Application of a High-Resolution Melt Assay for Monitoring SARS-CoV-2 Variants in Burkina Faso and Kenya (preprint)

The rapid emergence and global dissemination of SARS-CoV-2 highlighted a need for robust, adaptable surveillance systems. However, financial and infrastructure requirements for whole genome sequencing (WGS) mean most surveillance data have come from higher-resource geographies, despite unprecedented investment in sequencing in low-middle income countries (LMICs) throughout the SARS-CoV-2 pandemic. Consequently, the molecular epidemiology of SARS-CoV-2 in some LMICs is limited, and there is a need for more cost-accessible technologies to help close data gaps for surveillance of SARS-CoV-2 variants. To address this, we have developed two high-resolution melt curve (HRM) assays that target key variant-defining mutations in the SARS-CoV-2 genome, which give unique signature profiles that define different SARS-CoV-2 variants of concern (VOCs). Extracted RNA from SARS-CoV-2 positive samples collected from 205 participants (112 in Burkina Faso, 93 in Kenya) on the day of enrolment in the MALCOV study (Malaria as a Risk Factor for COVID-19) between February 2021 and February 2022 were analysed using our optimised HRM assays and compared to Next Generation Sequencing (NGS) on Oxford Nanopore MinION . With NGS as a reference, two HRM assays, HRM-VOC-1 and HRM-VOC-2, demonstrated sensitivity/specificity of 100%/99.29% and 92.86/99.39%, respectively, for detecting Alpha, 90.08%/100% and 92.31%/100% for Delta and 93.75%/100% and 100%/99.38% for Omicron. The assays described here provide a lower-cost approach (<$1 per sample) to conducting molecular epidemiology, capable of high-throughput testing. We successfully scaled up the HRM-VOC-2 assay to screen a total of 506 samples from which we were able to show the replacement of Alpha with the introduction of Delta and the replacement of Delta by the Omicron variant in this community in Kisumu, Kenya. These assays are readily adaptable and can focus on local epidemiological surveillance questions or be updated quickly to accommodate the emergence of a novel variant or adapt to novel and emerging pathogens.

Comparison of the analytical and clinical sensitivity of thirty-four rapid antigen tests with the most prevalent SARS-CoV-2 variants of concern during the COVID-19 pandemic in the UK (preprint)

Background The continued emergence of SARS-CoV-2 variants of concern (VOC) requires timely analytical and clinical evaluation of antigen-based rapid diagnostic tests (Ag-RDTs) especially those that are recommended for at home use. Methods The limit of detection (LOD) of 34 Ag-RDTs was evaluated using the most encountered SARS-CoV-2 VOC viral isolates (Alpha, Delta, Gamma, Omicron BA.1, Omicron BA.5) and the wild type (WT). Clinical sensitivity was further evaluated for five Ag-RDT utilising retrospective samples (Alpha, Delta, Omicron BA.1) and one Ag-RDT utilising prospective clinical samples (Delta and Omicron BA.1). Findings For the WT, Alpha, Delta, Gamma and Omicron (BA.1) variants 22, 32, 29, 31 and 32 of the 34 Ag-RDTs evaluated met the World Health Organisations (WHO) target product profile (TPP), respectively. Of the 31 Ag-RDTs included for Omicron BA.5 evaluation 29 met the WHO TPP. Additionally, the LODs for samples spiked with Omicron BA.5 were significantly lower than all other VOCs included (p<0.001). In the retrospective clinical evaluation when comparing RNA copies/mL, the Ag-RDTs detected Alpha and Omicron (BA.1) more sensitively than the Delta VOC. Samples with high RT-qPCR Cts (Ct>25) resulted in reduced test sensitivities across all variants. We used linear regression to model the 50% and 95% LOD of clinical samples and observed statistically similar results for all tests. In the prospective clinical samples, the sensitivity was statistically similar for the Delta VOC 71.9% (CI 95% 53.3-86.6%) and Omicron VOC 84.4% (CI95% 75.3-91.2%). Interpretation Test performance differs between SARS-CoV-2 VOCs, and high sensitivity was achieved when testing the Omicron BA.5 VOC compared to the WHO Ag-RDT requirements. Continuous evaluations must be performed to monitor test performance.

