High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing.

The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

An Observational Cohort Study on the Incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection and B.1.1.7 Variant Infection in Healthcare Workers by Antibody and Vaccination Status.

BACKGROUND: Natural and vaccine-induced immunity will play a key role in controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. SARS-CoV-2 variants have the potential to evade natural and vaccine-induced immunity. METHODS: In a longitudinal cohort study of healthcare workers (HCWs) in Oxfordshire, United Kingdom, we investigated the protection from symptomatic and asymptomatic polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection conferred by vaccination (Pfizer-BioNTech BNT162b2, Oxford-AstraZeneca ChAdOx1 nCOV-19) and prior infection (determined using anti-spike antibody status), using Poisson regression adjusted for age, sex, temporal changes in incidence and role. We estimated protection conferred after 1 versus 2 vaccinations and from infections with the B.1.1.7 variant identified using whole genome sequencing. RESULTS: In total, 13 109 HCWs participated; 8285 received the Pfizer-BioNTech vaccine (1407 two doses), and 2738 the Oxford-AstraZeneca vaccine (49 two doses). Compared to unvaccinated seronegative HCWs, natural immunity and 2 vaccination doses provided similar protection against symptomatic infection: no HCW vaccinated twice had symptomatic infection, and incidence was 98% lower in seropositive HCWs (adjusted incidence rate ratio 0.02 [95% confidence interval {CI} < .01-.18]). Two vaccine doses or seropositivity reduced the incidence of any PCR-positive result with or without symptoms by 90% (0.10 [95% CI .02-.38]) and 85% (0.15 [95% CI .08-.26]), respectively. Single-dose vaccination reduced the incidence of symptomatic infection by 67% (0.33 [95% CI .21-.52]) and any PCR-positive result by 64% (0.36 [95% CI .26-.50]). There was no evidence of differences in immunity induced by natural infection and vaccination for infections with S-gene target failure and B.1.1.7. CONCLUSIONS: Natural infection resulting in detectable anti-spike antibodies and 2 vaccine doses both provide robust protection against SARS-CoV-2 infection, including against the B.1.1.7 variant.

Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopedic Device Infection.

Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximize the amount of microbial DNA sequence data available for analysis. Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Of 114 samples generating sequence data, species-level positive percent agreement between metagenomic sequencing and culture was 50/65 (77%; 95% confidence interval [CI], 65 to 86%) and negative percent agreement was 103/114 (90%; 95% CI, 83 to 95%). Saponin treatment reduced the proportion of human bases sequenced in comparison to 5-µm filtration from a median (interquartile range [IQR]) of 98.1% (87.0% to 99.9%) to 11.9% (0.4% to 67.0%), improving reference genome coverage at a 10-fold depth from 18.7% (0.30% to 85.7%) to 84.3% (12.9% to 93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

Metagenomic Sequencing as a Pathogen-Agnostic Clinical Diagnostic Tool for Infectious Diseases: a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies.

Metagenomic sequencing is frequently claimed to have the potential to revolutionize microbiology through rapid species identification and antimicrobial resistance (AMR) prediction. We assess the progress toward these developments. We perform a systematic review and meta-analysis of all published literature on culture-independent metagenomic sequencing for pathogen-agnostic infectious disease diagnostics up to 12 August 2020. Methodologic bias and applicability were assessed using the tool Quadas-2. (Prospero CRD42020163777). A total of 2,023 clinical samples from 13/21 eligible diagnostic test accuracy studies were included in the meta-analysis. Reference standards were culture, molecular testing, clinical decision, or a composite measure. Sensitivity and specificity in the most widely investigated sample types were 90% (95% confidence interval [CI], 78% to 96%) and 86% (45% to 98%) for blood, 75% (54% to 89%) and 96% (72% to 100%) for cerebrospinal fluid (CSF), and 84% (79% to 88%) and 67% (38% to 87%) for orthopedic samples, respectively. We identified a limited use of controls, especially negative controls which were used in only 62% (13/21) of studies. AMR prediction and comparison to phenotypic results were undertaken in four studies; categorical agreement was 88%(80% to 97%), and very major and major error rates were 24% (8% to 40%) and 5% (0% to 12%), respectively. Better human DNA depletion methods are required; a median 91% (interquartile range [IQR], 82% to 98%; range, 76% to 98%) of sequences was classified as human. The median (IQR; range) time from sample to result was 29 hours (24 to 94; 4 to 144 hours). The reported consumable cost per sample ranged from $130 to $685. There is scope for improving the quality of reporting in clinical metagenomic studies. Although our results are limited by the heterogeneity displayed, our results reflect a promising outlook for clinical metagenomics. Methodological improvements and convergence around protocols and best practices may improve performance in the future.

Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples.

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

Detection in the United Kingdom of the Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, October to December 2018.

