ABSTRACT
: COVID-19 vaccination certificates (CVCs) have played a key role in safe reopening of borders for international travel and trade, so understanding key stakeholder perceptions of enablers and barriers for their effective use is critical. The COVID-19 Vaccination Policy Research and Deci-sion-Support Initiative in Asia (CORESIA) was established to address policy questions related to CVCs. We conducted two online surveys, i.e., one for the public and one for health and non-health sector experts, from June to October 2021 in nine Asian countries. Descriptive analysis identified participants, enablers, and barriers. Most participants (78% public, 89% experts) accepted the use of CVCs, primarily to resume international travel (76%). Most respondents in both surveys wanted the minimum vaccination coverage to be 60% before CVCs were implemented nation-wide. Most of the public (82%) agreed to maintain existing non-pharmaceutical interventions, while most experts wanted risk-based testing and quarantine policy for incoming travellers (51%) and both digital and paper format CVCs (64%). Support for CVCs for international travel remains high in Asia. Recognising key enablers and barriers for effective use of CVCs from COVID-19 pandemic may help policymakers draft effective border policies for future epidemics.
Subject(s)
COVID-19ABSTRACT
Background: Antibodies and T cells cooperate to control virus infections. The definition of the correlates of protection necessary to manage the COVID-19 pandemic, require both immune parameters but the complexity of traditional tests limits virus-specific T cell measurements. Methods: We test the sensitivity and performance of a simple and rapid SARS-CoV-2 Spike-specific T cell test based on stimulation of whole blood with peptides covering the SARS-CoV-2 Spike protein followed by cytokine (IFN-{gamma}, IL-2) measurement in different cohorts including BNT162b2 vaccinated (n=112; 201 samples), convalescent asymptomatic (n=62; 62 samples) and symptomatic (n=68; 115 samples) COVID-19 patients and SARS-CoV-1 convalescent individuals (n=12; 12 samples). Results: The sensitivity of the rapid cytokine whole blood test equates traditional methods of T cell analysis (ELISPOT, Activation Induced Markers). Utilizing this test we observed that Spike-specific T cells in vaccinated preferentially target the S2 region of Spike and that their mean magnitude is similar between them and SARS-CoV-2 convalescents at 3 months after vaccine or virus priming respectively. However, a wide heterogeneity of Spike-specific T cell magnitude characterizes the individual responses irrespective of the time of analysis. No correlation between neutralizing antibody levels and Spike-specific T cell magnitude were found. Conclusions: Rapid measurement of cytokine production in whole blood after peptide activation revealed a wide dynamic range of Spike-specific T cell response after vaccination that cannot be predicted from neutralizing antibody quantities. Both Spike-specific humoral and cellular immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.
Subject(s)
Severe Acute Respiratory Syndrome , Tumor Virus Infections , COVID-19ABSTRACT
From January 2020, Singapore implemented comprehensive measures to suppress SARS-CoV-2. Community transmission has been limited, although explosive outbreaks have occurred in migrant worker dormitories. We conducted longitudinal SARS-CoV-2 serology studies among 478 residents of a SARS-CoV-2 affected migrant worker dormitory between May and July 2020, and 937 community-dwelling adult Singapore residents with sera collected before September 2019 and in November/December 2020. By end 2020, <2 per 1000 (0.16%, 95% CrI: 0.008% - 0.72%) adult residents in the community were infected with SARS-CoV-2, approximately 4 times higher than the national notified case incidence. In contrast, in the migrant worker cohort, nearly two-thirds 63.8% (95% CrI: 57.9% - 70.3%) had been infected by July 2020; no symptoms were reported in >90% of these infections. SARS-CoV-2 suppression is feasible with rapid implementation of comprehensive control measures. However, the risk of large-scale epidemics in densely-populated environments requires specific consideration in preparedness planning.
ABSTRACT
Background: From January 2020, Singapore implemented comprehensive measures to suppress SARS-CoV-2. Community transmission has been limited, although explosive outbreaks have occurred in migrant worker dormitories.Methods: We conducted longitudinal SARS-CoV-2 serology studies among 478 residents of a SARS-CoV-2 affected migrant worker dormitory between May and July 2020, and 937 community-dwelling adult Singapore residents with sera collected before September 2019 and in November/December 2020. We tested sera for SARS-CoV-2 neutralising antibodies and used Bayesian methods to estimate infection incidence accounting for test sensitivity and specificity.Findings: We estimated that <2 per 1000 adult residents in the community were infected with SARS-CoV-2 in 2020 (cumulative seroprevalence: 0.16% (95% CrI: 0.008% - 0.72%), approximately 4 times higher than the national notified case incidence. In contrast, in the migrant worker cohort, nearly two-thirds had been infected by July 2020 (cumulative seroprevalence: 63.8% (95% CrI: 57.9% - 70.3%); no symptoms were reported in almost all of these infections.Conclusions: Our findings demonstrate that SARS-CoV-2 suppression is possible with rapid implementation of border restrictions, case isolation, contact tracing, quarantining and social distancing measures. However, the risk of large-scale epidemics in densely-populated environments requires specific consideration in preparedness planning. Prioritising these settings in vaccination strategies should minimise risk of future resurgences and potential for spillover to the wider community.Funding: This work was funded by the Wellcome Trust grant (221013/Z/20/Z), the NUS Efforts Against COVID-19 fund and the Singapore National Medical Research Council (STPRG-FY19-001 and COVID19RF-003).Declaration of Interests: L-FW and WNC are co-inventors of a patent application for the cPass test kit. The remaining authors declare no competing interests in relation to this work.Ethics Approval Statement: The community cohort study was approved by the National University of Singapore Institutional Review Board (reference H-20-032).
