Inference of Infectious Disease Transmission through a Relaxed Bottleneck Using Multiple Genomes Per Host.

In recent times, pathogen genome sequencing has become increasingly used to investigate infectious disease outbreaks. When genomic data is sampled densely enough amongst infected individuals, it can help resolve who infected whom. However, transmission analysis cannot rely solely on a phylogeny of the genomes but must account for the within-host evolution of the pathogen, which blurs the relationship between phylogenetic and transmission trees. When only a single genome is sampled for each host, the uncertainty about who infected whom can be quite high. Consequently, transmission analysis based on multiple genomes of the same pathogen per host has a clear potential for delivering more precise results, even though it is more laborious to achieve. Here, we present a new methodology that can use any number of genomes sampled from a set of individuals to reconstruct their transmission network. Furthermore, we remove the need for the assumption of a complete transmission bottleneck. We use simulated data to show that our method becomes more accurate as more genomes per host are provided, and that it can infer key infectious disease parameters such as the size of the transmission bottleneck, within-host growth rate, basic reproduction number, and sampling fraction. We demonstrate the usefulness of our method in applications to real datasets from an outbreak of Pseudomonas aeruginosa amongst cystic fibrosis patients and a nosocomial outbreak of Klebsiella pneumoniae.

Bayesian Inference of Clonal Expansions in a Dated Phylogeny.

Microbial population genetics models often assume that all lineages are constrained by the same population size dynamics over time. However, many neutral and selective events can invalidate this assumption and can contribute to the clonal expansion of a specific lineage relative to the rest of the population. Such differential phylodynamic properties between lineages result in asymmetries and imbalances in phylogenetic trees that are sometimes described informally but which are difficult to analyze formally. To this end, we developed a model of how clonal expansions occur and affect the branching patterns of a phylogeny. We show how the parameters of this model can be inferred from a given dated phylogeny using Bayesian statistics, which allows us to assess the probability that one or more clonal expansion events occurred. For each putative clonal expansion event, we estimate its date of emergence and subsequent phylodynamic trajectory, including its long-term evolutionary potential which is important to determine how much effort should be placed on specific control measures. We demonstrate the applicability of our methodology on simulated and real data sets. Inference under our clonal expansion model can reveal important features in the evolution and epidemiology of infectious disease pathogens. [Clonal expansion; genomic epidemiology; microbial population genomics; phylodynamics.].

Social risk factors for SARS-CoV-2 acquisition in university students: cross sectional survey.

The objectives of this study were to define risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in University of Cambridge (UoC) students during a period of increased incidence in October and November 2020. The study design was a survey.Routine public health surveillance identified an increase in the numbers of UoC students with confirmed SARS-CoV-2 positivity in the 10 days after a national lockdown was announced in the UK on 5th November 2020. Cases were identified both through symptom-triggered testing and a universal asymptomatic testing programme. An online questionnaire was sent to all UoC students on 25 November to investigate risk factors for testing positive in the period after 30th October 2020. This asked about symptoms, SARS-CoV-2 test results, aspects of university life, and attendance at social events in the week prior to lockdown. Univariate and multivariable analyses were undertaken evaluating potential risk factors for SARS-CoV-2 positivity.Among 3980 students responding to the questionnaire, 99 (2.5%) reported testing SARS-CoV-2 positive in the period studied; 28 (28%) were asymptomatic. We found strong independent associations with SARS-CoV-2 positivity and attendance at two social settings in the City of Cambridge (adjusted odds ratio favouring disease 13.0 (95% CI 6.2-26.9) and 14.2 (95% CI 2.9-70)), with weaker evidence of association with three further social settings. By contrast, we did not observe strong independent associations between disease risk and accommodation type or attendance at a range of activities associated with the university curriculum.To conclude attendance at social settings can facilitate widespread SARS-CoV-2 transmission in university students. Constraint of transmission in higher education settings needs to emphasise risks outside university premises, as well as a COVID-safe environment within university premises.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25 mM) reduced endplate potential (EPP) amplitudes by 10-40% and enhanced depression during repetitive (2-20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10-20 mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (-3.8 kcal/mol) to voltage-sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. KEY POINTS: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

Limited Capability for Testing Mycobacterium tuberculosis for Susceptibility to New Drugs.

