Myeloperoxidase inhibition may protect against endothelial glycocalyx shedding induced by COVID-19 plasma.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19, is a threat to public health. Evidence suggests increased neutrophil activation and endothelial glycocalyx (EG) damage are independently associated with severe COVID-19. Here, we hypothesised that an increased level of blood neutrophil myeloperoxidase (MPO) is associated with soluble EG breakdown, and inhibiting MPO activity may reduce EG damage. METHODS: Analysing a subset of acute and convalescent COVID-19 plasma, 10 from severe and 15 from non-severe COVID-19 cases, and 9 from pre-COVID-19 controls, we determined MPO levels, MPO activity and soluble EG proteins (syndecan-1 and glypican-1) levels by enzyme-linked immunosorbent assay. In vitro primary human aortic endothelial cells were cultured with plasma untreated or treated with specific MPO inhibitors (MPO-IN-28, AZD5904) to determine EG shedding. We then investigated whether inhibiting MPO activity decreased EG degradation. RESULTS: In COVID-19 plasma, MPO levels, MPO activity and levels of soluble EG proteins are significantly raised compared to controls, and concentrations increase in proportion to disease severity. Despite clinical recovery, protein concentrations remain significantly elevated. Interestingly, there is a trend of increasing MPO activity in convalescent plasma in both severe and non-severe groups. MPO levels and MPO activity correlate significantly with soluble EG levels and inhibiting MPO activity leads to reduced syndecan-1 shedding, in vitro. CONCLUSIONS: Neutrophil MPO may increase EG shedding in COVID-19, and inhibiting MPO activity may protect against EG degradation. Further research is needed to evaluate the utility of MPO inhibitors as potential therapeutics against severe COVID-19.

COVID-19 can result in severe disease and is potentially fatal. Neutrophils, the most abundant white blood cells in circulation, secrete antimicrobials that have been linked to severe COVID-19 development. The endothelial glycocalyx (EG) is a carbohydrate rich layer that coats the inner surface of the vasculature and damage to the EG is observed in severe COVID-19. Here, we investigate whether myeloperoxidase, an antimicrobial released by neutrophils, is associated with EG damage in COVID-19 patients. We also determine whether reducing myeloperoxidase activity prevents damage to the EG. Our results suggest myeloperoxidase is associated with EG damage and severe COVID-19. We also demonstrated that a reduction in myeloperoxidase activity may protect against EG degradation. Further studies to evaluate the utility of MPO inhibitors as a therapy against severe COVID-19 are warranted.

SARS-CoV-2 Omicron variant shedding during respiratory activities.

OBJECTIVES: As the world transitions to COVID-19 endemicity, studies focusing on aerosol shedding of highly transmissible SARS-CoV-2 variants of concern (VOCs) are vital for the calibration of infection control measures against VOCs that are likely to circulate seasonally. This follow-up Gesundheit-II aerosol sampling study aims to compare the aerosol shedding patterns of Omicron VOC samples with pre-Omicron variants analyzed in our previous study. DESIGN: Coarse and fine aerosol samples from 47 patients infected with SARS-CoV-2 were collected during various respiratory activities (passive breathing, talking, and singing) and analyzed using reverse transcription-quantitative polymerase chain reaction and virus culture. RESULTS: Compared with patients infected with pre-Omicron variants, comparable SARS-CoV-2 RNA copy numbers were detectable in aerosol samples of patients infected with Omicron despite being fully vaccinated. Patients infected with Omicron also showed a slight increase in viral aerosol shedding during breathing activities and were more likely to have persistent aerosol shedding beyond 7 days after disease onset. CONCLUSION: This follow-up study reaffirms the aerosol shedding properties of Omicron and should guide continued layering of public health interventions even in highly vaccinated populations.

Transmission modes of severe acute respiratory syndrome coronavirus 2 and implications for infection control: a review.

The complete picture regarding transmission modes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. This review summarises the available evidence on its transmission modes, our preliminary research findings and implications for infection control policy, and outlines future research directions. Environmental contamination has been reported in hospital settings occupied by infected patients, and is higher in the first week of illness. Transmission via environmental surfaces or fomites is likely, but decontamination protocols are effective in minimising this risk. The extent of airborne transmission is also unclear. While several studies have detected SARS-CoV-2 ribonucleic acid in air samples, none has isolated viable virus in culture. Transmission likely lies on a spectrum between droplet and airborne transmission, depending on the patient, disease and environmental factors. Singapore's current personal protective equipment and isolation protocols are sufficient to manage this risk.

