FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

A2B-COVID: A Tool for Rapidly Evaluating Potential SARS-CoV-2 Transmission Events.

Identifying linked cases of infection is a critical component of the public health response to viral infectious diseases. In a clinical context, there is a need to make rapid assessments of whether cases of infection have arrived independently onto a ward, or are potentially linked via direct transmission. Viral genome sequence data are of great value in making these assessments, but are often not the only form of data available. Here, we describe A2B-COVID, a method for the rapid identification of potentially linked cases of COVID-19 infection designed for clinical settings. Our method combines knowledge about infection dynamics, data describing the movements of individuals, and evolutionary analysis of genome sequences to assess whether data collected from cases of infection are consistent or inconsistent with linkage via direct transmission. A retrospective analysis of data from two wards at Cambridge University Hospitals NHS Foundation Trust during the first wave of the pandemic showed qualitatively different patterns of linkage between cases on designated COVID-19 and non-COVID-19 wards. The subsequent real-time application of our method to data from the second epidemic wave highlights its value for monitoring cases of infection in a clinical context.

The Removal of Airborne Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Other Microbial Bioaerosols by Air Filtration on Coronavirus Disease 2019 (COVID-19) Surge Units.

Airborne severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in a coronavirus disease 19 (COVID-19) ward before activation of HEPA-air filtration but not during filter operation; SARS-CoV-2 was again detected following filter deactivation. Airborne SARS-CoV-2 was infrequently detected in a COVID-19 intensive care unit. Bioaerosol was also effectively filtered.

Ventilator-associated pneumonia in critically ill patients with COVID-19.

BACKGROUND: Pandemic COVID-19 caused by the coronavirus SARS-CoV-2 has a high incidence of patients with severe acute respiratory syndrome (SARS). Many of these patients require admission to an intensive care unit (ICU) for invasive ventilation and are at significant risk of developing a secondary, ventilator-associated pneumonia (VAP). OBJECTIVES: To study the incidence of VAP and bacterial lung microbiome composition of ventilated COVID-19 and non-COVID-19 patients. METHODS: In this retrospective observational study, we compared the incidence of VAP and secondary infections using a combination of microbial culture and a TaqMan multi-pathogen array. In addition, we determined the lung microbiome composition using 16S RNA analysis in a subset of samples. The study involved 81 COVID-19 and 144 non-COVID-19 patients receiving invasive ventilation in a single University teaching hospital between March 15th 2020 and August 30th 2020. RESULTS: COVID-19 patients were significantly more likely to develop VAP than patients without COVID (Cox proportional hazard ratio 2.01 95% CI 1.14-3.54, p = 0.0015) with an incidence density of 28/1000 ventilator days versus 13/1000 for patients without COVID (p = 0.009). Although the distribution of organisms causing VAP was similar between the two groups, and the pulmonary microbiome was similar, we identified 3 cases of invasive aspergillosis amongst the patients with COVID-19 but none in the non-COVID-19 cohort. Herpesvirade activation was also numerically more frequent amongst patients with COVID-19. CONCLUSION: COVID-19 is associated with an increased risk of VAP, which is not fully explained by the prolonged duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the causative organisms of secondary pneumonia observed are similar to that seen in critically ill patients ventilated for other reasons.

Interleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.

A metagenomic prospective cohort study on gut microbiome composition and clinical infection in small bowel transplantation.

Two-thirds of small-bowel transplantation (SBT) recipients develop bacteremia, with the majority of infections occurring within 3 months post-transplant. Sepsis-related mortality occurs in 31% of patients and is commonly caused by bacteria of gut origin, which are thought to translocate across the implanted organ. Serial post-transplant surveillance endoscopies provide an opportunity to study whether the composition of the ileal and colonic microbiota can predict the emergence as well as the pathogen of subsequent clinical infections in the SBT patient population. Five participants serially underwent aspiration of ileal and colonic bowel effluents at transplantation and during follow-up endoscopy either until death or for up to 3 months post-SBT. We performed whole-metagenome sequencing (WMS) of 40 bowel effluent samples and compared the results with clinical infection episodes. Microbiome composition was concordant between participants and timepoint-matched ileal and colonic samples. Four out of five (4/5) participants had clinically significant infections thought to be of gut origin. Bacterial translocation from the gut was observed in 3/5 patients with bacterial infectious etiologies. In all three cases, the pathogens had demonstrably colonized the gut between 1-10 days prior to invasive clinical infection. Recipients with better outcomes received donor grafts with higher alpha diversity. There was an increase in the number of antimicrobial resistance genes associated with longer hospital stay for all participants. This metagenomic study provides preliminary evidence to support the pathogen translocation hypothesis of gut-origin sepsis in the SBT cohort. Ileal and colonic microbiome compositions were concordant; therefore, fecal metagenomic analysis could be a useful surveillance tool for impeding infection with specific gut-residing pathogens.