We describe detection in the United Kingdom (UK) of the drug-resistant Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate azithromycin resistance. Two female patients developed infection following contact with UK-resident men from the same sexual network linked to travel to Ibiza, Spain. One case failed treatment with ceftriaxone, and azithromycin and gentamicin, before successful treatment with ertapenem. Both isolates had indistinguishable whole-genome sequences. Urgent action is essential to contain this drug-resistant strain.

Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices.

BACKGROUND: Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections but potentially useful for any microbial sequencing, and compare detection by direct-from-clinical-sample metagenomic nanopore sequencing with Illumina sequencing and standard microbiological diagnostic techniques. RESULTS: DNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (> 90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. CONCLUSIONS: We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data and use of this pipeline to show initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. The high proportion of human DNA in prosthetic joint infection extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling. The nine samples sequenced in this pilot study have shown a proof of concept for sequencing and analysis that will enable us to investigate further sequencing to improve specificity and sensitivity.

Molecular Diagnosis of Orthopedic-Device-Related Infection Directly from Sonication Fluid by Metagenomic Sequencing.

Culture of multiple periprosthetic tissue samples is the current gold standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through culture of sonication fluid from explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of total DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97 (88%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis.

Atopic dermatitis (AD) is the most common dermatological disease of childhood. Many children with AD have asthma and AD shares regions of genetic linkage with psoriasis, another chronic inflammatory skin disease. We present here a genome-wide association study (GWAS) of childhood-onset AD in 1563 European cases with known asthma status and 4054 European controls. Using Illumina genotyping followed by imputation, we generated 268 034 consensus genotypes and in excess of 2 million single nucleotide polymorphisms (SNPs) for analysis. Association signals were assessed for replication in a second panel of 2286 European cases and 3160 European controls. Four loci achieved genome-wide significance for AD and replicated consistently across all cohorts. These included the epidermal differentiation complex (EDC) on chromosome 1, the genomic region proximal to LRRC32 on chromosome 11, the RAD50/IL13 locus on chromosome 5 and the major histocompatibility complex (MHC) on chromosome 6; reflecting action of classical HLA alleles. We observed variation in the contribution towards co-morbid asthma for these regions of association. We further explored the genetic relationship between AD, asthma and psoriasis by examining previously identified susceptibility SNPs for these diseases. We found considerable overlap between AD and psoriasis together with variable coincidence between allergic rhinitis (AR) and asthma. Our results indicate that the pathogenesis of AD incorporates immune and epidermal barrier defects with combinations of specific and overlapping effects at individual loci.

Recombination and population structure in Salmonella enterica.

Salmonella enterica is a bacterial pathogen that causes enteric fever and gastroenteritis in humans and animals. Although its population structure was long described as clonal, based on high linkage disequilibrium between loci typed by enzyme electrophoresis, recent examination of gene sequences has revealed that recombination plays an important evolutionary role. We sequenced around 10% of the core genome of 114 isolates of enterica using a resequencing microarray. Application of two different analysis methods (Structure and ClonalFrame) to our genomic data allowed us to define five clear lineages within S. enterica subspecies enterica, one of which is five times older than the other four and two thirds of the age of the whole subspecies. We show that some of these lineages display more evidence of recombination than others. We also demonstrate that some level of sexual isolation exists between the lineages, so that recombination has occurred predominantly between members of the same lineage. This pattern of recombination is compatible with expectations from the previously described ecological structuring of the enterica population as well as mechanistic barriers to recombination observed in laboratory experiments. In spite of their relatively low level of genetic differentiation, these lineages might therefore represent incipient species.

Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing.

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction.

Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to use nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, using Illumina and ONT's R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. 'Super accuracy' (sup) basecalled R10.4 reads - in particular duplex reads - have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically <10 %), with substantial implications for cost and throughput if relying on nanopore data only to enable bacterial genome reconstruction. In addition, recovery of small plasmids with the best-performing long-read assembler (Flye) was inconsistent. R10.4/Kit12 combined with sup basecalling holds promise as a singular sequencing technology in the reconstruction of commonly studied bacterial genomes, but hybrid assembly (Illumina+R9.4.1 hac) currently remains the highest throughput, most robust, and cost-effective approach to fully reconstruct these bacterial genomes.

Evaluation of sequence hybridization for respiratory viruses using the Twist Bioscience Respiratory Virus Research panel and the OneCodex Respiratory Virus sequence analysis workflow.