We surveyed availability of phenotypic drug susceptibility testing for drug-resistant Mycobacterium tuberculosis in Europe. Of 27 laboratories, 17 tested for linezolid, 11 for clofazimine, 9 for bedaquiline, and 6 for delamanid during 2019. Our findings indicate that testing capacity for newer and repurposed tuberculosis drugs exists, but its availability is limited.

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.

Loss of cerebellar glutamate transporters EAAT4 and GLAST differentially affects the spontaneous firing pattern and survival of Purkinje cells.

Loss of excitatory amino acid transporters (EAATs) has been implicated in a number of human diseases including spinocerebellar ataxias, Alzhiemer's disease and motor neuron disease. EAAT4 and GLAST/EAAT1 are the two predominant EAATs responsible for maintaining low extracellular glutamate levels and preventing neurotoxicity in the cerebellum, the brain region essential for motor control. Here using genetically modified mice we identify new critical roles for EAAT4 and GLAST/EAAT1 as modulators of Purkinje cell (PC) spontaneous firing patterns. We show high EAAT4 levels, by limiting mGluR1 signalling, are essential in constraining inherently heterogeneous firing of zebrin-positive PCs. Moreover mGluR1 antagonists were found to restore regular spontaneous PC activity and motor behaviour in EAAT4 knockout mice. In contrast, GLAST/EAAT1 expression is required to sustain normal spontaneous simple spike activity in low EAAT4 expressing (zebrin-negative) PCs by restricting NMDA receptor activation. Blockade of NMDA receptor activity restores spontaneous activity in zebrin-negative PCs of GLAST knockout mice and furthermore alleviates motor deficits. In addition both transporters have differential effects on PC survival, with zebrin-negative PCs more vulnerable to loss of GLAST/EAAT1 and zebrin-positive PCs more vulnerable to loss of EAAT4. These findings reveal that glutamate transporter dysfunction through elevated extracellular glutamate and the aberrant activation of extrasynaptic receptors can disrupt cerebellar output by altering spontaneous PC firing. This expands our understanding of disease mechanisms in cerebellar ataxias and establishes EAATs as targets for restoring homeostasis in a variety of neurological diseases where altered cerebellar output is now thought to play a key role in pathogenesis.

Genomic Epidemiology of Complex, Multispecies, Plasmid-Borne blaKPC Carbapenemase in Enterobacterales in the United Kingdom from 2009 to 2014.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the blaKPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of blaKPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, blaKPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of blaKPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 blaKPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of blaKPC (predominantly blaKPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), blaKPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-blaKPC and blaKPC plasmids and the common presence of multiple replicons within blaKPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control.

Reversal of proliferation deficits caused by chromosome 16p13.11 microduplication through targeting NFκB signaling: an integrated study of patient-derived neuronal precursor cells, cerebral organoids and in vivo brain imaging.

The molecular basis of how chromosome 16p13.11 microduplication leads to major psychiatric disorders is unknown. Here we have undertaken brain imaging of patients carrying microduplications in chromosome 16p13.11 and unaffected family controls, in parallel with iPS cell-derived cerebral organoid studies of the same patients. Patient MRI revealed reduced cortical volume, and corresponding iPSC studies showed neural precursor cell (NPC) proliferation abnormalities and reduced organoid size, with the NPCs therein displaying altered planes of cell division. Transcriptomic analyses of NPCs uncovered a deficit in the NFκB p65 pathway, confirmed by proteomics. Moreover, both pharmacological and genetic correction of this deficit rescued the proliferation abnormality. Thus, chromosome 16p13.11 microduplication disturbs the normal programme of NPC proliferation to reduce cortical thickness due to a correctable deficit in the NFκB signalling pathway. This is the first study demonstrating a biologically relevant, potentially ameliorable, signalling pathway underlying chromosome 16p13.11 microduplication syndrome in patient-derived neuronal precursor cells.

Familial t(1;11) translocation is associated with disruption of white matter structural integrity and oligodendrocyte-myelin dysfunction.

