There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control.
Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4% of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results.
We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples.
The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.
Background: There is an urgent need to understand the complex relationship between cross-reactive anti-viral immunity, disease susceptibility, and severity in the face of differential exposure to related, circulating Flaviviruses. Co-exposure to Dengue virus and Zika virus in Brazil is a case in point. A devastating aspect of the 2015-2016 South American Zika outbreak was the dramatic increase in numbers of infants born with microcephaly to mothers exposed to Zika virus during pregnancy. It has been proposed that this is more likely to ensue from Zika infection in women lacking cross-protective Dengue immunity. In this case series we measure the prevalence of Dengue immunity in a cohort of mothers exposed to Zika virus during pregnancy in the 2015-2016 Zika outbreak that gave birth to an infant affected by microcephaly and explore their adaptive immunity to Zika virus. Results: Fifty women from Sergipe, Brazil who gave birth to infants with microcephaly following Zika virus exposure during the 2015-16 outbreak were tested for serological evidence of Dengue exposure and IFNγ ELISpot spot forming cell (SFC) response to Zika virus. The majority (46/50) demonstrated Dengue immunity. IFNγ ELISpot responses to Zika virus antigens showed the following hierarchy: Env>NS1>NS3>C protein. Twenty T cell epitopes from Zika virus Env were identified. Responses to Zika virus antigens Env and NS1 were polyfunctional with cells making IFNγ, TNFα, IL-4, IL-13, and IL-10. In contrast, responses to NS5 only produced the immune regulatory TGFβ1 cytokine. There were SFC responses against Zika virus Env (1-20) and variant peptide sequences from West Nile virus, Dengue virus 1-4 and Yellow Fever virus. Conclusion: Almost all the women in our study showed serological evidence of Dengue immunity, suggesting that microcephaly can occur in DENV immune mothers. T cell Reynolds et al. CD4 Immunity in ZIKV Infection immunity to Zika virus showed a multifunctional response to the antigens Env and NS1 and immune regulatory responses to NS5 and C protein. Our data support an argument that different viral products may skew the antiviral response to a more pro or anti-inflammatory outcome, with an associated impact on immunopathogenesis.
Background. Transmission of SARS-CoV-2 by children and young people in school settings has not been directly evaluated, nor the main mechanisms of transmission identified. The study set out to undertake sequential longitudinal sampling of infected children, their contacts, and the environment.
Methods. Cases of COVID-19 were identified through statutory notification and matched to schools reporting cases. Cases of COVID-19 and their contacts from school and home were longitudinally sampled and tested for SARS-CoV-2. Surfaces and air in the home and school environment were also subject to longitudinal sampling and testing.
Results Onward transmission of virus to immediate classroom members who participated in the study was not detected. Evidence of more widespread transmission among children remaining in school was not identified with the exception of one unexpected cluster of three asymptomatic cases in one school. Children infected with SARS-CoV-2 in this study shed viral RNA for up to 10 days from symptom onset, with levels peaking at 5-8 days. Viral RNA was identified in the environment around children who were actively shedding virus in the home, but limited contamination was identified in schools. Variant of Concern B1.1.7 was identified in later cases studied.
Summary After 3 months, this small study has not found evidence to suggest COVID-19 is commonly transmitted by children within schools. A minority of infections may be subject to stochastic events that can lead to transmission. Further prospective and retrospective studies are required to identify factors associated with such events .
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