Molecular mechanisms of re-emerging chloramphenicol susceptibility in extended-spectrum beta-lactamase-producing Enterobacterales.

Infections with Enterobacterales (E) are increasingly difficult to treat due to antimicrobial resistance. After ceftriaxone replaced chloramphenicol (CHL) as empiric therapy for suspected sepsis in Malawi in 2004, extended-spectrum beta-lactamase (ESBL)-E rapidly emerged. Concurrently, resistance to CHL in Escherichia coli and Klebsiella spp. decreased, raising the possibility of CHL re-introduction. However, many phenotypically susceptible isolates still carry CHL acetyltransferase (cat) genes. To understand the molecular mechanisms and stability of this re-emerging CHL susceptibility we use a combination of genomics, phenotypic susceptibility assays, experimental evolution, and functional assays for CAT activity. Here, we show that of 840 Malawian E. coli and Klebsiella spp. isolates, 31% have discordant CHL susceptibility genotype-phenotype, and we select a subset of 42 isolates for in-depth analysis. Stable degradation of cat genes by insertion sequences leads to re-emergence of CHL susceptibility. Our study suggests that CHL could be reintroduced as a reserve agent for critically ill patients with ESBL-E infections in Malawi and similar settings and highlights the ongoing challenges in inferring antimicrobial resistance from sequence data.

Exploiting human immune repertoire transgenic mice for protective monoclonal antibodies against antimicrobial resistant Acinetobacter baumannii.

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.

High-Content Imaging to Phenotype Antimicrobial Effects on Individual Bacteria at Scale.

High-content imaging (HCI) is a technique for screening multiple cells in high resolution to detect subtle morphological and phenotypic variation. The method has been commonly deployed on model eukaryotic cellular systems, often for screening new drugs and targets. HCI is not commonly utilized for studying bacterial populations but may be a powerful tool in understanding and combatting antimicrobial resistance. Consequently, we developed a high-throughput method for phenotyping bacteria under antimicrobial exposure at the scale of individual bacterial cells. Imaging conditions were optimized on an Opera Phenix confocal microscope (Perkin Elmer), and novel analysis pipelines were established for both Gram-negative bacilli and Gram-positive cocci. The potential of this approach was illustrated using isolates of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus HCI enabled the detection and assessment of subtle morphological characteristics, undetectable through conventional phenotypical methods, that could reproducibly distinguish between bacteria exposed to different classes of antimicrobials with distinct modes of action (MOAs). In addition, distinctive responses were observed between susceptible and resistant isolates. By phenotyping single bacterial cells, we observed intrapopulation differences, which may be critical in identifying persistence or emerging resistance during antimicrobial treatment. The work presented here outlines a comprehensive method for investigating morphological changes at scale in bacterial populations under specific perturbation.IMPORTANCE High-content imaging (HCI) is a microscopy technique that permits the screening of multiple cells simultaneously in high resolution to detect subtle morphological and phenotypic variation. The power of this methodology is that it can generate large data sets comprised of multiple parameters taken from individual cells subjected to a range of different conditions. We aimed to develop novel methods for using HCI to study bacterial cells exposed to a range of different antibiotic classes. Using an Opera Phenix confocal microscope (Perkin Elmer) and novel analysis pipelines, we created a method to study the morphological characteristics of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus when exposed to antibacterial drugs with differing modes of action. By imaging individual bacterial cells at high resolution and scale, we observed intrapopulation differences associated with different antibiotics. The outlined methods are highly relevant for how we begin to better understand and combat antimicrobial resistance.

Inhibitory Concentrations of Ciprofloxacin Induce an Adaptive Response Promoting the Intracellular Survival of Salmonella enterica Serovar Typhimurium.

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fueled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of Salmonella enterica serovar Typhimurium to differing concentrations of ciprofloxacin, dependent on their MICs, and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium is able to recover from inhibitory concentrations of ciprofloxacin and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium has increased capacity for intracellular replication in comparison to that of untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. IMPORTANCE Antimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella enterica serovar Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin and that for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

Development and implementation of a customised rapid syndromic diagnostic test for severe pneumonia.

