Virology and immune dynamics reveal high household transmission of ancestral SARS-CoV-2 strain.

BACKGROUND: Household studies are crucial for understanding the transmission of SARS-CoV-2 infection, which may be underestimated from PCR testing of respiratory samples alone. We aim to combine the assessment of household mitigation measures; nasopharyngeal, saliva, and stool PCR testing; along with mucosal and systemic SARS-CoV-2-specific antibodies, to comprehensively characterize SARS-CoV-2 infection and transmission in households. METHODS: Between March and September 2020, we obtained samples from 92 participants in 26 households in Melbourne, Australia, in a 4-week period following the onset of infection with ancestral SARS-CoV-2 variants. RESULTS: The secondary attack rate was 36% (24/66) when using nasopharyngeal swab (NPS) PCR positivity alone. However, when respiratory and nonrespiratory samples were combined with antibody responses in blood and saliva, the secondary attack rate was 76% (50/66). SARS-CoV-2 viral load of the index case and household isolation measures were key factors that determine secondary transmission. In 27% (7/26) of households, all family members tested positive by NPS for SARS-CoV-2 and were characterized by lower respiratory Ct values than low transmission families (Median 22.62 vs. 32.91; IQR 17.06-28.67 vs. 30.37-34.24). High transmission families were associated with enhanced plasma antibody responses to multiple SARS-CoV-2 antigens and the presence of neutralizing antibodies. Three distinguishing saliva SARS-CoV-2 antibody features were identified according to age (IgA1 to Spike 1, IgA1 to nucleocapsid protein (NP)), suggesting that adults and children generate distinct mucosal antibody responses during the acute phase of infection. CONCLUSION: Utilizing respiratory and nonrespiratory PCR testing, along with the measurement of SARS-CoV-2-specific local and systemic antibodies, provides a more accurate assessment of infection within households and highlights some of the immunological differences in response between children and adults.

A case report describing the immune response of an infant with congenital heart disease and severe COVID-19.

Background: Children with SARS-CoV-2 infection generally present with milder symptoms or are asymptomatic in comparison with adults, however severe disease occurs in a subset of children. To date, the immune correlates of severe COVID-19 in young children have been poorly characterised. Methods: We report the kinetics of immune responses in relation to clinical and virological features in an infant with acute severe COVID-19 using high-dimensional flow cytometry and multiplex cytokine analysis. Results: Systemic cellular and cytokine profiling show an initial increase in neutrophils and monocytes with depletion of lymphoid cell populations (particularly CD8 + T and NK cells) and elevated inflammatory cytokines. Expansion of memory CD4 + T (but not CD8 + T) cells occurred over time, with a predominant Th2 bias. Marked activation of T cell populations observed during the acute infection gradually resolved as the child recovered. Substantial in vitro activation of T-cell populations and robust cytokine production, in response to inactivated SARS-CoV-2 stimulation, was observed 3 months after infection indicating durable, long-lived cellular immune memory. Conclusions: These findings provide important insights into the immune response of a young infant with severe COVID-19 and will help to inform future research into therapeutic targets for high-risk groups.

Comparison of Seroconversion in Children and Adults With Mild COVID-19.

Importance: The immune response in children with SARS-CoV-2 infection is not well understood. Objective: To compare seroconversion in nonhospitalized children and adults with mild SARS-CoV-2 infection and identify factors that are associated with seroconversion. Design, Setting, and Participants: This household cohort study of SARS-CoV-2 infection collected weekly nasopharyngeal and throat swabs and blood samples during the acute (median, 7 days for children and 12 days for adults [IQR, 4-13] days) and convalescent (median, 41 [IQR, 31-49] days) periods after polymerase chain reaction (PCR) diagnosis for analysis. Participants were recruited at The Royal Children's Hospital, Melbourne, Australia, from May 10 to October 28, 2020. Participants included patients who had a SARS-CoV-2-positive nasopharyngeal or oropharyngeal swab specimen using PCR analysis. Main Outcomes and Measures: SARS-CoV-2 immunoglobulin G (IgG) and cellular (T cell and B cell) responses in children and adults. Seroconversion was defined by seropositivity in all 3 (an in-house enzyme-linked immunosorbent assay [ELISA] and 2 commercial assays: a SARS-CoV-2 S1/S2 IgG assay and a SARS-CoV-2 antibody ELISA) serological assays. Results: Among 108 participants with SARS-CoV-2-positive PCR findings, 57 were children (35 boys [61.4%]; median age, 4 [IQR, 2-10] years) and 51 were adults (28 women [54.9%]; median age, 37 [IQR, 34-45] years). Using the 3 established serological assays, a lower proportion of children had seroconversion to IgG compared with adults (20 of 54 [37.0%] vs 32 of 42 [76.2%]; P < .001). This result was not associated with viral load, which was similar in children and adults (mean [SD] cycle threshold [Ct] value, 28.58 [6.83] vs 24.14 [8.47]; P = .09). In addition, age and sex were not associated with seroconversion within children (median age, 4 [IQR, 2-14] years for both seropositive and seronegative groups; seroconversion by sex, 10 of 21 girls [47.6%] vs 10 of 33 boys [30.3%]) or adults (median ages, 37 years for seropositive and 40 years for seronegative adults [IQR, 34-39 years]; seroconversion by sex, 18 of 24 women [75.0%] vs 14 of 18 men [77.8%]) (P > .05 for all comparisons between seronegative and seropositive groups). Symptomatic adults had 3-fold higher SARS-CoV-2 IgG levels than asymptomatic adults (median, 227.5 [IQR, 133.7-521.6] vs 75.3 [IQR, 36.9-113.6] IU/mL), whereas no differences were observed in children regardless of symptoms. Moreover, differences in cellular immune responses were observed in adults compared with children with seroconversion. Conclusions and Relevance: The findings of this cohort study suggest that among patients with mild COVID-19, children may be less likely to have seroconversion than adults despite similar viral loads. This finding has implications for future protection after SARS-CoV-2 infection in children and for interpretation of serosurveys that involve children. Further research to understand why seroconversion and development of symptoms are potentially less likely in children after SARS-CoV-2 infection and to compare vaccine responses may be of clinical and scientific importance.