An optimised method for recovery and quantification of laboratory generated SARS-CoV-2 aerosols by plaque assay. (preprint)

We present an optimised method for the recovery of laboratory generated SARS-CoV-2 aerosols and quantification by plaque assays. This method allows easy incorporation into existing standard operating procedures of biological containment level 3 (CL3) laboratories.

Multicentre diagnostic evaluation of OnSite COVID-19 Rapid Test (CTK Biotech) among symptomatic individuals in Brazil and The United Kingdom (preprint)

The COVID-19 pandemic has given rise to numerous commercially available antigen rapid diagnostic tests (Ag-RDTs). To generate and share accurate and independent data with the global community, multi-site prospective diagnostic evaluations of Ag-RDTs are required. This report describes the clinical evaluation of OnSite COVID-19 Rapid Test (CTK Biotech, California, USA) in Brazil and The United Kingdom. A total of 496 paired nasopharyngeal (NP) swabs were collected from symptomatic healthcare workers at Hospital das Clinicas in Sao Paulo, and 211 NP swabs were collected from symptomatic participants at a COVID-19 drive-through testing site in Liverpool, England. These swabs were analysed by Ag-RDT and results were compared to RT-qPCR. The overall clinical sensitivity of the OnSite COVID-19 Rapid test was 79.6% [95% Cl 70.8 - 86.8%] and specificity was 98.5% [95% Cl 97.2 - 99.3%]. Analytical evaluation of the Ag-RDT was assessed using direct culture supernatant of SARS-CoV-2 strains from Wild-Type (WT), Alpha, Delta, Gamma and Omicron lineages. Analytical limit of detection was 1.0x103 pfu/mL, 1.0x103 pfu/mL, 1.0x102 pfu/mL, 5.0x103 pfu/mL and 1.0x103 pfu/mL, giving a viral copy equivalent of approximately 2.1x105 copies/mL, 2.1x104 copies/mL, 1.6x104 copies/mL, 3.5x106 copies/mL and 8.7 x 104 for the Ag-RDT, when tested on the WT, Alpha, Delta, Gamma and Omicron lineages, respectively. Overall, the OnSite Ag-RDT demonstrates a lower clinical sensitivity than claimed by the manufacturers and narrowly missed out on the World Health Organization's minimum performance requirements for sensitivity of Ag-RDTs targeted to SARS-CoV-2. This study provides comparative performance of an Ag-RDT across two different settings, geographical areas, and population.

Head-to head comparison of anterior nares and nasopharyngeal swabs for SARS-CoV-2 antigen detection in a community drive-through test centre in the UK (preprint)

Objective: To conduct a head-to-head diagnostic accuracy evaluation of professionally taken anterior nares (AN) and nasopharyngeal (NP) swabs for SARS-CoV-2 antigen detection using rapid diagnostic tests (Ag-RDT). Methods: NP swabs for SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing and paired AN and NP swabs for the antigen detection were collected from symptomatic participants enrolled at a community drive-through COVID-19 test centre in Liverpool. Two Ag-RDT brands were evaluated: Sure-Status (PMC, India) and Biocredit (RapiGEN, South Korea). The visual read out of the Ag-RDT test band was quantitative scored and the 50% and 95% limit of detection (LoD) of both Ag-RDT brands using AN and NP swabs was calculated using a probabilistic logistic regression model. Results: A total of 604 participants were recruited of which 241 (40.3%) were SARS-CoV-2 positive by RT-qPCR. Sensitivity and specificity of AN swabs was equivalent to the obtained with NP swabs: 83.2% (75.2-89.4%) and 98.8% (96.5-99.6%) utilising NP swabs and 84.0% (76.2-90.1%) and 99.2% (97.0-99.8%) with AN swabs for Sure-Status and; 81.2% (73.1-87.7%) and 99.0% (94.7-86.5%) with NP swabs and 79.5% (71.3-86.3%) and 100% (96.5-100%) with AN swabs for Biocredit. The agreement of the AN and NP swabs was high for both brands with an inter-rater relatability (K) of 0.918 and 0.833 for Sure-Status and Biocredit, respectively. The overall 50% LoD and 95% LoD was 0.9-2.4 x 104 and 3.0-3.2 x 108 RNA copies/mL for NP swabs and 0.3- 1.1 x 105 and 0.7-7.9 x 107 RNA copies/mL and for AN swabs with no significant difference on LoD for any of the swabs types or test brands. Quantitative read-out of test line intensity was more often higher when using NP swabs with significantly higher scores for both Ag-RDT brands. Conclusions: the diagnostic accuracy of the two SARS-CoV-2 Ag-RDTs brands evaluated in this study was equivalent using AN swabs than NP swabs. However, test line intensity was lower when using AN swabs which could influence negatively the interpretation of the Ag-RDT results for lay users. Studies on Ag-RDT self-interpretation using AN and NP swabs are needed to ensure accurate test use in the wider community.