Background: The diagnosis of pneumonia has been hampered by a reliance on bacterial cultures which take several days to return a result, and are frequently negative. In critically ill patients this leads to the use of empiric, broad-spectrum antimicrobials and compromises good antimicrobial stewardship. The objective of this study was to establish the performance of a syndromic molecular diagnostic approach, using a custom TaqMan array card (TAC) covering 52 respiratory pathogens, and assess its impact on antimicrobial prescribing. Methods: The TAC was validated against a retrospective multi-centre cohort of broncho-alveolar lavage samples. The TAC was assessed prospectively in patients undergoing investigation for suspected pneumonia, with a comparator cohort formed of patients investigated when the TAC laboratory team were unavailable. Co-primary outcomes were sensitivity compared to conventional microbiology and, for the prospective study, time to result. Metagenomic sequencing was performed to validate findings in prospective samples. Antibiotic free days (AFD) were compared between the study cohort and comparator group. Results: 128 stored samples were tested, with sensitivity of 97% (95% confidence interval (CI) 88-100%). Prospectively, 95 patients were tested by TAC, with 71 forming the comparator group. TAC returned results 51 hours (interquartile range 41-69 hours) faster than culture and with sensitivity of 92% (95% CI 83-98%) compared to conventional microbiology. 94% of organisms identified by sequencing were detected by TAC. There was a significant difference in the distribution of AFDs with more AFDs in the TAC group (p=0.02). TAC group were more likely to experience antimicrobial de-escalation (odds ratio 2.9 (95%1.5-5.5)). Conclusions: Implementation of a syndromic molecular diagnostic approach to pneumonia led to faster results, with high sensitivity and impact on antibiotic prescribing.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

BACKGROUND: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden. METHODS: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples. FINDINGS: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13). INTERPRETATION: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. FUNDING: UK Medical Research Council.

Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections.

SARS-CoV-2 is notable both for its rapid spread, and for the heterogeneity of its patterns of transmission, with multiple published incidences of superspreading behaviour. Here, we applied a novel network reconstruction algorithm to infer patterns of viral transmission occurring between patients and health care workers (HCWs) in the largest clusters of COVID-19 infection identified during the first wave of the epidemic at Cambridge University Hospitals NHS Foundation Trust, UK. Based upon dates of individuals reporting symptoms, recorded individual locations, and viral genome sequence data, we show an uneven pattern of transmission between individuals, with patients being much more likely to be infected by other patients than by HCWs. Further, the data were consistent with a pattern of superspreading, whereby 21% of individuals caused 80% of transmission events. Our study provides a detailed retrospective analysis of nosocomial SARS-CoV-2 transmission, and sheds light on the need for intensive and pervasive infection control procedures.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, presents a global public health challenge. Hospitals have been at the forefront of this battle, treating large numbers of sick patients over several waves of infection. Finding ways to manage the spread of the virus in hospitals is key to protecting vulnerable patients and workers, while keeping hospitals running, but to generate effective infection control, researchers must understand how SARS-CoV-2 spreads. A range of factors make studying the transmission of SARS-CoV-2 in hospitals tricky. For instance, some people do not present any symptoms, and, amongst those who do, it can be difficult to determine whether they caught the virus in the hospital or somewhere else. However, comparing the genetic information of the SARS-CoV-2 virus from different people in a hospital could allow scientists to understand how it spreads. Samples of the genetic material of SARS-CoV-2 can be obtained by swabbing infected individuals. If the genetic sequences of two samples are very different, it is unlikely that the individuals who provided the samples transmitted the virus to one another. Illingworth, Hamilton et al. used this information, along with other data about how SARS-CoV-2 is transmitted, to develop an algorithm that can determine how the virus spreads from person to person in different hospital wards. To build their algorithm, Illingworth, Hamilton et al. collected SARS-CoV-2 genetic data from patients and staff in a hospital, and combined it with information about how SARS-CoV-2 spreads and how these people moved in the hospital . The algorithm showed that, for the most part, patients were infected by other patients (20 out of 22 cases), while staff were infected equally by patients and staff. By further probing these data, Illingworth, Hamilton et al. revealed that 80% of hospital-acquired infections were caused by a group of just 21% of individuals in the study, identifying a 'superspreader' pattern. These findings may help to inform SARS-CoV-2 infection control measures to reduce spread within hospitals, and could potentially be used to improve infection control in other contexts.

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.

A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities.

The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the method for a quantitative real-time reverse transcriptase PCR detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.

Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study.

