Intra-Host Mutation Rate of Acute SARS-CoV-2 Infection During the Initial Pandemic Wave (preprint)

Background: Our understanding of SARS-CoV-2 evolution and mutation rate is limited. The rate of SARS-CoV-2 evolution is minimized through a proofreading function encoded by NSP-14 and may be affected by patient comorbidity. Current understanding of SARS-CoV-2 mutational rate is through population based analysis while intra-host mutation rate remains poorly studied. Methods: Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) [≥]0.25, [≥]0.5 and [≥]0.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with ({Delta}NSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Results: Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI:90.8-96.4], 40.7 (95%CI:38.9-42.6) and 34.7 (95%CI:33.0-36.4) substitutions/genome/year at AF [≥]0.25, [≥]0.5, [≥]0.75 respectively. Mutation rate in {Delta}NSP-14 were significantly elevated at AF>0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Discussion: Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.

Longitudinal wastewater surveillance addressed public health priorities during the transition from "dynamic COVID-zero" to "opening up" in China: a population-based study (preprint)

Background Wastewater surveillance provides real-time, cost-effective monitoring of SARS-CoV-2 transmission. We developed the first city-level wastewater warning system in mainland China, located in Shenzhen. Our study aimed to reveal cryptic transmissions under the "dynamic COVID-zero" policy and characterize the dynamics of the infected population and variant prevalence, and then guide the allocation of medical resources during the transition to "opening up" in China. Methods In this population-based study, a total of 1,204 COVID-19 cases were enrolled to evaluate the contribution of Omicron variant-specific faecal shedding rates in wastewater. After that, wastewater samples from up to 334 sites distributed in communities and port areas in two districts of Shenzhen covering 1.74 million people were tested daily to evaluate the sensitivity and specificity of this approach and were validated against daily SARS-CoV-2 screening. After the public health policy was switched to "opening up" in December 7, 2022, we conducted wastewater surveillance at wastewater treatment plants and pump stations covering 3.55 million people to estimate infected populations using model prediction and detect the relative abundance of SARS-CoV-2 lineages using wastewater sequencing. Findings In total, 82.4% of SARS-CoV-2 Omicron cases tested positive for faecal viral RNA within the first four days after the diagnosis, which was far more than the proportion of the ancestral variant. A total of 27,759 wastewater samples were detected from July 26 to November 30 in 2022, showing a sensitivity of 73.8% and a specificity of 99.8%. We further found that wastewater surveillance played roles in providing early warnings and revealing cryptic transmissions in two communities. Based on the above results, we employed a prediction model to monitor the daily number of infected individuals in Shenzhen during the transition to "opening up" in China, with over 80% of the population infected in both Futian District and Nanshan District. Notably, the prediction of the daily number of hospital admission was consistent with the actual number. Further sequencing revealed that the Omicron subvariant BA.5.2.48 accounted for the most abundant SARS-CoV-2 RNA in wastewater, and BF.7.14 and BA.5.2.49 ranked second and third, respectively, which was consistent with the clinical sequencing. Interpretation This study provides a scalable solution for wastewater surveillance of SARS-CoV-2 to provide real-time monitoring of the new variants, infected populations and facilitate the precise prediction of hospital admission. This novel framework could be a One Health system for the surveillance of other infectious and emerging pathogens with faecal shedding and antibiotic resistance genes in the future. Funding Sanming Project of Medicine in Shenzhen, Shenzhen Key Medical Discipline Construction Fund.

Intramuscular Versus Intravenous SARS-CoV-2 Neutralizing Antibody Sotrovimab for Treatment of COVID-19 (COMET-TAIL): A Randomized Non-inferiority Clinical Trial (preprint)

Background: Convenient administration of coronavirus disease 2019 (COVID-19) treatment in community settings is desirable. Sotrovimab is a pan-sarbecovirus dual-action monoclonal antibody formulated for intravenous (IV) or intramuscular (IM) administration for early treatment of mild/moderate COVID-19. Methods: This phase 3, randomized, multicenter, open-label study tested non-inferiority of IM to IV administration using a 3.5% absolute non-inferiority margin. From June to August 2021, patients aged [≥]12 years with COVID-19, not hospitalized or receiving supplemental oxygen, and at high risk for progression were randomized 1:1:1 to a single 500-mg IV sotrovimab infusion or 500-mg or 250-mg IM sotrovimab injection. The primary composite endpoint was progression to all-cause hospitalization for >24 hours for acute management of illness or all-cause death through day 29. Results: Sotrovimab 500 mg IM was non-inferior to 500 mg IV: 10/376 (2.7%) participants in the sotrovimab 500-mg IM group versus 5/378 (1.3%) in the sotrovimab 500-mg IV group met the primary endpoint (absolute adjusted risk difference: 1.06% [95% confidence interval [CI]: -1.15%, 3.26%]). The CI upper limit was lower than the prespecified non-inferiority margin of 3.5%. 250-mg IM group enrollment was discontinued early because a greater proportion of hospitalizations was seen in that group versus the 500-mg groups. Serious adverse events occurred in <1% to 2% of participants across groups. Four participants experienced serious disease related events and died (500 mg IM: 2/393 [<1%]; 250 mg IM: 2/195 [1%]). Conclusions: Sotrovimab 500-mg IM injection was well tolerated and non-inferior to IV administration. IM administration could expand outpatient treatment access for COVID-19.