The aerobiology of SARS-CoV-2 in UK hospitals and the impact of aerosol generating procedures (preprint)

BackgroundSARS-CoV-2 nosocomial transmission to patients and healthcare workers (HCWs) has occurred throughout the COVID-19 pandemic. Aerosol generating procedures (AGPs) seemed particularly risky, and policies have restricted their use in all settings. We examined the prevalence of aerosolized SARS-CoV-2 in the rooms of COVID-19 patients requiring AGP or supplemental oxygen compared to those on room air. MethodsSamples were collected prospectively near to adults hospitalised with COVID-19 at two tertiary care hospitals in the UK from November 2020 - October 2021. The Sartorius MD8 AirPort air sampler was used to collect air samples at a minimum distance of 1.5 meters from patients. RT-qPCR was used following overnight incubation of membranes in culture media and extraction. ResultsWe collected 219 samples from patients rooms: individuals on room air (n=67), receiving oxygen (n=65) or AGP (n=67). Of these, 54 (24.6%) samples were positive for SARS-CoV-2 viral RNA. The highest prevalence was identified in the air around patients receiving oxygen (32.3%, n=21, CI95% 22.2 to 44.3%) with AGP and room air recording prevalence of (20.7%, n=18, CI95% 14.1 - 33.7%) and (22.3%, n=15, CI95% 13.5 - 30.4%) respectively. We did not detect a significant difference in the observed frequency of viral RNA between interventions. InterpretationSARS-CoV-2 viral RNA was detected in the air of hospital rooms of COVID-19 patients, and AGPs did not appear to impact the likelihood of viral RNA. Enhanced respiratory protection and appropriate infection prevention and control measures are required to be fully and carefully implemented for all COVID-19 patients to reduce risk of aerosol transmission.

Evaluation of Eight Lateral Flow Tests For The Detection Of Anti-SARS-CoV-2 Antibodies In A Vaccinated Population (preprint)

With the distribution of COVID-19 vaccinations across the globe and the limited access in many countries, quick determination of an individuals antibody status could be beneficial in allocating limited vaccine doses in low- and middle-income countries (LMIC). Antibody lateral flow tests (LFTs) have potential to address this need as a quick, point of care test, they also have a use case for identifying sero-negative individuals for novel therapeutics, and for epidemiology. Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n=60), AZD1222 (AstraZeneca, n=31), mRNA-1273 (Moderna, n=2) and Undeclared Vaccine Brand (n=2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. These tests are quick, low-cost point-of-care tools that can be used without prior training to establish antibody status and may prove valuable for allocating limited vaccine doses in LMICs to ensure those in at risk groups access the protection they need. Further investigation into their performance in vaccinated peoples is required before more widespread utilisation is considered.

A prospective diagnostic evaluation of accuracy of self-taken and healthcare worker-taken swabs for rapid COVID-19 testing (preprint)

