Viral Load of SARS-CoV-2 in Respiratory Aerosols Emitted by COVID-19 Patients while Breathing, Talking, and Singing (preprint)

Background: Multiple SARS-CoV-2 superspreading events suggest that aerosols play an important role in driving the COVID-19 pandemic. However, the detailed roles of coarse (>5m) and fine ([≤]5m) respiratory aerosols produced when breathing, talking, and singing are not well-understood. 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: Among the 22 study participants, 13 (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic patients and 1 presymptomatic patient. Viral loads ranged from 63 - 5,821 N gene copies per expiratory activity. Patients earlier in illness were more likely to emit detectable RNA, and loads differed significantly between breathing, talking, and singing. The largest proportion of SARS-CoV-2 RNA copies was emitted by singing (53%), followed by talking (41%) and breathing (6%). 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 the transmission of SARS-CoV-2. Exposure to fine aerosols should be mitigated, especially in indoor environments where airborne transmission of SARS-CoV-2 is likely to occur. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging, and whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an important enquiry for future studies.

Anosmia in SARS-CoV-2 Patients: a Retrospective Study (preprint)

BackgroundPost viral anosmia has been reported in human coronavirus infections. In this current pandemic, olfactory dysfunction (OD) has emerged as a common key presenting symptom of COVID-19 infection. In this study, we describe OD assessment in the inpatient setting of patients both suspected of and with confirmed COVID-19 infection via University of Pennsylvania Smell Identification Test (UPSIT) objective assessment and a simple self-reported 3-item questionnaire.MethodsThirty patients admitted to the isolation wards of the National University Hospital, Singapore for either suspected or confirmed COVID-19 infection from April to May 2020 were recruited to this study. 10 patients who tested negative for SARS-CoV-2 were recruited as control subjects. The 20 patients with COVID-19 infection were divided into two groups (10 had olfactory testing performed during the first week of illness, 10 in the second week of illness). A simple 3-question survey was administered to each participant - to rank the severity, state the onset and duration of their hyposmia. Olfactory testing was performed using an English version of the UPSIT.ResultsLoss of smell was reported in 2 participants from the control group, 6 participants from the in the first week of illness and 5 participants from the second week of illness. Two COVID-19 patients have anosmia on the UPSIT. COVID-19 patients were more likely to have severe hyposmia or anosmia by objective assessment, a difference that was statistically significant (P = 0.0485). The differences in degree of OD among COVID-19 patients in their first and second week of illness were not statistically significant (P= 0.6563).ConclusionSelf-reported anosmia was higher among COVID-19 patients compared to controls who were admitted to isolation wards for respiratory symptoms but were tested negative for SARS-CoV-2 infection. On objective assessment by the UPSIT, COVID-19 patients were found to have higher rates of severe hyposmia or anosmia, a difference that was statistically significant (P = 0.0485). A limitation of this study is the odorants used in UPSIT which may be less familiar to the primarily Asian participants in this study, owing to cultural differences. 

Addressing anti-syncytin antibody levels, and fertility and breastfeeding concerns, following BNT162B2 COVID-19 mRNA vaccination (preprint)

Objective To determine whether antibodies against the SARS-CoV-2 spike protein following BNT162B2 (Pfizer-BioNTech) COVID-19 mRNA vaccination cross-react with human syncytin-1 protein, and if BNT162B2 mRNA enters breast milk. Methods In this observational cohort study of female front-line workers with no history of COVID-19 infection, we amplified BNT162B2 mRNA in plasma and breast milk and assayed anti-SARS-CoV-2 neutralising antibodies and anti-human syncytin-1 binding antibodies in plasma, at early (1-4 days) and late (4-7 weeks) time points following first-dose vaccination. Results Fifteen consented participants (mean age 40.4 years, various ethnicities) who received at least one dose of BNT162B2, including five breast-feeding women and two women who were inadvertently vaccinated in early pregnancy, were recruited. BNT162B2 mRNA, detected by amplifying part of the spike-encoding region, was detected in plasma 1-4 days following the first dose (n=13), but not 4-5 weeks later (n=2), nor was the mRNA isolated from aqueous or lipid breast milk fractions collected 0-7 days post-vaccination (n=5). Vaccine recipients demonstrated strong SARS-CoV-2 neutralising activity by at least four weeks after the first dose (n=15), including the two pregnant women. None had placental anti-syncytin-1 binding antibodies at either time-point following vaccination. Conclusions BNT162B2-vaccinated women did not transmit vaccine mRNA to breast milk, and did not produce a concurrent humoral response to syncytin-1, suggesting that cross-reactivity to syncytin-1 on the developing trophoblast, or other adverse effects in the breast-fed infant from vaccine mRNA ingestion, are unlikely. What are the novel findings of this work? COVID-19 vaccination with BNT162B2 did not elicit a cross-reacting humoral response to human syncytin-1 despite robust neutralising activity to the SARS-CoV2 spike protein, and while vaccine mRNA was isolated from plasma, it was not found in breast milk. What are the clinical implications of this work? Our work directly addresses the fertility and breastfeeding concerns fuelling vaccine hesitancy among reproductive-age women, by suggesting that BNT162B2 vaccination is unlikely to cause adverse effects on the developing trophoblast, via cross-reacting anti-syncytin-1 antibodies, or to the breastfed neonate, via mRNA breast milk transmission.