Validation of ISARIC 4C Mortality and Deterioration Scores in a Mixed Vaccination Status Cohort of Hospitalized Coronavirus Disease 2019 (COVID-19) Patients in Singapore.

In this cross-sectional study, we studied performance of the International Severe Acute Respiratory and Emerging Infections Consortium mortality and deterioration scores in a cohort of 410 hospitalized patients (51.2% fully vaccinated). area under the receiver operating characteristic curves were 0.778 and 0.764, respectively, comparable to originally published validation cohorts. Subgroup analysis showed equally good performance in vaccinated and partially or unvaccinated patients.

Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Respiratory Aerosols Emitted by Patients With Coronavirus Disease 2019 (COVID-19) While Breathing, Talking, and Singing.

BACKGROUND: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 µm) and fine (≤5 µm) respiratory aerosols produced when breathing, talking, and singing. METHODS: Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing. RESULTS: Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63-5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative. CONCLUSIONS: Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies.

Clinical and Virological Features of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants of Concern: A Retrospective Cohort Study Comparing B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta).

BACKGROUND: The impact of SARS-CoV-2 variants of concern (VOCs) on disease severity is unclear. In this retrospective study, we compared the outcomes of patients infected with B.1.1.7, B.1.351, and B.1.617.2 with wild-type strains from early 2020. METHODS: National surveillance data from January to May 2021 were obtained and outcomes in relation to VOCs were explored. Detailed patient-level data from all patients with VOC infection admitted to our center between December 2020 and May 2021 were analyzed. Clinical outcomes were compared with a cohort of 846 patients admitted from January to April 2020. RESULTS: A total of 829 patients in Singapore in the study period were infected with these 3 VOCs. After adjusting for age and sex, B.1.617.2 was associated with higher odds of oxygen requirement, intensive care unit admission, or death (adjusted odds ratio [aOR], 4.90; 95% confidence interval [CI]: 1.43-30.78). Of these patients, 157 were admitted to our center. After adjusting for age, sex, comorbidities, and vaccination, the aOR for pneumonia with B.1.617.2 was 1.88 (95% CI: .95-3.76) compared with wild-type. These differences were not seen with B.1.1.7 and B.1.351. Vaccination status was associated with decreased severity. B.1.617.2 was associated with significantly lower polymerase chain reaction cycle threshold (Ct) values and longer duration of Ct value ≤30 (median duration 18 days for B.1.617.2, 13 days for wild-type). CONCLUSIONS: B.1.617.2 was associated with increased severity of illness, and with lower Ct values and longer viral shedding. These findings provide impetus for the rapid implementation of vaccination programs.

External validation of the PRIORITY model in predicting COVID-19 critical illness in vaccinated and unvaccinated patients.

OBJECTIVES: Predictive scores are important tools for the triage of patients with coronavirus disease 2019. The PRIORITY score is advantageous because it does not require laboratory and radiologic information. However, the original development and validation cohorts studied only unvaccinated patients in early 2020. We aimed to externally validate the PRIORITY score in a cohort of patients with the novel delta and omicron variants of coronavirus disease 2019 and mixed vaccination status. METHODS: A total of 410 patients were included in a cross-sectional sampling of all patients admitted to the National Centre of Infectious Diseases on October 27, 2021. A further 102 and 136 patients with vaccine-breakthrough Delta and Omicron variant infection from April to August and December 2021, respectively, were also included. Variables at the time of admission were collected retrospectively from medical records and used to calculate the probability of deterioration using the PRIORITY model. RESULTS: Of the total 648 included patients, 447 (69.0%) were vaccinated. The mean age was 61.6 years (standard deviation ± 19.0 years), and 268 patients (41.4%) were female. A total of 112 patients (17.3%) met the primary outcome of developing critical illness or mortality. The performance of the score in this cohort was comparable with the original cohorts, with an area under the receiver operating characteristic curve for all patients of 0.794 (95% CI, 0.752-0.835; p < 0.001), regression coefficient of 1.069, and intercept of 0.04. Subgroup analysis of unvaccinated and vaccinated patients showed that performance was superior in vaccinated individuals, with an area under the receiver operating characteristic curve of 0.684 (95% CI, 0.608-0.760; p < 0.0001) and 0.831 (95% CI, 0.772-0.891; p < 0.0001), respectively. DISCUSSION: Our data support the continued use of the PRIORITY score in this era of novel variants and increased vaccination uptake.

