Evaluation of the impact of pre-analytical conditions on sample stability for the detection of SARS-CoV-2 RNA

Demand for accurate SARS-CoV-2 diagnostics is high. Most samples in the UK are collected in the community and rely on the postal service for delivery to the laboratories. The current recommendation remains that swabs should be collected in Viral Transport Media (VTM) and transported with a cold chain to the laboratory for RNA extraction and RT-qPCR. This is not always possible. We aimed to test the stability of SARS-CoV-2 RNA subjected to different pre-analytical conditions. Swabs were dipped into PBS containing cultured SARS-CoV-2 and placed in either a dry tube or a tube containing either normal saline or VTM. The tubes were then stored at different temperatures (20–50 °C) for variable periods (8 h to 5 days). Samples were tested by RT-qPCR targeting SARS-CoV-2 E gene. VTM outperformed swabs in saline and dry swabs in all conditions. Samples in VTM were stable, independent of a cold chain, for 5 days, with a maximum increase in cycle threshold (Ct) of 1.34 when held at 40 °C. Using normal saline as the transport media resulted in a loss of sensitivity (increased Ct) over time and with increasing temperature (up to 7.8 cycles compared to VTM). SARS-CoV-2 was not detected in 3/9 samples in normal saline when tested after 120 h incubation. Transportation of samples in VTM provides a high level of confidence in the results despite the potential for considerable, uncontrolled variation in temperature and longer transportation periods. False negative results may be seen after 96 h in saline and viral loads will appear lower

Risk factors and vectors for SARS-CoV-2 household transmission: a prospective, longitudinal cohort study

BACKGROUND: Despite circumstantial evidence for aerosol and fomite spread of SARS-CoV-2, empirical data linking either pathway with transmission are scarce. Here we aimed to assess whether the presence of SARS-CoV-2 on frequently-touched surfaces and residents' hands was a predictor of SARS-CoV-2 household transmission. METHODS: In this longitudinal cohort study, during the pre-alpha (September to December, 2020) and alpha (B.1.1.7; December, 2020, to April, 2021) SARS-CoV-2 variant waves, we prospectively recruited contacts from households exposed to newly diagnosed COVID-19 primary cases, in London, UK. To maximally capture transmission events, contacts were recruited regardless of symptom status and serially tested for SARS-CoV-2 infection by RT-PCR on upper respiratory tract (URT) samples and, in a subcohort, by serial serology. Contacts' hands, primary cases' hands, and frequently-touched surface-samples from communal areas were tested for SARS-CoV-2 RNA. SARS-CoV-2 URT isolates from 25 primary case-contact pairs underwent whole-genome sequencing (WGS). FINDINGS: From Aug 1, 2020, until March 31, 2021, 620 contacts of PCR-confirmed SARS-CoV-2-infected primary cases were recruited. 414 household contacts (from 279 households) with available serial URT PCR results were analysed in the full household contacts' cohort, and of those, 134 contacts with available longitudinal serology data and not vaccinated pre-enrolment were analysed in the serology subcohort. Household infection rate was 28·4% (95% CI 20·8-37·5) for pre-alpha-exposed contacts and 51·8% (42·5-61·0) for alpha-exposed contacts (p=0·0047). Primary cases' URT RNA viral load did not correlate with transmission, but was associated with detection of SARS-CoV-2 RNA on their hands (p=0·031). SARS-CoV-2 detected on primary cases' hands, in turn, predicted contacts' risk of infection (adjusted relative risk [aRR]=1·70 [95% CI 1·24-2·31]), as did SARS-CoV-2 RNA presence on household surfaces (aRR=1·66 [1·09-2·55]) and contacts' hands (aRR=2·06 [1·57-2·69]). In six contacts with an initial negative URT PCR result, hand-swab (n=3) and household surface-swab (n=3) PCR positivity preceded URT PCR positivity. WGS corroborated household transmission. INTERPRETATION: Presence of SARS-CoV-2 RNA on primary cases' and contacts' hands and on frequently-touched household surfaces associates with transmission, identifying these as potential vectors for spread in households. FUNDING: National Institute for Health Research Health Protection Research Unit in Respiratory Infections, Medical Research Council

Transmission of SARS-CoV-2 by children to contacts in schools and households: a prospective cohort and environmental sampling study in London.