BNT162b2 vaccination induces SARS-CoV-2 specific antibody secretion into human milk with minimal transfer of vaccine mRNA (preprint)

ImportanceTo examine the impact of SARS-CoV-2 vaccination of lactating mothers on human milk Objective(1) To quantify SARS-CoV-2-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in human milk of lactating mothers who received the BNT162b2 vaccine, with reference to a cohort convalescent from antenatal COVID-19, and healthy lactating mothers. (2) To detect and quantify vaccine mRNA in human milk after BNT162b2 vaccination. DesignGestational Immunity For Transfer 2 (GIFT-2) is a prospective cohort study of lactating mothers who were due to receive two doses of BNT162b2 vaccine, recruited between 5th February 2021 and 9th February 2021. SettingLactating healthcare workers living in Singapore ParticipantsConvenience sample of ten lactating healthcare workers. Human milk samples were collected at four time points: pre-vaccination, 1-3 days after dose one, 7-10 days after dose one, and 3-7 days after dose two of the BNT162b2 vaccine. ExposureTwo doses of the BNT162b2 vaccine 21 days apart. Main Outcome and Measure(i) SARS-CoV-2-specific IgA and IgG in human milk of lactating mothers who received BNT162b2 vaccine, (ii) Detection and quantification of vaccine mRNA in human milk after BNT162b2 vaccination. ResultsTen lactating healthcare workers aged 32.5 years (range 29 - 42) were recruited, with 40 human milk samples collected and analysed. SARS-CoV-2-specific IgA was predominant in human milk of lactating mothers who received BNT162b2 vaccine. The sharpest rise in antibody production was 3 -7 days after dose two of the BNT162b2 vaccine, with medians of 1110 picomolar of anti-SARS-CoV-2 spike and 374 picomolar of anti-Receptor Binding Domain IgA. Vaccine mRNA was detected only on rare occasions, at a maximum concentration of 2 ng/mL. Conclusions and RelevanceIn this cohort of ten lactating mothers following BNT162b2 vaccination, nine (90%) produced SARS-CoV-2 IgA, and ten (100%) produced IgG in human milk with minimal amounts of vaccine mRNA. Lactating individuals should continue breastfeeding in an uninterrupted manner after receiving mRNA vaccination for SARS-CoV-2. Trial RegistrationRegistered at clinicaltrials.gov (NCT04802278). Key PointsO_ST_ABSQuestionC_ST_ABSDoes BNT162b2 (i) induce the production and secretion of SARS-CoV-2 specific antibodies into human milk, and/or (ii) get secreted into human milk? FindingsIn this cohort that included ten lactating healthcare workers following BNT162b2 vaccination, 90% produced SARS-CoV-2 immunoglobulin A, and 100% produced immunoglobulin G in human milk, with minimal amounts of vaccine mRNA transfer. MeaningLactating individuals should continue breastfeeding in an uninterrupted manner after receiving SARS-CoV-2 mRNA vaccination.

Haematological profile of hospitalised COVID-19 patients from a centre in Singapore (preprint)

Background: Haematological markers such as absolute lymphopenia has been associated with severe COVID-19 infection. However, the described cohorts were generally unwell with a large proportion of patients requiring intensive care stay. It is uncertain if these markers apply to a population with less severe illness. We sought to describe the haematological profile of patients with mild disease with COVID-19 that were admitted to a single centre in Singapore. Methods: We examined 554 consecutive PCR positive SARS-COV-2 patients who were admitted to a single tertiary healthcare institution from Feb 2020 to April 2020 2020. We examined patients based on their haematological profile based on full blood count obtained within 24h of presentation. Results: Patients with pneumonia had higher neutrophil percentages (66.5±11.6 vs 55.2±12.6%, p<0.001), lower absolute lymphocyte count (1.5±1.1 vs 1.9±2.1 x109/L, p<0.011) and absolute eosinophil count (0.2±0.9 vs 0.7±1.8 x109/L, p=0.002). Platelet counts (210±56 vs 230±61, p=0.020) were slightly lower in the group with pneumonia. We did not demonstrate significant differences in the neutrophil-lymphocyte ratio, lymphocyte-monocyte ratio and platelet-lymphocyte ratio in patients with or without pneumonia. Sixty-eight patients (12.3%) had peripheral eosinophilia. This was more common in migrant workers living in dormitories. Conclusion: Neutrophilia and lymphopenia were found to be markers associated with severe COVID-19 illness. We did not find that combined haematological parameters: NLR, MLR and PLR, had any association with disease severity in our cohort of patients with mild-moderate disease. Migrant workers living in dormitories had eosinophilia which may reflect concurrent chronic parasitic infection.