Children and Adults in a Household Cohort Study Have Robust Longitudinal Immune Responses Following SARS-CoV-2 Infection or Exposure.

Children have reduced severity of COVID-19 compared to adults and typically have mild or asymptomatic disease. The immunological mechanisms underlying these age-related differences in clinical outcomes remain unexplained. Here, we quantify 23 immune cell populations in 141 samples from children and adults with mild COVID-19 and their PCR-negative close household contacts at acute and convalescent time points. Children with COVID-19 displayed marked reductions in myeloid cells during infection, most prominent in children under the age of five. Recovery from infection in both children and adults was characterised by the generation of CD8 TCM and CD4 TCM up to 9 weeks post infection. SARS-CoV-2-exposed close contacts also had immunological changes over time despite no evidence of confirmed SARS-CoV-2 infection on PCR testing. This included an increase in low-density neutrophils during convalescence in both exposed children and adults, as well as increases in CD8 TCM and CD4 TCM in exposed adults. In comparison to children with other common respiratory viral infections, those with COVID-19 had a greater change in innate and T cell-mediated immune responses over time. These findings provide new mechanistic insights into the immune response during and after recovery from COVID-19 in both children and adults.

Adding saliva testing to oropharyngeal and deep nasal swab testing increases PCR detection of SARS-CoV-2 in primary care and children.

OBJECTIVE: To compare the concordance and acceptability of saliva testing with standard-of-care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in children and in general practice. DESIGN: Prospective multicentre diagnostic validation study. SETTING: Royal Children's Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July-October 2020. PARTICIPANTS: 1050 people who provided paired saliva and oropharyngeal-nasal swabs for SARS-CoV-2 testing. MAIN OUTCOME MEASURES: Numbers of cases in which SARS-CoV-2 was detected in either specimen type by real-time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. RESULTS: SARS-CoV-2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58-84%) for saliva and 63% (95% CI, 49-76%) for oropharyngeal-nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70-95%) for saliva and 63% (95% CI, 45-79%) for oropharyngeal-nasal swabs. Adding saliva testing to standard-of-care oropharyngeal-nasal swab testing increased overall case detection by 59% (95% CI, 29-95%). Providing saliva was preferred to an oropharyngeal-nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). CONCLUSION: In children over 10 years of age and adults, saliva testing alone may be suitable for SARS-CoV-2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal-nasal swab testing for increasing case detection.

COVID-19 public health measures and respiratory viruses in children in Melbourne.

AIM: To describe the epidemiology of respiratory viruses in children before and during the 2020 SARS-CoV-2 pandemic and the relationship to public health measures instituted by the Victorian government. METHODS: Retrospective audit of respiratory viruses at a tertiary paediatric hospital in Melbourne from January 2015 up to week 47, 2020 in children under 18 years of age. The proportion of positive cases in weeks 1-47 in 2015-2019 (period 1) were compared to weeks 1-47, 2020 (period 2), and reviewed in the context of public health restrictions in Victoria. RESULTS: An annual average of 4636 tests were performed in period 1 compared to 3659 tests in period 2. Proportions of positive influenza A virus, influenza B virus, respiratory syncytial virus (RSV) and human parainfluenza virus were significantly reduced in period 2 compared to period 1: 77.3, 89.4, 68.6 and 66.9% reductions, respectively (all P < 0.001). From week 12-47, 2020, 28 893 SARS-CoV-2 tests were performed with a 0.64% positivity rate. Influenza viruses were not detected after week 17, RSV was not detected after week 35. CONCLUSIONS: Strict public health measures and border closures were successful in eliminating community transmission of SARS-CoV-2 in Melbourne. This was associated with a significant reduction in other respiratory virus infections in children. Identifying sustainable and effective ongoing public health interventions to reduce transmission of RSV and influenza could result in reduced morbidity and mortality in children and requires further research.