Respiratory viral infections are a major global clinical problem, and rapid, cheap, scalable and agnostic diagnostic tests that capture genome-level information on viral variation are urgently needed. Metagenomic approaches would be ideal, but remain currently limited in that much of the genetic content in respiratory samples is human, and amplifying and sequencing the viral/pathogen component in an unbiased manner is challenging. PCR-based tests, including those which detect multiple pathogens, are already widely used, but do not capture information on strain-level variation; tests with larger viral repertoires are also expensive on a per-test basis. One intermediate approach is the use of large panels of viral probes or 'baits', which target or 'capture' sequences representing complete genomes amongst several different common viral pathogens; these are then amplified, sequenced and analysed with a sequence analysis workflow. Here we evaluate one such commercial bait capture method (the Twist Bioscience Respiratory Virus Research Panel) and sequence analysis workflow (OneCodex), using control (simulated) and patient samples head-to-head with a validated multiplex PCR clinical diagnostic test (BioFire FilmArray). We highlight the limited sensitivity and specificity of the joint Twist Bioscience/OneCodex approach, which are further reduced by shortening workflow times and increasing sample throughput to reduce per-sample costs. These issues with performance may be driven by aspects of both the laboratory (e.g. capacity to enrich for viruses present in low numbers), bioinformatics methods used (e.g. a limited viral reference database) and thresholds adopted for calling a virus as present or absent. As a result, this workflow would require further optimization prior to any implementation for respiratory virus characterization in a routine diagnostic healthcare setting.

Identifying Bacterial Airways Infection in Stable Severe Asthma Using Oxford Nanopore Sequencing Technologies.

Previous metagenomic studies in asthma have been limited by inadequate sequencing depth for species-level bacterial identification and by heterogeneity in clinical phenotyping. We hypothesize that chronic bacterial airways infection is a key "treatable trait" whose prevalence, clinical phenotype and reliable biomarkers need definition. In this study, we have applied a method for Oxford Nanopore sequencing for the unbiased metagenomic characterization of severe asthma. We optimized methods to compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total sputum DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from highly phenotyped severe asthma during clinical stability. In participants with severe asthma (n = 23) H. influenzae was commonly cultured (n = 8) and identified as the dominant bacterial species by metagenomic sequencing using an optimized method for Illumina MiSeq and Oxford Nanopore. Alongside superior operational characteristics, Oxford Nanopore achieved near complete genome coverage of H. influenzae and demonstrated a high level of agreement with Illumina MiSeq data. Clinically significant infection was confirmed with validated H. influenzae plasmid-based quantitative PCR assay. H. influenzae positive patients were found to have sputum neutrophilia and lower FeNO. In conclusion, using an optimized method of direct sequencing of induced sputum samples, H. influenzae was identified as a clinically relevant pathogen in severe asthma and was identified reliably using metagenomic sequencing. Application of these protocols in ongoing analysis of large patient cohorts will allow full characterization of this clinical phenotype. IMPORTANCE The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood. Traditional culture lacks sensitivity and current sequencing techniques are limited by operational problems and limited ability to identify pathogens at species level. We optimized a new sequencing technique-Oxford Nanopore technologies (ONT)-for use on human sputum samples and compared it with existing methods. We found ONT was effective for rapidly analyzing samples and could identify bacteria at the species level. We used this to show Haemophilus influenzae was a dominant bacterium in the airways in people with severe asthma. The presence of Haemophilus was associated with a "neutrophilic" form of asthma - a subgroup for which we currently lack specific treatments. Therefore, this technique could be used to target chronic antibiotic therapy and in research to characterize the full breadth of bacteria in the airways.

Epidemiological data and genome sequencing reveals that nosocomial transmission of SARS-CoV-2 is underestimated and mostly mediated by a small number of highly infectious individuals.

OBJECTIVES: Despite robust efforts, patients and staff acquire SARS-CoV-2 infection in hospitals. We investigated whether whole-genome sequencing enhanced the epidemiological investigation of healthcare-associated SARS-CoV-2 acquisition. METHODS: From 17-November-2020 to 5-January-2021, 803 inpatients and 329 staff were diagnosed with SARS-CoV-2 infection at four Oxfordshire hospitals. We classified cases using epidemiological definitions, looked for a potential source for each nosocomial infection, and evaluated genomic evidence supporting transmission. RESULTS: Using national epidemiological definitions, 109/803(14%) inpatient infections were classified as definite/probable nosocomial, 615(77%) as community-acquired and 79(10%) as indeterminate. There was strong epidemiological evidence to support definite/probable cases as nosocomial. Many indeterminate cases were likely infected in hospital: 53/79(67%) had a prior-negative PCR and 75(95%) contact with a potential source. 89/615(11% of all 803 patients) with apparent community-onset had a recent hospital exposure. Within 764 samples sequenced 607 genomic clusters were identified (>1 SNP distinct). Only 43/607(7%) clusters contained evidence of onward transmission (subsequent cases within ≤ 1 SNP). 20/21 epidemiologically-identified outbreaks contained multiple genomic introductions. Most (80%) nosocomial acquisition occurred in rapid super-spreading events in settings with a mix of COVID-19 and non-COVID-19 patients. CONCLUSIONS: Current surveillance definitions underestimate nosocomial acquisition. Most nosocomial transmission occurs from a relatively limited number of highly infectious individuals.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2.

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage.

BACKGROUND: Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. PRINCIPAL FINDINGS: We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). SIGNIFICANCE: This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.