Airway-resident T cells from unexposed individuals cross-recognize SARS-CoV-2.

T cells can contribute to clearance of respiratory viruses that cause acute-resolving infections such as SARS-CoV-2, helping to provide long-lived protection against disease. Recent studies have suggested an additional role for T cells in resisting overt infection: pre-existing cross-reactive responses were preferentially enriched in healthcare workers who had abortive infections1, and in household contacts protected from infection2. We hypothesize that such early viral control would require pre-existing cross-reactive memory T cells already resident at the site of infection; such airway-resident responses have been shown to be critical for mediating protection after intranasal vaccination in a murine model of SARS-CoV3. Bronchoalveolar lavage samples from the lower respiratory tract of healthy donors obtained before the COVID-19 pandemic revealed airway-resident, SARS-CoV-2-cross-reactive T cells, which correlated with the strength of human seasonal coronavirus immunity. We therefore demonstrate the potential to harness functional airway-resident SARS-CoV-2-reactive T cells in next-generation mucosal vaccines.

Inflammation induced by influenza virus impairs human innate immune control of pneumococcus.

Colonization of the upper respiratory tract by pneumococcus is important both as a determinant of disease and for transmission into the population. The immunological mechanisms that contain pneumococcus during colonization are well studied in mice but remain unclear in humans. Loss of this control of pneumococcus following infection with influenza virus is associated with secondary bacterial pneumonia. We used a human challenge model with type 6B pneumococcus to show that acquisition of pneumococcus induced early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function was associated with the clearance of pneumococcus. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate immune function and altered genome-wide nasal gene responses to the carriage of pneumococcus. Levels of the cytokine CXCL10, promoted by viral infection, at the time pneumococcus was encountered were positively associated with bacterial load.

A Randomized Controlled Clinical Trial of Nasal Immunization with Live Virulence Attenuated Streptococcus pneumoniae Strains Using Human Infection Challenge.

Rationale: Pneumococcal pneumonia remains a global health problem. Pneumococcal colonization increases local and systemic protective immunity, suggesting that nasal administration of live attenuated Streptococcus pneumoniae (Spn) strains could help prevent infections. Objectives: We used a controlled human infection model to investigate whether nasopharyngeal colonization with attenuated S. pneumoniae strains protected against recolonization with wild-type (WT) Spn (SpnWT). Methods: Healthy adults aged 18-50 years were randomized (1:1:1:1) for nasal administration twice (at a 2-wk interval) with saline solution, WT Spn6B (BHN418), or one of two genetically modified Spn6B strains, SpnA1 (Δfhs/piaA) or SpnA3 (ΔproABC/piaA) (Stage I). After 6 months, participants were challenged with SpnWT to assess protection against the homologous serotype (Stage II). Measurements and Main Results: 125 participants completed both study stages per intention to treat. No serious adverse events were reported. In Stage I, colonization rates were similar among groups: SpnWT, 58.1% (18 of 31); SpnA1, 60% (18 of 30); and SpnA3, 59.4% (19 of 32). Anti-Spn nasal IgG levels after colonization were similar in all groups, whereas serum IgG responses were higher in the SpnWT and SpnA1 groups than in the SpnA3 group. In colonized individuals, increases in IgG responses were identified against 197 Spn protein antigens and serotype 6 capsular polysaccharide using a pangenome array. Participants given SpnWT or SpnA1 in Stage I were partially protected against homologous challenge with SpnWT (29% and 30% recolonization rates, respectively) at stage II, whereas those exposed to SpnA3 achieved a recolonization rate similar to that in the control group (50% vs. 47%, respectively). Conclusions: Nasal colonization with genetically modified live attenuated Spn was safe and induced protection against recolonization, suggesting that nasal administration of live attenuated Spn could be an effective strategy for preventing pneumococcal infections. Clinical trial registered with the ISRCTN registry (ISRCTN22467293).

Human Infection Challenge with Serotype 3 Pneumococcus.

Rationale: Streptococcus pneumoniae serotype 3 (SPN3) is a cause of invasive pneumococcal disease and associated with low carriage rates. Following the introduction of pediatric 13-valent pneumococcal conjugate vaccine (PCV13) programs, SPN3 declines are less than other vaccine serotypes and incidence has increased in some populations coincident with a shift in predominant circulating SPN3 clade, from I to II. A human challenge model provides an effective means for assessing the impact of PCV13 on SPN3 in the upper airway. Objectives: To establish SPN3's ability to colonize the nasopharynx using different inoculum clades and doses, and the safety of an SPN3 challenge model. Methods: In a human challenge study involving three well-characterized and antibiotic-sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade], and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, and 160,000 cfu/100 µl) were escalated until maximal colonization rates were achieved, with concurrent acceptable safety. Measurement and Main Results: Presence and density of experimental SPN3 nasopharyngeal colonization in nasal wash samples, assessed using microbiological culture and molecular methods, on Days 2, 7, and 14 postinoculation. A total of 96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n = 6-10 per dose group, 10 groups). Colonization rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% [24/29] developed a sore throat); one developed otitis media requiring antibiotics. No serious adverse events occurred. Conclusions: An SPN3 human challenge model is feasible and safe with comparable carriage rates to an established Serotype 6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms.