Subject(s)
COVID-19ABSTRACT
The viral load of SARS-CoV-2 in clinical samples as measured by the primary diagnostic tool of RT-PCR is an imperfect readout for infection potential as most targeted assays designed for sensitivity, indiscriminately detect short and long RNA fragments, although infectivity is embodied only in the whole virus and its intact genome. Here, we used next-generation sequencing (NGS) to characterize 155 clinical samples and show sensitive and quantitative detection of viral RNA which confirmed subgenomic RNA in 57.6% of samples and provided a novel method to determine relative integrity of viral RNA in samples. The relative abundance of long fragments quantified as a viral fragmentation score was positively associated with viral load and inversely related to time from disease onset. An empirically determined score cut-off for presence of substantially fragmented RNA was able to identify 100% of samples collected after 8 days of illness with poor infection potential in line with current clinical understanding of infectiousness of SARS-CoV-2. The quantification of longer fragments in addition to existing short targets in an NGS or RT-PCR-based assay could provide a valuable readout of infection potential simultaneous to the detection of any fragments of SARS-CoV-2 RNA in test samples.
ABSTRACT
Mass testing has been proposed as a strategy to address and contain the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We have developed MassMark, a novel and highly scalable multiplex method that employs next generation sequencing for high-throughput, accurate and sensitive detection of SARS-CoV-2, while minimizing handling complexity and resources by utilizing a serial pooling strategy to accommodate over 9,000 samples per workflow. Analytical validation showed that MassMark was able to detect SARS-CoV-2 RNA down to a level of 100 copies per reaction. We evaluated the clinical performance of MassMark in a simulated screening testing with 22 characterized samples from three different sources (nasopharyngeal swabs, nasal swabs and saliva), comprising of 12 SARS-CoV-2 positive samples with mid to late Ct values (range: 22.98-32.72) and 10 negative samples. There was one false negative and no false positives, giving an overall sensitivity and specificity of 91.67% and 100% respectively, when compared against an optimized RT-PCR test with a target size within 70 bp (CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel).
Subject(s)
Coronavirus InfectionsABSTRACT
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.
Subject(s)
Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
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.
Subject(s)
COVID-19ABSTRACT
Background Active cases of COVID-19 has primarily been diagnosed via RT-PCR of nasopharyngeal (NP) swabs. Saliva and self-administered nasal (SN) swabs can be collected safely without trained staff. We aimed to test the sensitivity of naso-oropharyngeal saliva and SN swabs compared to NP swabs in a large cohort of migrant workers in Singapore. Methods We recruited 200 male adult subjects: 45 with acute respiratory infection, 104 asymptomatic close contacts, and 51 confirmed COVID-19 cases. Each subject underwent NP swab, SN swab and saliva collection for RT-PCR testing at 1 to 3 timepoints. We additionally used a direct-from-sample amplicon-based next-generation sequencing (NGS) workflow to establish phylogeny. Results Of 200 subjects, 91 and 46 completed second and third rounds of testing, respectively. Of 337 sets of tests, there were 150 (44.5%) positive NP swabs, 127 (37.7%) positive SN swabs, and 209 (62.0%) positive saliva. Test concordance between different sample sites was good, with a kappa statistic of 0.616 for NP and SN swabs, and 0.537 for NP and saliva. In confirmed symptomatic COVID-19 subjects, the likelihood of a positive test from any sample fell beyond 14 days of symptom onset. NGS was conducted on 18 SN and saliva samples, with phylogenetic analyses demonstrating lineages for all samples tested were Clade O (GISAID nomenclature) and lineage B.6 (PANGOLIN nomenclature). Conclusion This study supports saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further delineates the role of oropharyngeal secretions in increasing the sensitivity of testing. However, SN swabs were inferior as an alternate sample type. Our study also provides evidence that a straightforward next-generation sequencing workflow can provide direct-from-sample phylogenetic analysis for public health decision-making.