SARS-CoV-2 convalescence and hybrid immunity elicits mucosal immune responses (preprint)

Mucosal antibodies play a key role in the protection against SARS-CoV-2 infection in the upper respiratory tract, and potentially in limiting virus replication and therefore onward transmission. While systemic immunity to SARS-CoV-2 is well understood, little is known about the antibodies present on the nasal mucosal surfaces. In this study, we evaluated SARS-CoV-2 mucosal antibodies in response to infection, vaccination, or a combination of both. Paired nasal fluid and serum samples were collected from 136 individuals, which include convalescent, vaccinated, or breakthrough infections. We detected a high correlation between IgG responses in serum and nasal fluids, which were higher in both compartments in vaccinated compared to convalescent participants. Contrary, nasal and systemic SARS-CoV-2 IgA responses were weakly correlated, indicating a compartmentalization between the local and systemic IgA responses. SARS-CoV-2 secretory component IgA (s-IgA) antibodies, present exclusively on mucosal surfaces, were detected in the nasal fluid only in a minority of vaccinated subjects and were significantly higher in previously infected individuals. s-IgA binding antibodies showed significant correlation with neutralizing activity of nasal fluids against SARS-CoV-2 ancestral B.1 and Omicron-BA.5 variant, indicating that s-IgA is the crucial contributor to neutralization in the nasal mucosa. Neutralization against both SARS-CoV-2 strains was higher in the mucosa of subjects with previous SARS-CoV-2 infections compared to vaccinated participants. In summary, we demonstrate that currently available vaccines elicit strong systemic antibody responses, but SARS-CoV-2 infection generates more potent binding and neutralizing mucosal antibodies. Our results support the importance to develop SARS-CoV-2 vaccines that elicit mucosal antibodies.

Analysis of the risk and pre-emptive control of viral outbreaks accounting for within-host dynamics: SARS-CoV-2 antigen testing as a case study (preprint)

In the era of living with COVID-19, the risk of localised SARS-CoV-2 outbreaks remains. Here, we develop a multi-scale modelling framework for estimating the local outbreak risk for a viral disease (the probability that a major outbreak results from a single case introduced into the population), accounting for within-host viral dynamics. Compared to population-level models previously used to estimate outbreak risks, our approach enables more detailed analysis of how the risk can be mitigated through pre-emptive interventions such as antigen testing. Considering SARS-CoV-2 as a case study, we quantify the within-host dynamics using data from individuals with omicron variant infections. We demonstrate that regular antigen testing reduces, but may not eliminate, the outbreak risk, depending on characteristics of local transmission. In our baseline analysis, daily antigen testing reduces the outbreak risk by 45% compared to a scenario without antigen testing. Additionally, we show that accounting for heterogeneity in within-host dynamics between individuals affects outbreak risk estimates and assessments of the impact of antigen testing. Our results therefore highlight important factors to consider when using multi-scale models to design pre-emptive interventions against SARS-CoV-2 and other viruses.

Morbidity and mortality burden of COVID-19 in rural Madagascar: results from a longitudinal cohort and nested seroprevalence study (preprint)

Introduction: Three years into the pandemic, there remains significant uncertainty about the true infection and mortality burden of COVID-19 in the WHO-Africa region. High quality, population-representative studies in Africa are rare and tend to be conducted in national capitals or large cities, leaving a substantial gap in our understanding of the impact of COVID-19 in rural, low-resource settings. Here, we estimated the spatio-temporal morbidity and mortality burden associated with COVID-19 in a rural health district of Madagascar until the first half of 2021. Methods: We integrated a nested seroprevalence study within a pre-existing longitudinal cohort conducted in a representative sample of 1600 households in Ifanadiana District, Madagascar. Socio-demographic and health information was collected in combination with dried blood spots for about 6500 individuals of all ages, which were analysed to detect IgG and IgM antibodies against four specific proteins of SARS-CoV2 in bead-based multiplex immunoassay. We evaluated spatio-temporal patterns in COVID-19 infection history and its associations with several geographic, socio-economic and demographic factors via logistic regressions. Results: Eighteen percent of people had been infected by April-June 2021, with seroprevalence increasing with individuals age. COVID-19 primarily spread along the only paved road and in major towns during the first epidemic wave, subsequently spreading along secondary roads during the second wave to more remote areas. Wealthier individuals and those with occupations such as commerce and formal employment were at higher risk of being infected in the first wave. Adult mortality increased in 2020, particularly for older men for whom it nearly doubled up to nearly 40 deaths per 1000. Less than 10% of mortality in this period could be directly attributed to COVID-19 deaths given known infection fatality ratios and observed seroprevalence in the district. Conclusion: Our study provides a very granular understanding on COVID-19 transmission and mortality in a rural population of sub-Saharan Africa and suggests that the disease burden in these areas may have been substantially underestimated.