ObjectivesTo compare self-taken and healthcare worker (HCW)-taken throat/nasal swabs to perform rapid diagnostic tests (RDT) for SARS-CoV-2, and how these compare to RT-PCR. We hypothesised that self-taken samples are non-inferior for use with RDTs and in clinical and research settings could have substantial individual and public health benefit. DesignA prospective diagnostic accuracy evaluation as part of the Facilitating Accelerated Clinical Evaluation of Novel Diagnostic Tests for COVID -19 (FALCON C-19), workstream C (undifferentiated community testing). SettingNHS Test and Trace drive-through community PCR testing site (Liverpool, UK). Participants Eligible participants 18 years or older with symptoms of COVID-19. 250 participants recruited; one withdrew before analysis. SamplingSelf-administered throat/nasal swab for the Covios(R) RDT, a trained HCW taken throat/nasal sample for PCR and HCW comparison throat/nasal swab for RDT. Main outcome measuresSensitivity, specificity, and positive and negative predictive values (PPV, NPV) were calculated; comparisons between self-taken and HCW-taken samples used McNemars test. ResultsSeventy-five participants (75/249, 30.1%) were positive by RT-PCR. RDTs with self-taken swabs had a sensitivity of 90.5% (67/74, 95% CI: 83.9-97.2), compared to 78.4% (58/74, 95% CI: 69.0-87.8) for HCW-taken swabs (absolute difference 12.2%, 95% CI: 4.7-19.6, p=0.003). Specificity for self-taken swabs was 99.4% (173/174, 95% CI: 98.3-100.0), versus 98.9% (172/174, 95% CI: 97.3-100.0) for HCW-taken swabs (absolute difference 0.6%, 95% CI: 0.5-1.7, p=0.317). The PPV of self-taken RDTs (98.5%, 67/68, 95% CI: 95.7-100.0) and HCW-taken RDTs (96.7%, 58/60, 95% CI 92.1-100.0) were not significantly different (p=0.262). However, the NPV of self-taken swab RDTs was significantly higher (96.1%, 173/180, 95% CI: 93.2-98.9) than HCW-taken RDTs (91.5%, 172/188, 95% CI 87.5-95.5, p=0.003). ConclusionSelf-taken swabs for COVID-19 testing offer substantial individual benefits in terms of convenience, accuracy, and reduced risk of transmitting infection. Our results demonstrate that self-taken throat/nasal samples can be used by lay individuals as part of rapid testing programmes for symptomatic adults. Trial RegistrationIRAS ID:28422, clinical trial ID: NCT04408170 SummaryO_ST_ABSWhat is already known on this topic?C_ST_ABSO_LIRapid diagnostic tests (RDTs)for SARS-CoV-2 Ag are a cheaper point-of-care alternative to RT-PCR for diagnosing COVID-19 disease. C_LIO_LIThe accuracy of tests can vary dependent on sampling technique, test processing and reading of results. C_LI What this study adds?O_LISelf-taken throat-nasal swabs for RDTs can be used by symptomatic adults to give reliable results to diagnose SARS-CoV-2. C_LIO_LISelf-sampling can be implemented with little training and no assistance. C_LI

Field performance of NowCheck Rapid antigen test for SARS-CoV-2 in Kisumu County, western Kenya (preprint)

BackgroundLow- and middle-income countries (LMICs) are increasingly adopting low-cost Coronavirus disease 2019 (COVID-19) rapid antigen tests to meet the high demand for SARS-CoV-2 testing. Whilst testing using real-time polymerase chain reaction (RT-PCR) is the current gold standard, its widespread use in LMICs is limited by high costs, turnaround times and is not readily available in most places. COVID-19 antigen tests (Ag-RDT) provide a suitable alternative due to their low cost, rapid turnaround time and easy to set up and use. This study aimed to assess the field performance of the NowCheck COVID-19 antigen kit (Ag-RDT) as a point of care test (POCT) in select healthcare facilities in western Kenya. MethodsWe conducted a prospective multi-facility field evaluation study of the NowCheck COVID-19 rapid antigen test (Ag-RDT) compared to SARS-CoV-2 RT-PCR (RT-PCR). After obtaining informed consent, trained laboratory technicians collected two pairs of oropharyngeal and nasopharyngeal swabs, both antigen and RT-PCR testing, first for Ag-RDT and next for RT-PCR. We performed Ag-RDTs on-site and shared the results with both the study participants and their healthcare providers within 15-30 minutes. We carried out all RT-PCR tests in a central referral laboratory. The turnaround time for RT-PCR results was typically 24-48 hours. We captured the results of both methods using an electronic digital application. FindingsBetween December 2020 and March 2021, we enrolled 997 participants who met the Kenyan Ministry of Health COVID-19 case definition. The median age of study participants was 39 years (range one to 80 years), with 54% male. Ag-RDT had a sensitivity of 84.5% (76.0-90.8) and a specificity of 94.4% (95% CI: 92.7-95.8) with an accuracy of 94.2% (92.5-95.6) when a cycle threshold value (Ct value) of [≤]35 was used. The highest sensitivity of 87.7% (77.2-94.5) was observed in samples with Ct values [≤] 30 and the highest specificity of 97.5% (96.2-98.5) at Ct value of <40. InterpretationThe NowCheck COVID-19 Ag-RDT showed good performance in field evaluation in multiple healthcare facilities in a developing country. The sensitivity of the kit exceeded the minimum recommended cut-off of 80% as recommended by WHO1. The high specificity of this kit at 94.4% at Ct values [≤]33 and 97.5% at Ct values <40 matched that of real-time PCR, making it a good rule-out test for symptomatic patients with COVID-19-like symptoms. The faster turnaround time to results, lower cost, simple analytical steps requiring no equipment or infrastructure makes antigen testing an attractive field-screening method to meet the high demand for COVID-19 testing. FundingAchmea Foundation, Pfizer Foundation, Dimagi and the Netherlands Ministry of Foreign Affairs supported this project. The funding sources did not have any role in study design, data collection, analysis, interpretation, summarizing the data or decision to submit the manuscript for publication.