Virological and serological kinetics of SARS-CoV-2 Delta variant vaccine breakthrough infections: a multicentre cohort study.

OBJECTIVES: Highly effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed but variants of concerns are worrisome, especially B.1.617.2 (Delta) which has rapidly spread across the world. We aim to study if vaccination alters virological and serological kinetics in breakthrough infections. METHODS: We conducted a multicentre retrospective cohort study of patients in Singapore who had received a licensed mRNA vaccine and been admitted to hospital with B.1.617.2 SARS-CoV-2 infection. We compared clinical features, virological and serological kinetics (anti-nucleocapsid, anti-spike and surrogate virus neutralization titres) between fully vaccinated and unvaccinated individuals. RESULTS: Out of 218 individuals with B.1.617.2 infection, 84 received an mRNA vaccine of which 71 were fully vaccinated, 130 were unvaccinated and four received a non-mRNA vaccine. Despite significantly older age in the vaccine breakthrough group, only 2.8% (2/71) developed severe COVID-19 requiring oxygen supplementation compared with 53.1% (69/130) in the unvaccinated group (p < 0.001). Odds of severe COVID-19 following vaccination were significantly lower (adjusted odds ratio 0.07 95% CI 0.015-0.335, p 0.001). PCR cycle threshold values were similar between vaccinated and unvaccinated groups at diagnosis, but viral loads decreased faster in vaccinated individuals. Early, robust boosting of anti-spike protein antibodies was observed in vaccinated patients; however, these titres were significantly lower against B.1.617.2 than the wildtype vaccine strain. DISCUSSION: The mRNA vaccines are highly effective at preventing symptomatic and severe COVID-19 associated with B.1.617.2 infection. Vaccination is associated with faster decline in viral RNA load and a robust serological response. Vaccination remains a key strategy for control of the COVID-19 pandemic.

Lack of viable severe acute respiratory coronavirus virus 2 (SARS-CoV-2) among PCR-positive air samples from hospital rooms and community isolation facilities.

BACKGROUND: Understanding the extent of aerosol-based transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for tailoring interventions for control of the coronavirus disease 2019 (COVID-19) pandemic. Multiple studies have reported the detection of SARS-CoV-2 nucleic acid in air samples, but only one study has successfully recovered viable virus, although it is limited by its small sample size. OBJECTIVE: We aimed to determine the extent of shedding of viable SARS-CoV-2 in respiratory aerosols from COVID-19 patients. METHODS: In this observational air sampling study, air samples from airborne-infection isolation rooms (AIIRs) and a community isolation facility (CIF) housing COVID-19 patients were collected using a water vapor condensation method into liquid collection media. Samples were tested for presence of SARS-CoV-2 nucleic acid using quantitative real-time polymerase chain reaction (qRT-PCR), and qRT-PCR-positive samples were tested for viability using viral culture. RESULTS: Samples from 6 (50%) of the 12 sampling cycles in hospital rooms were positive for SARS-CoV-2 RNA, including aerosols ranging from <1 µm to >4 µm in diameter. Of 9 samples from the CIF, 1 was positive via qRT-PCR. Viral RNA concentrations ranged from 179 to 2,738 ORF1ab gene copies per cubic meter of air. Virus cultures were negative after 4 blind passages. CONCLUSION: Although SARS-CoV-2 is readily captured in aerosols, virus culture remains challenging despite optimized sampling methodologies to preserve virus viability. Further studies on aerosol-based transmission and control of SARS-CoV-2 are needed.

Persistent Symptoms and Association With Inflammatory Cytokine Signatures in Recovered Coronavirus Disease 2019 Patients.