Syndromic surveillance as a predictive tool for health-related school absences in COVID-19 Sentinel Schools in Catalonia, Spain. (preprint)

Monitoring influenza-like illness through syndromic surveillance could be an important strategy in the COVID-19 emergence scenario. The study aims to implement syndromic surveillance for children aged 6-11 years in COVID-19 sentinel schools in Catalonia. Data collection was made by self-applied survey to collect daily health status and symptoms. We proceed logistic mixed models and a Latent Class Analysis to investigate associations with syndromes and school absence. Were enrolled 135 students (2163 person-days) that filled 1536 surveys and 60 participants reported illness (29.52 by 100 person/day) and registered 189 absence events, 62 of them (32.8%) related to health reasons. Subgroups of influenza-like illness were founded such as a significantly and positively association with school absences. The findings of this study can be applied to the detection of health events, and association with school absences, offering an opportunity for quick action, or simply for monitoring and understanding the students' health situation.

"Its no use saying it in English": A qualitative study exploring community leaders perceptions of the challenges and opportunities with translating and interpreting COVID-19 related public health messaging to reach ethnic minorities in Australia (preprint)

Background: The Australian Government implemented a range of public health response strategies and communication approaches to reduce the spread of COVID-19; however, concerns have been raised around a failure to consider culturally and linguistically diverse (CaLD) communities sufficiently in these processes. This research aimed to understand the factors that have impacted COVID-19 communication and engagement efforts during the pandemic from the perspective of key CaLD community stakeholders and community members. A further aim was to understand the processes that could be adopted to support future communication strategies, including the promotion of pandemic-related vaccines. Approach: This study included 29 key informant interviews with community and faith-based leaders in New South Wales, Australia. Results: The overwhelming message from community leaders was a sense of shared responsibility between their organisations and the government in communicating pertinent and accurate COVID-19 related information to CaLD communities. They expressed a sense of duty to keep their community members safe. While acknowledging this shared responsibility, community leaders and others shouldered significant costs related to resources and time that need to be acknowledged by governments in preparing for future disease outbreaks. They felt that governments should consider: 1) improving communication between governments and CaLD organisations; 2) responding to the specific CaLD needs with greater agility; 3) foregrounding social media in their communication strategy; 4) reinvesting in local public health units to know their population; 5) investing in a health ambassadors model program; 6) preparing a hybrid model of translators/interpreters to fill the gap; and, 7) reimagining vaccine information campaigns to better target CaLD communities. Conclusion: Given the technical details about the COVID-19 virus conveyed in government information campaigns and the media, ensuring the most vulnerable populations, including people from CaLD backgrounds, access clear, concise and timely public health messaging from both governments and community organisations requires further attention.

Occupational differences in the prevalence and severity of long-COVID: Analysis of the ONS Coronavirus (COVID-19) Infection Survey (preprint)

Objectives: To establish whether prevalence and severity of long-COVID symptoms vary by industry and occupation. Methods: We utilised ONS Coronavirus Infection Survey (CIS) data (February 2021-April 2022) of working-age participants (16-65 years). Exposures were industrial sector, occupation and major Standard Occupational Classification (SOC) group. Outcomes were self-reported: (1) long-COVID symptoms; and (2) reduced function due to long-COVID. Binary (outcome 1) and ordered (outcome 2) logistic regression were used to estimate odds ratios (OR) and prevalence (marginal means) for all exposures. Results: Public facing industries, including teaching and education, social care, healthcare, civil service, retail and transport industries and occupations had highest odds ratios for long-COVID. By major SOC group, those in caring, leisure and other services (OR 1.44, CIs: 1.38-1.52) had substantially elevated odds than average. For almost all exposures, the pattern of odds ratios for long-COVID symptoms followed that for SARS-CoV-2 infections, except for professional occupations (OR<1 for infection; OR>1 for long-COVID). The probability of reporting long-COVID for industry ranged from 7.7% (financial services) to 11.6% (teaching and education); whereas the prevalence of reduced function by a lot ranged from 17.1% (arts, entertainment and recreation) to 22-23% (teaching and education and armed forces ) and to 27% (those not working). Conclusions: The risk and prevalence of long-COVID differs across industries and occupations. Generally, it appears that likelihood of developing long-COVID symptoms follows likelihood of SARS-CoV-2 infection, except for professional occupations. These findings highlight sectors and occupations where further research is needed to understand the occupational factors resulting in long-COVID.

