClinicalTrials.gov , NCT01099930. Findings Between diagnosis and aHSCT, 24 patients had 167 clinical relapses over 140 patient-years with 188 Gd-enhancing lesions on 48 pre-aHSCT MRI scans. Median follow-up was 6·7 years (range 3·9–12·7). The primary outcome, multiple sclerosis activity-free survival at 3 years after transplantation was 69·6% (95% CI 46·6–84·2). With up to 13 years of follow-up after aHSCT, no relapses occurred and no Gd enhancing lesions or new T2 lesions were seen on 314 MRI sequential scans. The rate of brain atrophy decreased to that expected for healthy controls. One of 24 patients died of transplantation-related complications. 35% of patients had a sustained improvement in their Expanded Disability Status Scale score. Interpretation We describe the first treatment to fully halt all detectable CNS inflammatory activity in patients with multiple sclerosis for a prolonged period in the absence of any ongoing disease-modifying drugs. Furthermore, many of the patients had substantial recovery of neurological function despite their disease's aggressive nature. Funding Multiple Sclerosis Scientific Research Foundation.
Summary Modelling is valuable in the planning and evaluation of interventions, especially when a controlled trial is ethically or logistically impossible. Models are often used to calculate the ...expected course of events in the absence of more formal assessments. They are also used to derive estimates of rare or future events from recorded intermediate points. When developing models, decisions are needed about the appropriate level of complexity to be represented and about model structure and assumptions. The degree of rigor in model development and assessment can vary greatly, and there is a danger that existing beliefs inappropriately influence judgments about model assumptions and results.
A major challenge for kidney transplantation is balancing the need for immunosuppression to prevent rejection, while minimizing drug‐induced toxicities.
We used DNA microarrays (HG‐U95Av2 GeneChips, ...Affymetrix) to determine gene expression profiles for kidney biopsies and peripheral blood lymphocytes (PBLs) in transplant patients including normal donor kidneys, well‐functioning transplants without rejection, kidneys undergoing acute rejection, and transplants with renal dysfunction without rejection. We developed a data analysis schema based on expression signal determination, class comparison and prediction, hierarchical clustering, statistical power analysis and real‐time quantitative PCR validation. We identified distinct gene expression signatures for both biopsies and PBLs that correlated significantly with each of the different classes of transplant patients. This is the most complete report to date using commercial arrays to identify unique expression signatures in transplant biopsies distinguishing acute rejection, acute dysfunction without rejection and well‐functioning transplants with no rejection history. We demonstrate for the first time the successful application of high density DNA chip analysis of PBL as a diagnostic tool for transplantation. The significance of these results, if validated in a multicenter prospective trial, would be the establishment of a metric based on gene expression signatures for monitoring the immune status and immunosuppression of transplanted patients.
Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized ...broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.