Although the underlying neurobiology of major mental illness (MMI) remains unknown, emerging evidence implicates a role for oligodendrocyte-myelin abnormalities. Here, we took advantage of a large family carrying a balanced t(1;11) translocation, which substantially increases risk of MMI, to undertake both diffusion tensor imaging and cellular studies to evaluate the consequences of the t(1;11) translocation on white matter structural integrity and oligodendrocyte-myelin biology. This translocation disrupts among others the DISC1 gene which plays a crucial role in brain development. We show that translocation-carrying patients display significant disruption of  white matter integrity compared with familial controls. At a cellular level, we observe dysregulation of key pathways controlling oligodendrocyte development and morphogenesis in induced pluripotent stem cell (iPSC) derived case oligodendrocytes. This is associated with reduced proliferation and a stunted morphology in vitro. Further, myelin internodes in a humanized mouse model that recapitulates the human translocation as well as after transplantation of t(1;11) oligodendrocyte progenitors were significantly reduced when  compared with controls. Thus we provide evidence that the t(1;11) translocation has biological effects at both the systems and cellular level that together suggest oligodendrocyte-myelin dysfunction.

GRIN2A-related disorders: genotype and functional consequence predict phenotype.

Alterations of the N-methyl-d-aspartate receptor (NMDAR) subunit GluN2A, encoded by GRIN2A, have been associated with a spectrum of neurodevelopmental disorders with prominent speech-related features, and epilepsy. We performed a comprehensive assessment of phenotypes with a standardized questionnaire in 92 previously unreported individuals with GRIN2A-related disorders. Applying the criteria of the American College of Medical Genetics and Genomics to all published variants yielded 156 additional cases with pathogenic or likely pathogenic variants in GRIN2A, resulting in a total of 248 individuals. The phenotypic spectrum ranged from normal or near-normal development with mild epilepsy and speech delay/apraxia to severe developmental and epileptic encephalopathy, often within the epilepsy-aphasia spectrum. We found that pathogenic missense variants in transmembrane and linker domains (misTMD+Linker) were associated with severe developmental phenotypes, whereas missense variants within amino terminal or ligand-binding domains (misATD+LBD) and null variants led to less severe developmental phenotypes, which we confirmed in a discovery (P = 10-6) as well as validation cohort (P = 0.0003). Other phenotypes such as MRI abnormalities and epilepsy types were also significantly different between the two groups. Notably, this was paralleled by electrophysiology data, where misTMD+Linker predominantly led to NMDAR gain-of-function, while misATD+LBD exclusively caused NMDAR loss-of-function. With respect to null variants, we show that Grin2a+/- cortical rat neurons also had reduced NMDAR function and there was no evidence of previously postulated compensatory overexpression of GluN2B. We demonstrate that null variants and misATD+LBD of GRIN2A do not only share the same clinical spectrum (i.e. milder phenotypes), but also result in similar electrophysiological consequences (loss-of-function) opposing those of misTMD+Linker (severe phenotypes; predominantly gain-of-function). This new pathomechanistic model may ultimately help in predicting phenotype severity as well as eligibility for potential precision medicine approaches in GRIN2A-related disorders.

Functional assessment of triheteromeric NMDA receptors containing a human variant associated with epilepsy.

KEY POINTS: NMDA receptors are neurotransmitter-gated ion channels that are critically involved in brain cell communication Variations in genes encoding NMDA receptor subunits have been found in a range of neurodevelopmental disorders. We investigated a de novo genetic variant found in patients with epileptic encephalopathy that changes a residue located in the ion channel pore of the GluN2A NMDA receptor subunit. We found that this variant (GluN2AN615K ) impairs physiologically important receptor properties: it markedly reduces Mg2+ blockade and channel conductance, even for receptors in which one GluN2AN615K is co-assembled with one wild-type GluN2A subunit. Our findings are consistent with the GluN2AN615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers. ABSTRACT: NMDA receptors are ionotropic calcium-permeable glutamate receptors with a voltage-dependence mediated by blockade by Mg2+ . Their activation is important in signal transduction, as well as synapse formation and maintenance. Two unrelated individuals with epileptic encephalopathy carry a de novo variant in the gene encoding the GluN2A NMDA receptor subunit: a N615K missense variant in the M2 pore helix (GRIN2AC1845A ). We hypothesized that this variant underlies the neurodevelopmental disorders in carriers and explored its functional consequences by electrophysiological analysis in heterologous systems. We focused on GluN2AN615K co-expressed with wild-type GluN2 subunits in physiologically relevant triheteromeric NMDA receptors containing two GluN1 and two distinct GluN2 subunits, whereas previous studies have investigated the impact of the variant in diheteromeric NMDA receptors with two GluN1 and two identical GluN2 subunits. We found that GluN2AN615K -containing triheteromers showed markedly reduced Mg2+ blockade, with a value intermediate between GluN2AN615K diheteromers and wild-type NMDA receptors. Single-channel conductance was reduced by four-fold in GluN2AN615K diheteromers, again with an intermediate value in GluN2AN615K -containing triheteromers. Glutamate deactivation rates were unaffected. Furthermore, we expressed GluN2AN615K in cultured primary mouse cortical neurons, observing a decrease in Mg2+ blockade and reduction in current density, confirming that the variant continues to have significant functional impact in neuronal systems. Our results demonstrate that the GluN2AN615K variant has substantial effects on NMDA receptor properties fundamental to the roles of the receptor in synaptic plasticity, even when expressed alongside wild-type subunits. This work strengthens the evidence indicating that the GluN2AN615K variant underlies the disabling neurodevelopmental phenotype in carriers.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation.