Immune responses and disease biomarker long-term changes following COVID-19 mRNA vaccination in a cohort of rheumatic disease patients (preprint)

Objective. To evaluate seroreactivity and disease biomarkers after 2 or 3 doses of COVID-19 mRNA vaccines in a cohort of patients with rheumatic diseases. Methods. We collected biological samples longitudinally before and after 2-3 doses of COVID-19 mRNA vaccines from a cohort of patients with systemic lupus erythematosus (SLE), psoriatic arthritis, Sjogrens syndrome, ankylosing spondylitis, and inflammatory myositis. Anti-SARS-CoV-2 spike IgG and IgA and anti-dsDNA concentration were measured by ELISA. A surrogate neutralization assay was utilized to measure antibody neutralization ability. Lupus disease activity was measured by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Expression of type I interferon signature was measured by real-time PCR. The frequency of extrafollicular double negative 2 (DN2) B cells was measured by flow cytometry. Results. Most of the patients generated high SARS-CoV-2 spike-specific neutralizing antibodies comparable to those in healthy controls after 2 doses of mRNA vaccines. The antibody level declined over time but recovered after the third dose of the vaccine. Rituximab treatment substantially reduced antibody level and neutralization ability. Among SLE patients, no consistent increase in SLEDAI scores was observed post-vaccination. The changes in anti-dsDNA antibody concentration and expression of type I IFN signature genes were highly variable but did not show consistent or significant increases. Frequency of DN2 B cells remained largely stable. Conclusion. Rheumatic disease patients without rituximab treatment have robust antibody responses toward COVID-19 mRNA vaccination. Disease activity and disease-associated biomarkers remain largely stable over 3 doses of vaccines, suggesting that COVID-19 mRNA vaccines may not exacerbate rheumatic diseases.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 are Susceptible SARS-CoV-2Virus Infection in vitro (preprint)

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry. Because there is limited evidence of active oral infection by SARS-CoV-2 viruses, we assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Viral dynamics in hTERT TIGKs were different from those in A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells at day 3 after infection. Analysis of oral epithelial cells infected by replication competent SARS-CoV-2 viruses expressing GFP showed that the signals of GFP and SARS-CoV-2 mRNAs were not evenly distributed. Taken together, our results demonstrated oral epithelial cells were susceptible to SARS-CoV-2 viruses despite of low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for development of future vaccines and therapeutics.

Impact of COVID-19 Infection on Cardiorespiratory Fitness, Sleep, and Psychology of Endurance Athletes– CAESAR Study (preprint)

COVID-19 has harmful impact on health. It is especially important for endurance athletes (EAs). Sleep and psychology influence sport performance. Aims of this study were: (1) investigation of the consequences of mild COVID-19 on sleep and psychology and (2) assessment of the conse-quences of the infection on cardiopulmonary exercise test (CPET) results. 49 EAs (males= 43; 87.76%, females= 6; 12.24%, age= 39.9±7.8 years, height= 178.4±6.8 cm, weight= 76.3±10.4 kg; BMI= 24.0±2.6 kg·m−2) underwent maximal cycling or running CPET pre- and post- COVID-19 and completed a survey. Exercise performance was deteriorated after COVID-19 (maximal oxy-gen uptake; VO2max= 47.81±7.81 vs 44.97±7.00 ml·kg·min−1 respectively pre- and post- infection; p<0.001). Waking up at night affected heart rate (HR) at the respiratory compensation point (RCP) (p=0.028). Sleep time influenced pulmonary ventilation (p=0.013), breathing frequency (p=0.010), and blood lactate concentration (Lac) (p=0.013) at RCP. Maximal power/speed (p=0.046) and HR (p=0.070) linked with the quality of sleep. Stress management and relaxation techniques linked with VO2max (p=0.046), maximal power/speed (p=0.033), and maximal Lac (p=0.045). Cardiorespiratory fitness was deteriorated after mild COVID-19 and was correlated with sleep and mental health. Medical Professionals should encourage EAs to maintain proper mental health and sleep after COVID-19 infection to facilitate recovery.