Delayed Acute Ischemic Stroke as a Late Manifestation of Asymptomatic COVID-19: The Silent 'Long Haul' Wave of the Pandemic? (preprint)

Background: Multiple neurological manifestations have been associated with acute Coronavirus disease 2019 (COVID-19) infections, however the late sequelae of COVID-19 are less well described. We describe a series of consecutive young men who present with ischemic stroke in the convalescent phase of asymptomatic COVID-19 infection.Methods: We included consecutive young acute ischemic stroke patients (≤50 years old) with laboratory confirmed COVID-19 infections based on positive SARS-CoV-2 serology, without any respiratory symptoms. We reviewed their clinical course, imaging, and laboratory findings.Findings: Eighteen young male patients, aged 35-50 years old, who were diagnosed with asymptomatic COVID-19 based on positive SARS-CoV-2 serology, presented with acute ischemic stroke from 21 May 2020 to 14 October 2020. The median time from positive serology to stroke was 54·5 days (range 0-130). The median age of the patients was 41 (range 35-50), with a median National Institutes of Health Stroke Scale of five (range 1-25). Ten (55·6%) patients presented with large vessel occlusions, of which 6 patients underwent acute recanalization treatment. Only three patients had a possible cardiac source of embolus. The annual incidence rate ischemic stroke was 2·16 (95% CI 1·36 – 3·48, p<0.001) times more compared to an age, sex, ethnicity-matched historical cohort.Interpretation: Eighteen young patients, with laboratory confirmed COVID-19, presented with acute ischemic strokes in the convalescent period. We report what could be the next wave of complications from the COVID-19 pandemic. Larger prospective studies are needed to identify young patients with cryptogenic stroke who may have had COVID-19 infections.Funding: There was no funding for this study.Declaration of Interests: No authors report any conflict of interest related to this study.Ethics Approval Statement: The study was approved by our institutional ethics review board and a waiver of consent was permitted in view of the observational nature of the study.

Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection (preprint)

The efficacy of virus-specific T cells in clearing pathogens involves a fine balance between their antiviral and inflammatory features. SARS-CoV-2-specific T cells in individuals who clear SARS-CoV-2 infection without symptoms or disease could reveal non-pathological yet protective characteristics. We therefore compared the quantity and function of SARS-CoV-2-specific T cells in a cohort of asymptomatic individuals (n=85) with that of symptomatic COVID-19 patients (n=76), at different time points after antibody seroconversion. We quantified T cells reactive to structural proteins (M, NP and Spike) using ELISpot assays, and measured the magnitude of cytokine secretion (IL-2, IFN-{gamma}, IL-4, IL-6, IL-1{beta}, TNF- and IL-10) in whole blood following T cell activation with SARS-CoV-2 peptide pools as a functional readout. Frequencies of T cells specific for the different SARS-CoV-2 proteins in the early phases of recovery were similar between asymptomatic and symptomatic individuals. However, we detected an increased IFN-{gamma} and IL-2 production in asymptomatic compared to symptomatic individuals after activation of SARS-CoV-2-specific T cells in blood. This was associated with a proportional secretion of IL-10 and pro-inflammatory cytokines (IL-6, TNF- and IL-1{beta}) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2-specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2 infected individuals are not characterized by a weak antiviral immunity; on the contrary, they mount a robust and highly functional virus-specific cellular immune response. Their ability to induce a proportionate production of IL-10 might help to reduce inflammatory events during viral clearance.

Unheeded SARS-CoV-2 protein? Look deep into negative-sense RNA (preprint)

SARS-CoV-2 is a novel ssRNA+ virus from the Coronaviridae family, which has caused the global COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest of RNA viruses, comprising of 26 known protein-coding loci. This study aimed to explore the coding potential of negative-strand RNA intermediate for its potential to contain additional protein coding-loci. Surprisingly, we have found several putative ORFs and one brandt new functional SARS-CoV-2 protein-coding loci and called it Avo1 (Ambient viral ORF1). This sequence is located on negative-sense RNA intermediate and bona fide coding for 81 amino acid residues long protein and contains strong Kozak sequence for translation on eukaryotic ribosomes. In silico translated protein Avo1 has a predominantly alpha-helical structure. The existence of Avo1 gene is supported also by its evolutionarily and structural conservation in RaTG13 bat coronavirus. The nucleotide sequence of Avo1 also contains a unique SREBP2 binding site which is closely related to the so-called cytokine storm in severe COVID-19 patients. Altogether, our results suggest the existence of still undescribed SARS-CoV-2 protein, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.