Thirteen-Valent Pneumococcal Conjugate Vaccine-Induced Immunoglobulin G (IgG) Responses in Serum Associated With Serotype-Specific IgG in the Lung.

Pneumococcal conjugate vaccine (PCV) efficacy is lower for noninvasive pneumonia than invasive disease. In this study, participants were immunized with 13-valent PCV (PCV13) or hepatitis A vaccine (control). Bronchoalveolar lavage samples were taken between 2 and 6 months and serum at 4 and 7 weeks postvaccination. In the lung, anti-capsular immunoglobulin G (IgG) levels were higher in the PCV13 group compared to controls for all serotypes, except 3 and 6B. Systemically, IgG levels were elevated in the PCV13 group at 4 weeks for all serotypes, except serotype 3. IgG in bronchoalveolar lavage and serum positively correlated for nearly all serotypes. PCV13 shows poor immunogenicity to serotype 3, implying lack of protective efficacy. Clinical Trials Registration. ISRCTN 45340436.

Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

Experimental Human Pneumococcal Colonization in Older Adults Is Feasible and Safe, Not Immunogenic.

Rationale: Pneumococcal colonization is key to the pathogenesis of invasive disease but is also immunogenic in young adults, protecting against recolonization. Colonization is rarely detected in older adults, despite high rates of pneumococcal disease.Objectives: To establish experimental human pneumococcal colonization in healthy adults aged 50-84 years, to measure the immune response to pneumococcal challenge, and to assess the protective effect of prior colonization against autologous strain rechallenge.Methods: Sixty-four participants were inoculated with Streptococcus pneumoniae (serotype 6B; 80,000 cfu in each nostril). Colonization was determined by bacterial culture of nasal wash, and humoral immune responses were assessed by anticapsular and antiprotein IgG concentrations.Measurements and Main Results: Experimental colonization was established in 39% of participants (25/64) with no adverse events. Colonization occurred in 47% (9/19) of participants aged 50-59 compared with 21% (3/14) in those aged ≥70 years. Previous pneumococcal polysaccharide vaccination did not protect against colonization. Colonization did not confer serotype-specific immune boosting, with a geometric mean titer (95% confidence interval) of 2.7 µg/ml (1.9-3.8) before the challenge versus 3.0 (1.9-4.7) 4 weeks after colonization (P = 0.53). Furthermore, pneumococcal challenge without colonization led to a drop in specific antibody concentrations from 2.8 µg/ml (2.0-3.9) to 2.2 µg/ml (1.6-3.0) after the challenge (P = 0.006). Antiprotein antibody concentrations increased after successful colonization. Rechallenge with the same strain after a median of 8.5 months (interquartile range, 6.7-10.1) led to recolonization in 5/16 (31%).Conclusions: In older adults, experimental pneumococcal colonization is feasible and safe but demonstrates different immunological outcomes compared with younger adults in previous studies.

Nasal Pneumococcal Density Is Associated with Microaspiration and Heightened Human Alveolar Macrophage Responsiveness to Bacterial Pathogens.

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited.Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells.Methods: We collected BAL from healthy pneumococcal-challenged participants aged 18-49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations.Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density (r = 0.71; P = 0.029). Similarly, AM-heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r = 0.61, P = 0.025).Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.

Saliva Alternative to Upper Respiratory Swabs for SARS-CoV-2 Diagnosis.

PCR of upper respiratory specimens is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 infection. However, saliva sampling is an easy alternative to nasal and throat swabbing. We found similar viral loads in saliva samples and in nasal and throat swab samples from 110 patients with coronavirus disease.

Intrapulmonary Pharmacokinetics of Cefepime and Enmetazobactam in Healthy Volunteers: Towards New Treatments for Nosocomial Pneumonia.