Abstract Background Epidemiological investigations into Mycobacterium tuberculosis outbreaks use 24-locus genotyping (MIRU-VNTR typing). Where no epidemiological link can be found between patients, ...the importance of shared genotypes remains unclear. This issue is especially problematic and time-consuming when tracing contacts within some social groups at high tuberculosis risk, in which unwillingness to volunteer information is common. We investigated whether whole-genome sequencing (WGS) could delineate outbreaks with greater resolution than MIRU-VNTR typing has done. Methods We sequenced 390 M tuberculosis isolates from 254 patients from the UK Midlands (1994–2011) using Illumina technology ( appendix ). We estimated the expected genomic diversity between isolates within a transmission chain by measuring pairwise nucleotide differences between genomes within hosts (79 individuals with pulmonary and extrapulmonary disease, or multiple pulmonary episodes) and between hosts within 25 household outbreaks (63 individuals). We then investigated 11 MIRU-VNTR-based community clusters (168 patients, 157 transmission events) to assess whether WGS could delineate outbreaks more effectively. For each cluster we reconstructed the most plausible transmission chain based on epidemiological data collected by tuberculosis nurses, pairwise nucleotide distances, and times of diagnosis, and compared the genomic diversity across these constructed links with that within individuals and within household outbreaks. Findings 109 (96%) of 114 isolates were within five SNPs of another isolate taken from the same individual or from an individual in the same household outbreak. On the basis of longitudinal isolates from individuals or households, we estimated an evolutionary rate of 0·5 SNPs per genome per year, consistent with a maximum of five SNPs between related isolates 3 years or less apart. Using a greater than five SNP threshold to assess 11 MIRU-VNTR-based community clusters, we found that none of 69 epidemiologically related pairs of MIRU-VNTR-matched cases plausibly related by transmission, two of 13 possibly related pairs, and 13 of 75 pairs with no known epidemiological relation were separated by more than five SNPs (p<0·0001). Seven MIRU-VNTR-matched pairs with no epidemiological relation had more than 30 SNPs, five of seven belonging to the same immigrant community cluster. WGS also showed that 62 of 75 MIRU-VNTR-matched pairs for which no epidemiological relation had been identified from contact tracing were highly likely to indicate transmission: in one substance misuse cluster, 38 individuals were linked by five or fewer SNPs without a single epidemiological link having been established previously. Further analysis suggested that microevolutionary divergence of lineages within outbreaks could signal possible super-spreaders, corroborated by clinical and epidemiological data in two clusters. Interpretation WGS can delineate tuberculosis outbreaks with greater resolution than has previously been possible. These findings offer public health teams the potential to limit outbreak investigations to patients who are likely to be linked by recent transmission, irrespective of whether it has been possible to identify epidemiological links, and to save resources where they are not, even in the context of matched MIRU-VNTR genotypes. Uniquely, WGS also provides information about the genetic structure of outbreak clusters, thereby providing the potential to direct public health resources towards individuals most likely to have infected the largest number of secondary cases. As a consequence, the Health Protection Agency is considering introduction of WGS technology for routine tuberculosis public health practice in England. Funding NIHR Oxford Biomedical Research Centre and the UKCRC Modernising Medical Microbiology Consortium (UKCRC Translational Infection Research Initiative supported by MRC, Biotechnology and Biological Sciences Research Council, and NIHR on behalf of the Department of Health grant G0800778 and the Wellcome Trust 087646/Z/08/Z ).
Study objective: The rhythm detection algorithms of automated external defibrillators have been derived from adult rhythms, and their ability to discriminate between shockable and nonshockable ...rhythms in children is largely unknown. This study evaluates the performance of 1 automated external defibrillator algorithm in infants and children and evaluates algorithm performance with anterior-posterior versus sternal-apex lead placement. Methods: We enrolled pediatric patients in a critical care unit, an electrophysiology laboratory, and a cardiac operating room. A monitor-defibrillator recorded ECGs by means of standard defibrillation-monitor pads. Selected 15-second rhythm samples were played into a LIFEPAK 500 automated external defibrillator, and the automated external defibrillator “shock/no shock” decision was documented. To determine sensitivity and specificity, the automated external defibrillator decision was compared with the “shockable” versus “nonshockable” rhythm classification provided by 3 expert clinicians who were blinded to the automated external defibrillator decision. Results: We recorded 1,561 rhythm samples from 203 pediatric patients (median age 11 months; range, day of birth to 7 years). The automated external defibrillator recommended a shock for 72 of 73 rhythm samples classified as coarse ventricular fibrillation by expert review (sensitivity 99%; 95% confidence interval CI 93% to 100%); and correctly reached a “no shock advised” decision for 1,465 of 1,472 rhythm samples classified as nonshockable by experts (specificity 99.5%). Specificity was 99.1% (95% CI 97.8% to 99.8%) with the sternal-apex lead and 99.4% (95% CI 98.1% to 99.9%) with the anterior-posterior lead. Conclusion: This automated external defibrillator algorithm has high specificity and sensitivity when used in infants and children with either sternal-apex or anterior-posterior lead placement. Ann Emerg Med. 2003;42:185-196.
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