Innate cell profiles during the acute and convalescent phase of SARS-CoV-2 infection in children.

Children have mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) confirmed disease (COVID-19) compared to adults and the immunological mechanisms underlying this difference remain unclear. Here, we report acute and convalescent innate immune responses in 48 children and 70 adults infected with, or exposed to, SARS-CoV-2. We find clinically mild SARS-CoV-2 infection in children is characterised by reduced circulating subsets of monocytes (classical, intermediate, non-classical), dendritic cells and natural killer cells during the acute phase. In contrast, SARS-CoV-2-infected adults show reduced proportions of non-classical monocytes only. We also observe increased proportions of CD63+ activated neutrophils during the acute phase to SARS-CoV-2 in infected children. Children and adults exposed to SARS-CoV-2 but negative on PCR testing display increased proportions of low-density neutrophils that we observe up to 7 weeks post exposure. This study characterises the innate immune response during SARS-CoV-2 infection and household exposure in children.

Maintaining human milk bank services throughout the COVID-19 pandemic: A global response.

If maternal milk is unavailable, the World Health Organization recommends that the first alternative should be pasteurised donor human milk (DHM). Human milk banks (HMBs) screen and recruit milk donors, and DHM principally feeds very low birth weight babies, reducing the risk of complications and supporting maternal breastfeeding where used alongside optimal lactation support. The COVID-19 pandemic has presented a range of challenges to HMBs worldwide. This study aimed to understand the impacts of the pandemic on HMB services and develop initial guidance regarding risk limitation. A Virtual Collaborative Network (VCN) comprising over 80 HMB leaders from 36 countries was formed in March 2020 and included academics and nongovernmental organisations. Individual milk banks, national networks and regional associations submitted data regarding the number of HMBs, volume of DHM produced and number of recipients in each global region. Estimates were calculated in the context of missing or incomplete data. Through open-ended questioning, the experiences of milk banks from each country in the first 2 months of the pandemic were collected and major themes identified. According to data collected from 446 individual HMBs, more than 800,000 infants receive DHM worldwide each year. Seven pandemic-related specific vulnerabilities to service provision were identified, including sufficient donors, prescreening disruption, DHM availability, logistics, communication, safe handling and contingency planning, which were highly context-dependent. The VCN now plans a formal consensus approach to the optimal response of HMBs to new pathogens using crowdsourced data, enabling the benchmarking of future strategies to support DHM access and neonatal health in future emergencies.

SARS-CoV-2 in human milk is inactivated by Holder pasteurisation but not cold storage.

AIM: As the COVID-19 pandemic evolves, human milk banks world-wide continue to provide donor human milk to vulnerable infants who lack access to mother's own milk. Under these circumstances, ensuring the safety of donor human milk is paramount, as the risk of vertical transmission of SARS-CoV-2 is not fully understood. Here, we investigate the inactivation of SARS-CoV-2 in human milk by pasteurisation and the stability of SARS-CoV-2 in human milk under cold storage. METHODS: SARS-CoV-2 was experimentally inoculated into human milk samples from healthy donors or into a control medium. Triplicates of each sample were layered onto uninfected cells after Holder pasteurisation (63°C for 30 min), heating to 56°C for 30 min, or after 48 h of storage at 4°C or -30°C. Infectious titres of virus were determined at 72 h post-infection by endpoint titration. RESULTS: Following heating to 63°C or 56°C for 30 min, replication competent (i.e. live) SARS-CoV-2 was undetected in both human milk and the control medium. Cold storage of SARS-CoV-2 in human milk (either at 4°C or -30°C) did not significantly impact infectious viral load over a 48 h period. CONCLUSION: SARS-CoV-2 is effectively inactivated by Holder pasteurisation, suggesting that existing milk bank processes will effectively mitigate the risk of transmission of SARS-COV-2 to vulnerable infants through pasteurised donor human milk. The demonstrated stability of SARS-CoV-2 in refrigerated or frozen human milk may assist in the development of guidelines around safe expressing and storing of milk from COVID-19 infected mothers.