Cefepime-enmetazobactam is a novel ß-lactam-ß-lactamase inhibitor combination with broad-spectrum antimicrobial activity against a range of multidrug-resistant Enterobacteriaceae This agent is being developed for a range of serious hospital infections. An understanding of the extent of partitioning of ß-lactam-ß-lactamase inhibitor combinations into the human lung is required to better understand the potential role of cefepime-enmetazobactam for the treatment of nosocomial pneumonia. A total of 20 healthy volunteers were used to study the intrapulmonary pharmacokinetics of a regimen of 2 g cefepime-1 g enmetazobactam every 8 h intravenously (2 g/1 g q8h i.v.). Each volunteer contributed multiple plasma samples and a single epithelial lining fluid (ELF) sample, obtained by bronchoalveolar lavage. Concentrations of cefepime and enmetazobactam were quantified using liquid chromatography-tandem mass spectrometry. The pharmacokinetic data were modeled using a population methodology, and Monte Carlo simulations were performed to assess the attainment of pharmacodynamic targets defined in preclinical models. The concentration-time profiles of both agents in plasma and ELF were similar. The mean ± standard deviation percentage of partitioning of total drug concentrations of cefepime and enmetazobactam between plasma and ELF was 60.59% ± 28.62% and 53.03% ± 21.05%, respectively. Using pharmacodynamic targets for cefepime of greater than the MIC and free enmetazobactam concentrations of >2 mg/liter in ELF of 20% of the dosing interval, a regimen of cefepime-enmetazobactam of 2 g/0.5 g q8h i.v. infused over 2 h resulted in a probability of target attainment of ≥90% for Enterobacteriaceae with cefepime-enmetazobactam MICs of ≤8 mg/liter. This result provides a rationale to further consider cefepime-enmetazobactam for the treatment of nosocomial pneumonia caused by multidrug-resistant Enterobacteriaceae.

Pneumococcal Colonization in Healthy Adult Research Participants in the Conjugate Vaccine Era, United Kingdom, 2010-2017.

Pneumococcal colonization is rarely studied in adults, except as part of family surveys. We report the outcomes of colonization screening in healthy adults (all were nonsmokers without major comorbidities or contact with children aged <5 years) who had volunteered to take part in clinical research. Using nasal wash culture, we detected colonization in 6.5% of volunteers (52 of 795). Serotype 3 was the commonest serotype (10 of 52 isolates). The majority of the remaining serotypes (35 of 52 isolates) were nonvaccine serotypes, but we also identified persistent circulation of serotypes 19A and 19F. Resistance to at least 1 of 6 antibiotics tested was found in 8 of 52 isolates.

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state.

Alveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differential expression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). We investigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographical locations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously, with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2 features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybrid phenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functions allowing for appropriate responses to stimuli and tissue environment.

Polysaccharide-Specific Memory B Cells Predict Protection against Experimental Human Pneumococcal Carriage.

RATIONALE: We have previously demonstrated that experimental pneumococcal carriage enhances immunity and protects healthy adults against carriage reacquisition after rechallenge with a homologous strain. OBJECTIVES: To investigate the role of naturally acquired pneumococcal protein and polysaccharide (PS)-specific immunity in protection against carriage acquisition using a heterologous challenge model. METHODS: We identified healthy volunteers that were naturally colonized with pneumococcus and, after clearance of their natural carriage episode, challenged them with a heterologous 6B strain. In another cohort of volunteers we assessed 6BPS-specific, PspA-specific, and PspC-specific IgG and IgA plasma and memory B-cell populations before and 7, 14, and 35 days after experimental pneumococcal inoculation. MEASUREMENTS AND MAIN RESULTS: Heterologous challenge with 6B resulted in 50% carriage among volunteers with previous natural pneumococcal carriage. Protection from carriage was associated with a high number of circulating 6BPS IgG-secreting memory B cells at baseline. There were no associations between protection from carriage and baseline levels of 6BPS IgG in serum or nasal wash, PspA-specific, or PspC-specific memory B cells or plasma cells. In volunteers who did not develop carriage, the number of circulating 6BPS memory B cells decreased and the number of 6BPS plasma cells increased postinoculation. CONCLUSIONS: Our data indicate that naturally acquired PS-specific memory B cells, but not levels of circulating IgG at time of pneumococcal exposure, are associated with protection against carriage acquisition.

Single use and conventional bronchoscopes for Broncho alveolar lavage (BAL) in research: a comparative study (NCT 02515591).