BACKGROUND: The burden and influence of health-care associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unknown. We aimed to examine the use of rapid SARS-CoV-2 sequencing combined with detailed epidemiological analysis to investigate health-care associated SARS-CoV-2 infections and inform infection control measures. METHODS: In this prospective surveillance study, we set up rapid SARS-CoV-2 nanopore sequencing from PCR-positive diagnostic samples collected from our hospital (Cambridge, UK) and a random selection from hospitals in the East of England, enabling sample-to-sequence in less than 24 h. We established a weekly review and reporting system with integration of genomic and epidemiological data to investigate suspected health-care associated COVID-19 cases. FINDINGS: Between March 13 and April 24, 2020, we collected clinical data and samples from 5613 patients with COVID-19 from across the East of England. We sequenced 1000 samples producing 747 high-quality genomes. We combined epidemiological and genomic analysis of the 299 patients from our hospital and identified 35 clusters of identical viruses involving 159 patients. 92 (58%) of 159 patients had strong epidemiological links and 32 (20%) patients had plausible epidemiological links. These results were fed back to clinical, infection control, and hospital management teams, leading to infection-control interventions and informing patient safety reporting. INTERPRETATION: We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and showed the benefit of combined genomic and epidemiological analysis for the investigation of health-care associated COVID-19. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection-control interventions to further reduce health-care associated infections. Our findings have important implications for national public health policy as they enable rapid tracking and investigation of infections in hospital and community settings. FUNDING: COVID-19 Genomics UK funded by the Department of Health and Social Care, UK Research and Innovation, and the Wellcome Sanger Institute.

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission.

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff.

Patients admitted to NHS hospitals are now routinely screened for SARS-CoV-2 (the virus that causes COVID-19), and isolated from other patients if necessary. Yet healthcare workers, including frontline patient-facing staff such as doctors, nurses and physiotherapists, are only tested and excluded from work if they develop symptoms of the illness. However, there is emerging evidence that many people infected with SARS-CoV-2 never develop significant symptoms: these people will therefore be missed by 'symptomatic-only' testing. There is also important data showing that around half of all transmissions of SARS-CoV-2 happen before the infected individual even develops symptoms. This means that much broader testing programs are required to spot people when they are most infectious. Rivett, Sridhar, Sparkes, Routledge et al. set out to determine what proportion of healthcare workers was infected with SARS-CoV-2 while also feeling generally healthy at the time of testing. Over 1,000 staff members at a large UK hospital who felt they were well enough to work, and did not fit the government criteria for COVID-19 infection, were tested. Amongst these, 3% were positive for SARS-CoV-2. On closer questioning, around one in five reported no symptoms, two in five very mild symptoms that they had dismissed as inconsequential, and a further two in five reported COVID-19 symptoms that had stopped more than a week previously. In parallel, healthcare workers with symptoms of COVID-19 (and their household contacts) who were self-isolating were also tested, in order to allow those without the virus to quickly return to work and bolster a stretched workforce. Finally, the rates of infection were examined to probe how the virus could have spread through the hospital and among staff ­ and in particular, to understand whether rates of infection were greater among staff working in areas devoted to COVID-19 patients. Despite wearing appropriate personal protective equipment, healthcare workers in these areas were almost three times more likely to test positive than those working in areas without COVID-19 patients. However, it is not clear whether this genuinely reflects greater rates of patients passing the infection to staff. Staff may give the virus to each other, or even acquire it at home. Overall, this work implies that hospitals need to be vigilant and introduce broad screening programmes across their workforces. It will be vital to establish such approaches before 'lockdown' is fully lifted, so healthcare institutions are prepared for any second peak of infections.

Effective control of SARS-CoV-2 transmission between healthcare workers during a period of diminished community prevalence of COVID-19.

Previously, we showed that 3% (31/1032)of asymptomatic healthcare workers (HCWs) from a large teaching hospital in Cambridge, UK, tested positive for SARS-CoV-2 in April 2020. About 15% (26/169) HCWs with symptoms of coronavirus disease 2019 (COVID-19) also tested positive for SARS-CoV-2 (Rivett et al., 2020). Here, we show that the proportion of both asymptomatic and symptomatic HCWs testing positive for SARS-CoV-2 rapidly declined to near-zero between 25th April and 24th May 2020, corresponding to a decline in patient admissions with COVID-19 during the ongoing UK 'lockdown'. These data demonstrate how infection prevention and control measures including staff testing may help prevent hospitals from becoming independent 'hubs' of SARS-CoV-2 transmission, and illustrate how, with appropriate precautions, organizations in other sectors may be able to resume on-site work safely.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens.

The intestinal 'organoid' (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection. We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 °C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.