Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination, and Drug Susceptibility Prediction.

Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

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.

Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing.

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/µl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies.

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative 'backbone' of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

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.

Constructing custom-made radiotranscriptomic signatures of vascular inflammation from routine CT angiograms: a prospective outcomes validation study in COVID-19.

BACKGROUND: Direct evaluation of vascular inflammation in patients with COVID-19 would facilitate more efficient trials of new treatments and identify patients at risk of long-term complications who might respond to treatment. We aimed to develop a novel artificial intelligence (AI)-assisted image analysis platform that quantifies cytokine-driven vascular inflammation from routine CT angiograms, and sought to validate its prognostic value in COVID-19. METHODS: For this prospective outcomes validation study, we developed a radiotranscriptomic platform that uses RNA sequencing data from human internal mammary artery biopsies to develop novel radiomic signatures of vascular inflammation from CT angiography images. We then used this platform to train a radiotranscriptomic signature (C19-RS), derived from the perivascular space around the aorta and the internal mammary artery, to best describe cytokine-driven vascular inflammation. The prognostic value of C19-RS was validated externally in 435 patients (331 from study arm 3 and 104 from study arm 4) admitted to hospital with or without COVID-19, undergoing clinically indicated pulmonary CT angiography, in three UK National Health Service (NHS) trusts (Oxford, Leicester, and Bath). We evaluated the diagnostic and prognostic value of C19-RS for death in hospital due to COVID-19, did sensitivity analyses based on dexamethasone treatment, and investigated the correlation of C19-RS with systemic transcriptomic changes. FINDINGS: Patients with COVID-19 had higher C19-RS than those without (adjusted odds ratio [OR] 2·97 [95% CI 1·43-6·27], p=0·0038), and those infected with the B.1.1.7 (alpha) SARS-CoV-2 variant had higher C19-RS values than those infected with the wild-type SARS-CoV-2 variant (adjusted OR 1·89 [95% CI 1·17-3·20] per SD, p=0·012). C19-RS had prognostic value for in-hospital mortality in COVID-19 in two testing cohorts (high [≥6·99] vs low [<6·99] C19-RS; hazard ratio [HR] 3·31 [95% CI 1·49-7·33], p=0·0033; and 2·58 [1·10-6·05], p=0·028), adjusted for clinical factors, biochemical biomarkers of inflammation and myocardial injury, and technical parameters. The adjusted HR for in-hospital mortality was 8·24 (95% CI 2·16-31·36, p=0·0019) in patients who received no dexamethasone treatment, but 2·27 (0·69-7·55, p=0·18) in those who received dexamethasone after the scan, suggesting that vascular inflammation might have been a therapeutic target of dexamethasone in COVID-19. Finally, C19-RS was strongly associated (r=0·61, p=0·00031) with a whole blood transcriptional module representing dysregulation of coagulation and platelet aggregation pathways. INTERPRETATION: Radiotranscriptomic analysis of CT angiography scans introduces a potentially powerful new platform for the development of non-invasive imaging biomarkers. Application of this platform in routine CT pulmonary angiography scans done in patients with COVID-19 produced the radiotranscriptomic signature C19-RS, a marker of cytokine-driven inflammation driving systemic activation of coagulation and responsible for adverse clinical outcomes, which predicts in-hospital mortality and might allow targeted therapy. FUNDING: Engineering and Physical Sciences Research Council, British Heart Foundation, Oxford BHF Centre of Research Excellence, Innovate UK, NIHR Oxford Biomedical Research Centre, Wellcome Trust, Onassis Foundation.

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.

Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae.

Escherichia coli and other Enterobacteriaceae are diverse species with "open" pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

Genomic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients.

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonized with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole-genome sequencing of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies, which may vary as a result of different inputs and selection pressures. Whole-genome sequencing of 46 contemporaneous patient isolates identified one (2 %; 95 % CI 0.05-11 %) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10 % of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including blaCTX-M, blaSHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention. This article contains data hosted by Microreact.

In vitro colonization of an experimental silicone by Candida albicans.

Denture soft-lining materials are exposed to the oral cavity for long periods and are in continuous contact with saliva, subject to inhibition of liquid molecules, and susceptible to colonization by microorganisms. The opportunist yeast Candida albicans is of particular concern in this context, being associated with denture plaque and denture-related stomatitis. In this study, penetration of C. albicans into an experimental silicone elastomer soft-lining material was investigated under batch and continuous culture conditions. A model "denture plaque" microcosm was also used. Increasing the filler concentration within the elastomer decreased penetration under both batch and continuous culture conditions. In continuous culture, C. albicans penetration was significantly greater in pure culture than in the presence of bacteria after 72-h incubation (p < 0.05).

An assessment of air as a source of DNA contamination encountered when performing PCR.

Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.