BACKGROUND: Broncho alveolar lavage (BAL) is widely used for investigative research to study innate, cellular and humoral immune responses, and in early phase drug trials. Conventional (multiple use) flexible bronchoscopes have time and monetary costs associated with cleaning, and carries a small risk of cross infection. Single use bronchoscopes may provide an alternative, but have not been evaluated in this context. METHODS: Healthy volunteers underwent bronchoscopy at a day-case clinical research unit using the Ambu® aScopeTM single-use flexible intubation bronchoscope. Broncho alveolar lavage was performed from a sub segmental bronchus within the right middle lobe; a total of 200 ml of warmed normal saline was instilled then aspirated using handheld suction. BAL volume yield, cell yield and viability were recorded. RESULTS: Ten volunteers, (mean age 23 years, six male) participated. Bronchoscopies were carried out by one of two senior bronchoscopists, experienced in the technique of obtaining BAL for research purposes. The results were compared to 50 (mean age 23, 14 male) procedures performed using the conventional scope by the same two bronchoscopists. The total volume yield was significantly higher in the disposable group median 152 ml (IQR 141-166 ml) as compared to conventional 124 ml (110-135 ml), p = <0.01. The total cell yield and viability were similar in both groups, with no significant differences. CONCLUSIONS: With single use bronchoscopes, we achieved a larger BAL volume yield than conventional bronchoscopes, with comparable cell yield and viability. Better volume yields can potentially reduce post procedure side effects such as pleuritic chest pain and cough. The risk of cross infection can be eliminated, providing reassurance to researchers and participants. Reduced maintenance requirements can be cost effective. These could potentially be used for early phase drug development studies. TRIAL REGISTRATION: This trial was registered prospectively in July 2015 with the National Clinical Trials register, with the following registration number assigned: NCT 02515591 .

First human challenge testing of a pneumococcal vaccine. Double-blind randomized controlled trial.

RATIONALE: New vaccines are urgently needed to protect the vulnerable from bacterial pneumonia. Clinical trials of pneumonia vaccines are slow and costly, requiring tens of thousands of patients. Studies of pneumococcal vaccine efficacy against colonization have been proposed as a novel method to down-select between vaccine candidates. OBJECTIVES: Using our safe and reproducible experimental human pneumococcal colonization model, we aimed to determine the effect of 13-valent pneumococcal conjugate vaccine (PCV) on colonization. METHODS: A total of 100 healthy participants aged 18-50 years were recruited into this double-blind randomized placebo-controlled trial. They were randomly assigned to PCV (n = 49) or hepatitis A (control, n = 50) vaccination and inoculated with 80,000 CFU/100 µl of Streptococcus pneumoniae (6B) per naris. MEASUREMENTS AND MAIN RESULTS: Participants were followed up for 21 days to determine pneumococcal colonization by culture of nasal wash. The PCV group had a significantly reduced rate of 6B colonization (10% [5 of 48]) compared with control subjects (48% [23 of 48]) (risk ratio, 0.22; confidence interval, 0.09-0.52; P < 0.001). Density of colonization was reduced in the PCV group compared with the control group following inoculation. The area under the curve (density vs. day) was significantly reduced in the PCV compared with control group (geometric mean, 259 vs. 11,183; P = 0.017). CONCLUSIONS: PCV reduced pneumococcal colonization rate, density, and duration in healthy adults. The experimental human pneumococcal colonization model is a safe, cost-effective, and efficient method to determine the protective efficacy of new vaccines on pneumococcal colonization; PCV provides a gold standard against which to test these novel vaccines. Clinical trial registered with ISRCTN: 45340436.

Serotype 3 Experimental Human Pneumococcal Challenge (EHPC) study protocol: dose ranging and reproducibility in a healthy volunteer population (challenge 3).

INTRODUCTION: Since the introduction of pneumococcal conjugate vaccines, pneumococcal disease rates have declined for many vaccine-type serotypes. However, serotype 3 (SPN3) continues to cause significant disease and is identified in colonisation epidemiological studies as one of the top circulating serotypes in adults in the UK. Consequently, new vaccines that provide greater protection against SPN3 colonisation/carriage are urgently needed. The Experimental Human Pneumococcal Challenge (EHPC) model is a unique method of determining pneumococcal colonisation rates, understanding acquired immunity, and testing vaccines in a cost-effective manner. To enhance the development of effective pneumococcal vaccines against SPN3, we aim to develop a new relevant and safe SPN3 EHPC model with high attack rates which could be used to test vaccines using small sample size. METHODS AND ANALYSIS: This is a human challenge study to establish a new SPN3 EHPC model, consisting of two parts. In the dose-ranging/safety study, cohorts of 10 healthy participants will be challenged with escalating doses of SPN3. If first challenge does not lead into colonisation, participants will receive a second challenge 2 weeks after. Experimental nasopharyngeal (NP) colonisation will be determined using nasal wash sampling. Using the dose that results in ≥50% of participants being colonised, with a high safety profile, we will complete the cohort with another 33 participants to check for reproducibility of the colonisation rate. The primary outcome of this study is to determine the optimal SPN3 dose and inoculation regime to establish the highest rates of NP colonisation in healthy adults. Secondary outcomes include determining density and duration of experimental SPN3 NP colonisation and characterising mucosal and systemic immune responses to SPN3 challenge. ETHICS AND DISSEMINATION: This study is approved by the NHS Research and Ethics Committee (reference 22/NW/0051). Findings will be published in peer-reviewed journals and reports will be made available to participants.