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of ß-III spectrin and GLAST.

Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking ß-III spectrin (ß-III-/-). One function of ß-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In ß-III-/- mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of ß-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with ß-III-/- mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young ß-III-/- mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of ß-III-/- mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

Mortality risks associated with emergency admissions during weekends and public holidays: an analysis of electronic health records.

BACKGROUND: Weekend hospital admission is associated with increased mortality, but the contributions of varying illness severity and admission time to this weekend effect remain unexplored. METHODS: We analysed unselected emergency admissions to four Oxford University National Health Service hospitals in the UK from Jan 1, 2006, to Dec 31, 2014. The primary outcome was death within 30 days of admission (in or out of hospital), analysed using Cox models measuring time from admission. The primary exposure was day of the week of admission. We adjusted for multiple confounders including demographics, comorbidities, and admission characteristics, incorporating non-linearity and interactions. Models then considered the effect of adjusting for 15 common haematology and biochemistry test results or proxies for hospital workload. FINDINGS: 257 596 individuals underwent 503 938 emergency admissions. 18 313 (4·7%) patients admitted as weekday energency admissions and 6070 (5·1%) patients admitted as weekend emergency admissions died within 30 days (p<0·0001). 9347 individuals underwent 9707 emergency admissions on public holidays. 559 (5·8%) died within 30 days (p<0·0001 vs weekday). 15 routine haematology and biochemistry test results were highly prognostic for mortality. In 271 465 (53·9%) admissions with complete data, adjustment for test results explained 33% (95% CI 21 to 70) of the excess mortality associated with emergency admission on Saturdays compared with Wednesdays, 52% (lower 95% CI 34) on Sundays, and 87% (lower 95% CI 45) on public holidays after adjustment for standard patient characteristics. Excess mortality was predominantly restricted to admissions between 1100 h and 1500 h (pinteraction=0·04). No hospital workload measure was independently associated with mortality (all p values >0·06). INTERPRETATION: Adjustment for routine test results substantially reduced excess mortality associated with emergency admission at weekends and public holidays. Adjustment for patient-level factors not available in our study might further reduce the residual excess mortality, particularly as this clustered around midday at weekends. Hospital workload was not associated with mortality. Together, these findings suggest that the weekend effect arises from patient-level differences at admission rather than reduced hospital staffing or services. FUNDING: NIHR Oxford Biomedical Research Centre.

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.

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.

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.

BugMat and FindNeighbour: command line and server applications for investigating bacterial relatedness.

BACKGROUND: Large scale bacterial sequencing has made the determination of genetic relationships within large sequence collections of bacterial genomes derived from the same microbial species an increasingly common task. Solutions to the problem have application to public health (for example, in the detection of possible disease transmission), and as part of divide-and-conquer strategies selecting groups of similar isolates for computationally intensive methods of phylogenetic inference using (for example) maximal likelihood methods. However, the generation and maintenance of distance matrices is computationally intensive, and rapid methods of doing so are needed to allow translation of microbial genomics into public health actions. RESULTS: We developed, tested and deployed three solutions. BugMat is a fast C++ application which generates one-off in-memory distance matrices. FindNeighbour and FindNeighbour2 are server-side applications which build, maintain, and persist either complete (for FindNeighbour) or sparse (for FindNeighbour2) distance matrices given a set of sequences. FindNeighbour and BugMat use a variation model to accelerate computation, while FindNeighbour2 uses reference-based compression. Performance metrics show scalability into tens of thousands of sequences, with options for scaling further. CONCLUSION: Three applications, each with distinct strengths and weaknesses, are available for distance-matrix based analysis of large bacterial collections. Deployed as part of the Public Health England solution for M. tuberculosis genomic processing, they will have wide applicability.