BACKGROUND: The complications and sequelae of coronavirus disease 2019 (COVID-19) and their effect on long-term health are unclear, and the trajectory of associated immune dysregulation is poorly understood. METHODS: We conducted a prospective longitudinal multicenter cohort study at 4 public hospitals in Singapore. Patients with COVID-19 were monitored for a median of 6 months after recovery from acute infection. Clinical symptoms and radiologic data were collected, along with plasma samples for quantification of immune mediators. The relationship between clinical symptoms and immune cytokine profiles was investigated. RESULTS: Two hundred eighty-eight participants were recruited, and follow-up data were available for 183, 175, and 120 participants at days 30, 90, and 180 postsymptom onset, respectively. Symptoms related to COVID-19 were present in 31 (16.9%), 13 (7.4%), and 14 (11.7%) at days 30, 90, and 180. In a multivariable model, age >65 years, non-Chinese ethnicity, and the severity of acute infection were associated with increased likelihood of persistent symptoms. Recovered COVID-19 patients had elevated levels of proinflammatory interleukin (IL)-17A, stem cell factor, IL-12p70, and IL-1ß and pro-angiogenic macrophage inflammatory protein 1ß, brain-derived neurotrophic factor, and vascular endothelial growth factor at day 180 compared with healthy controls. Higher levels of monocyte chemoattractant protein-1 and platelet-derived growth factor-BB were detected in patients with persistent symptoms, versus symptom-free patients. CONCLUSIONS: Approximately 10% of recovered patients had persistent symptoms 6 months after initial infection. Immune cytokine signatures of the recovered patients reflected ongoing chronic inflammation and angiogenesis. Patients with COVID-19 should be monitored closely for emerging long-term health consequences.

Dynamics of SARS-CoV-2 neutralising antibody responses and duration of immunity: a longitudinal study.

BACKGROUND: Studies have found different waning rates of neutralising antibodies compared with binding antibodies against SARS-CoV-2. The impact of neutralising antibody waning rate at the individual patient level on the longevity of immunity remains unknown. We aimed to investigate the peak levels and dynamics of neutralising antibody waning and IgG avidity maturation over time, and correlate this with clinical parameters, cytokines, and T-cell responses. METHODS: We did a longitudinal study of patients who had recovered from COVID-19 up to day 180 post-symptom onset by monitoring changes in neutralising antibody levels using a previously validated surrogate virus neutralisation test. Changes in antibody avidities and other immune markers at different convalescent stages were determined and correlated with clinical features. Using a machine learning algorithm, temporal change in neutralising antibody levels was classified into five groups and used to predict the longevity of neutralising antibody-mediated immunity. FINDINGS: We approached 517 patients for participation in the study, of whom 288 consented for outpatient follow-up and collection of serial blood samples. 164 patients were followed up and had adequate blood samples collected for analysis, with a total of 546 serum samples collected, including 128 blood samples taken up to 180 days post-symptom onset. We identified five distinctive patterns of neutralising antibody dynamics as follows: negative, individuals who did not, at our intervals of sampling, develop neutralising antibodies at the 30% inhibition level (19 [12%] of 164 patients); rapid waning, individuals who had varying levels of neutralising antibodies from around 20 days after symptom onset, but seroreverted in less than 180 days (44 [27%] of 164 patients); slow waning, individuals who remained neutralising antibody-positive at 180 days post-symptom onset (46 [28%] of 164 patients); persistent, although with varying peak neutralising antibody levels, these individuals had minimal neutralising antibody decay (52 [32%] of 164 patients); and delayed response, a small group that showed an unexpected increase of neutralising antibodies during late convalescence (at 90 or 180 days after symptom onset; three [2%] of 164 patients). Persistence of neutralising antibodies was associated with disease severity and sustained level of pro-inflammatory cytokines, chemokines, and growth factors. By contrast, T-cell responses were similar among the different neutralising antibody dynamics groups. On the basis of the different decay dynamics, we established a prediction algorithm that revealed a wide range of neutralising antibody longevity, varying from around 40 days to many decades. INTERPRETATION: Neutralising antibody response dynamics in patients who have recovered from COVID-19 vary greatly, and prediction of immune longevity can only be accurately determined at the individual level. Our findings emphasise the importance of public health and social measures in the ongoing pandemic outbreak response, and might have implications for longevity of immunity after vaccination. FUNDING: National Medical Research Council, Biomedical Research Council, and A*STAR, Singapore.

High-risk chest radiographic features associated with COVID-19 disease severity.