Inflammation of the Nasal Mucosa is Associated with Susceptibility to Experimental Pneumococcal Challenge in Older Adults.

Streptococcus pneumoniae colonization in the upper respiratory tract is linked to pneumococcal disease development, predominantly affecting young children and older adults. As the global population ages and comorbidities increase, there is a heightened concern about this infection. We investigated the immunological responses of older adults to pneumococcal controlled human infection by analysing the cellular composition and gene expression in the nasal mucosa. Our comparative analysis with data from a concurrent study in younger adults revealed distinct gene expression patterns in older individuals susceptible to colonization, highlighted by neutrophil activation and elevated levels of CXCL9 and CXCL10. Unlike younger adults challenged with pneumococcus, older adults did not show recruitment of monocytes into the nasal mucosa following nasal colonization. However, older adults who were protected from colonization showed increased degranulation of CD8+ T cells, both before and after pneumococcal challenge. These findings suggest age-associated cellular changes, in particular enhanced mucosal inflammation, that may predispose older adults to pneumococcal colonization.

Respiratory mucosal immune memory to SARS-CoV-2 after infection and vaccination.

Respiratory mucosal immunity induced by vaccination is vital for protection from coronavirus infection in animal models. In humans, the capacity of peripheral vaccination to generate sustained immunity in the lung mucosa, and how this is influenced by prior SARS-CoV-2 infection, is unknown. Here we show using bronchoalveolar lavage samples that donors with history of both infection and vaccination have more airway mucosal SARS-CoV-2 antibodies and memory B cells than those only vaccinated. Infection also induces populations of airway spike-specific memory CD4+ and CD8+ T cells that are not expanded by vaccination alone. Airway mucosal T cells induced by infection have a distinct hierarchy of antigen specificity compared to the periphery. Spike-specific T cells persist in the lung mucosa for 7 months after the last immunising event. Thus, peripheral vaccination alone does not appear to induce durable lung mucosal immunity against SARS-CoV-2, supporting an argument for the need for vaccines targeting the airways.

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: An observational cohort study.

BACKGROUND: The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the United Kingdom National Health Service (NHS). We conducted an observational cohort study of SARS-CoV-2 infection in frontline healthcare workers (HCW) working in an acute NHS Trust during the first wave of the pandemic, to answer emerging questions surrounding SARS-CoV-2 infection, diagnosis, transmission and control. METHODS: Using self-collected weekly saliva and twice weekly combined oropharyngeal/nasopharyngeal (OP/NP) samples, in addition to self-assessed symptom profiles and isolation behaviours, we retrospectively compared SARS-CoV-2 detection by RT-qPCR of saliva and OP/NP samples. We report the association with contemporaneous symptoms and isolation behaviour. RESULTS: Over a 12-week period from 30th March 2020, 40·0% (n = 34/85, 95% confidence interval 31·3-51·8%) HCW had evidence of SARS-CoV-2 infection by surveillance OP/NP swab and/or saliva sample. Symptoms were reported by 47·1% (n = 40) and self-isolation by 25·9% (n = 22) participants. Only 44.1% (n = 15/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of a positive result and only 29·4% (n = 10/34) reported self-isolation periods. Overall agreement between paired saliva and OP/NP swabs was 93·4% (n = 211/226 pairs) but rates of positive concordance were low. In paired samples with at least one positive result, 35·0% (n = 7/20) were positive exclusively by OP/NP swab, 40·0% (n = 8/20) exclusively by saliva and in only 25·0% (n = 5/20) were the OP/NP and saliva result both positive. CONCLUSIONS: HCW are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections. Without routine asymptomatic SARS-CoV-2 screening, it is likely that HCW with SARS-CoV-2 infection would continue to attend work. Saliva, in addition to OP/NP swab testing, facilitated ascertainment of symptomatic and asymptomatic SARS-CoV-2 infections. Combined saliva and OP/NP swab sampling would improve detection of SARS-CoV-2 for surveillance and is recommended for a high sensitivity strategy.