BACKGROUND: Assessing transmission of SARS-CoV-2 by children in schools is of crucial importance to inform public health action. We assessed frequency of acquisition of SARS-CoV-2 by contacts of pupils with COVID-19 in schools and households, and quantified SARS-CoV-2 shedding into air and onto fomites in both settings. METHODS: We did a prospective cohort and environmental sampling study in London, UK in eight schools. Schools reporting new cases of SARS-CoV-2 infection to local health protection teams were invited to take part if a child index case had been attending school in the 48 h before a positive SARS-CoV-2 PCR test. At the time of the study, PCR testing was available to symptomatic individuals only. Children aged 2-14 years (extended to <18 years in November, 2020) with a new nose or throat swab SARS-CoV-2 positive PCR from an accredited laboratory were included. Incidents involving exposure to at least one index pupil with COVID-19 were identified (the prevailing variants were original, α, and δ). Weekly PCR testing for SARS-CoV-2 was done on immediate classroom contacts (the so-called bubble), non-bubble school contacts, and household contacts of index pupils. Testing was supported by genome sequencing and on-surface and air samples from school and home environments. FINDINGS: Between October, 2020, and July, 2021 from the eight schools included, secondary transmission of SARS-CoV-2 was not detected in 28 bubble contacts, representing ten bubble classes (participation rate 8·8% [IQR 4·6-15·3]). Across eight non-bubble classes, 3 (2%) of 62 pupils tested positive, but these were unrelated to the original index case (participation rate 22·5% [9·7-32·3]). All three were asymptomatic and tested positive in one setting on the same day. In contrast, secondary transmission to previously negative household contacts from infected index pupils was found in six (17%) of 35 household contacts rising to 13 (28%) of 47 household contacts when considering all potential infections in household contacts. Environmental contamination with SARS-CoV-2 was rare in schools: fomite SARS-CoV-2 was identified in four (2%) of 189 samples in bubble classrooms, two (2%) of 127 samples in non-bubble classrooms, and five (4%) of 130 samples in washrooms. This contrasted with fomites in households, where SARS-CoV-2 was identified in 60 (24%) of 248 bedroom samples, 66 (27%) of 241 communal room samples, and 21 (11%) 188 bathroom samples. Air sampling identified SARS-CoV-2 RNA in just one (2%) of 68 of school air samples, compared with 21 (25%) of 85 air samples taken in homes. INTERPRETATION: There was no evidence of large-scale SARS-CoV-2 transmission in schools with precautions in place. Low levels of environmental contamination in schools are consistent with low transmission frequency and suggest adequate cleaning and ventilation in schools during the period of study. The high frequency of secondary transmission in households associated with evident viral shedding throughout the home suggests a need to improve advice to households with infection in children to prevent onward community spread. The data suggest that SARS-CoV-2 transmission from children in any setting is very likely to occur when precautions are reduced. FUNDING: UK Research and Innovation and UK Department of Health and Social Care, National Institute for Health and Care Research

Transmission of SARS-CoV-2 by children to contacts in schools and households: a prospective cohort and environmental sampling study in London

Background: Assessing transmission of SARS-CoV-2 by children in schools is of critical importance to inform public health action. We assessed frequency of acquisition of SARS-CoV-2 by contacts of pupils with COVID-19 in schools and households, and quantified SARS-CoV-2 shed into air and onto fomites in both settings. Methods: Incidents involving exposure to at least one index pupil with COVID-19 in 8 schools were identified between October 2020-July 2021 (prevailing variants, original, alpha and delta). Weekly PCR testing for SARS-CoV-2 was undertaken on immediate classroom contacts (the “bubble”), non-bubble school contacts, and household contacts of index pupils, supported by genome sequencing, and on surface and air samples from school and home environments. Findings: Secondary transmission of SARS-CoV-2 was not detected in 28 bubble contacts, representing 10 bubble classes (participation rate 8.8%, IQR 4.6-15.3%). Across 8 non-bubble classes, 3/62 pupils tested positive but these were unrelated to the original index case (participation rate 22.5%, IQR 9.7-32.3%). All three were asymptomatic and tested positive in one setting on the same day. In contrast, secondary transmission to previously-negative household contacts from infected index pupils was 17.1% (6/35) rising to 27.7% (13/47) when considering all potential infections in household contacts. Environmental contamination with SARS-CoV-2 was rare in schools; fomite SARS-CoV-2 was identified in 4/189 (2.1%) samples in bubble classrooms, 2/127 (1.6%) samples in non-bubble classrooms, and 5/130 (3.8%) samples in washrooms. This contrasted with fomites in households, where SARS-CoV-2 was identified in 60/248 (24.2%) bedroom samples, 66/241 (27.4%) communal room samples, and 21/188 (11.2%) bathroom samples. Air sampling identified SARS-CoV-2 RNA in just 1/68 (1.5%) of school air samples, compared with 21/85 (24.7%) of air samples taken in homes. Interpretation: There was no evidence of large scale SARS-CoV-2 transmission in schools with precautions in place. Low levels of environmental contamination in schools are consistent with low transmission frequency and suggest adequate cleaning and ventilation in schools during the period of study. The high frequency of secondary transmission in households associated with evident viral shedding throughout the home suggests a need to improve advice to households with infection in children in order to prevent onward community spread. The data suggest that SARS-CoV-2 transmission from children in any setting is very likely to occur when precautions are reduced