OBJECTIVES: High-risk CXR features in COVID-19 are not clearly defined. We aimed to identify CXR features that correlate with severe COVID-19. METHODS: All confirmed COVID-19 patients admitted within the study period were screened. Those with suboptimal baseline CXR were excluded. CXRs were reviewed by three independent radiologists and opacities recorded according to zones and laterality. The primary endpoint was defined as hypoxia requiring supplemental oxygen, and CXR features were assessed for association with this endpoint to identify high-risk features. These features were then used to define criteria for a high-risk CXR, and clinical features and outcomes of patients with and without baseline high-risk CXR were compared using logistic regression analysis. RESULTS: 109 patients were included. In the initial analysis of 40 patients (36.7%) with abnormal baseline CXR, presence of bilateral opacities, multifocal opacities, or any upper or middle zone opacity were associated with supplemental oxygen requirement. Of the entire cohort, 29 patients (26.6%) had a baseline CXR with at least one of these features. Having a high-risk baseline CXR was significantly associated with requiring supplemental oxygen in univariate (odds ratio 14.0, 95% confidence interval 3.90-55.60) and multivariate (adjusted odds ratio 8.38, 95% CI 2.43-28.97, P = 0.001) analyses. CONCLUSION: We identified several high-risk CXR features that are significantly associated with severe illness. The association of upper or middle zone opacities with severe illness has not been previously emphasized. Recognition of these specific high-risk CXR features is important to prioritize limited healthcare resources for sicker patients.

Predictors for development of critical illness amongst older adults with COVID-19: Beyond age to age-associated factors.

INTRODUCTION: Older adults with COVID-19 have disproportionately higher rates of severe disease and mortality. It is unclear whether this is attributable to age or attendant age-associated risk factors. This retrospective cohort study aims to characterize hospitalized older adults and examine if comorbidities, frailty and acuity of clinical presentation exert an age-independent effect on COVID-19 severity. METHODS: We studied 275 patients admitted to the National Centre of Infectious Disease, Singapore. We measured: 1)Charlson Comorbidity Index(CCI) as burden of comorbidities; 2)Clinical Frailty Scale(CFS) and Frailty Index(FI); and 3)initial acuity. We studied characteristics and outcomes of critical illness, stratified by age groups (50-59,60-69 and ≥70). We conducted hierarchical logistic regression in primary model(N = 262, excluding direct admissions to intensive care unit) and sensitivity analysis(N = 275): age and gender in base model, entering CCI, frailty (CFS or FI) and initial acuity sequentially. RESULTS: The ≥70 age group had highest CCI(p<.001), FI(p<.001) and CFS(p<.001), and prevalence of geriatric syndromes (polypharmacy,53.5%; urinary symptoms,37.5%; chronic pain,23.3% and malnutrition,23.3%). Thirty-two (11.6%) developed critical illness. In the primary regression model, age was not predictive for critical illness when a frailty predictor was added. Significant predictors in the final model (AUC 0.809) included male gender (p=.012), CFS (p=.038), and high initial acuity (p=.021) but not CCI or FI. In sensitivity analysis, FI (p=.028) but not CFS was significant. CONCLUSIONS: In hospitalized older adults with COVID-19, geriatric syndromes are not uncommon. Acuity of clinical presentation and frailty are important age-independent predictors of disease severity. CFS and FI provide complimentary information in predicting interval disease progression and rapid disease progression respectively.

Environmental contamination in a coronavirus disease 2019 (COVID-19) intensive care unit-What is the risk?

BACKGROUND: The risk of environmental contamination by severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in the intensive care unit (ICU) is unclear. We evaluated the extent of environmental contamination in the ICU and correlated this with patient and disease factors, including the impact of different ventilatory modalities. METHODS: In this observational study, surface environmental samples collected from ICU patient rooms and common areas were tested for SARS-CoV-2 by polymerase chain reaction (PCR). Select samples from the common area were tested by cell culture. Clinical data were collected and correlated to the presence of environmental contamination. Results were compared to historical data from a previous study in general wards. RESULTS: In total, 200 samples from 20 patient rooms and 75 samples from common areas and the staff pantry were tested. The results showed that 14 rooms had at least 1 site contaminated, with an overall contamination rate of 14% (28 of 200 samples). Environmental contamination was not associated with day of illness, ventilatory mode, aerosol-generating procedures, or viral load. The frequency of environmental contamination was lower in the ICU than in general ward rooms. Eight samples from the common area were positive, though all were negative on cell culture. CONCLUSION: Environmental contamination in the ICU was lower than in the general wards. The use of mechanical ventilation or high-flow nasal oxygen was not associated with greater surface contamination, supporting their use and safety from an infection control perspective. Transmission risk via environmental surfaces in the ICUs is likely to be low. Nonetheless, infection control practices should be strictly reinforced, and transmission risk via droplet or airborne spread remains.