Streptococcus pneumoniae colonisation associates with impaired adaptive immune responses against SARS-CoV-2 (preprint)

Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, secondary pneumococcal pneumonia has been reported as infrequent. This apparent contradiction may be explained by interactions of SARS-CoV-2 and pneumococcus in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses. Here, we investigated the relationship of these two respiratory pathogens in two distinct cohorts of a) healthcare workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and b) patients with moderate to severe disease who presented to hospital. We assessed the effect of co-infection on host antibody, cellular and inflammatory responses to the virus. In both cohorts, pneumococcal colonisation was associated with diminished anti-viral immune responses, which affected primarily mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients. Our findings suggest that S. pneumoniae modulates host immunity to SARS-CoV-2 and raises the question if pneumococcal carriage also enables immune escape of other respiratory viruses through a similar mechanism and facilitates reinfection occurrence.

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: an observational cohort study. (preprint)

Background The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the UK National Health Service (NHS). During the first wave of SARS-CoV-2 transmission in UK, SARS-CoV-2 NHS diagnostic test availability was limited to self-isolating symptomatic staff. The burden of symptomatic and asymptomatic infection in healthcare workers (HCW) attending work was unknown. Methods We conducted an observational cohort study of SARS-CoV-2 infection in HCW working in an acute NHS Trust during the first wave of the COVID-19 pandemic, using serial self-collected saliva and nasopharyngeal (NP) samples. We also collected self-assessed symptom profiles and isolation behaviours. We retrospectively compared SARS-CoV-2 detection by RT-PCR from saliva (weekly) and NP swabs (twice weekly) from 85 individuals in this cohort and evaluated the association with symptoms. Findings Over a 12-week period from 30th March 2020, 40% (n=34/85, CI95% 31.3-51.8%) HCWs had evidence of SARS-CoV-2 infection by surveillance NP swab and/or saliva RT-qPCR. Agreement between paired saliva and NP swabs was poor (28.6%, CI95% 13.2-48.7%) with both methods detecting symptomatic and asymptomatic infections. Symptoms were reported by 47.1% (n=40) and self-isolation by 25.9% participants (n=22). Only 41.2% (n=14/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of the infection. Interpretation HCWs are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections in HCW. Saliva is an easily accessible fluid sample for screening for SARS-CoV-2 infection and in addition to NP swab, facilitated ascertainment of symptomatic and asymptomatic cases in this setting. Combined saliva and NP testing would improve detection of SARS-CoV-2 for surveillance. Better understanding of transmissibility from asymptomatic staff using transmission-based infection precautions, is required to inform policy.

Limit of detection in different matrices of nineteen commercially available rapid antigen tests for the detection of SARS-CoV-2 (preprint)

In the context of the coronavirus disease 2019 (COVID-19) pandemic there has been an increase of the use of antigen-detection rapid diagnostic tests (Ag-RDT). The performance of Ag-RDT vary greatly between manufacturers and evaluating their analytical limit of detection (LOD) has become high priority. Here we describe a manufacturer-independent evaluation of the LOD of 19 marketed Ag-RDT using live SARS-CoV-2 spiked in different matrices: direct culture supernatant, a dry swab, and a swab in Amies. Additionally, the LOD using dry swab was investigated after 7 days storage at -80{degrees}C of the SARS-CoV-2 serial dilutions. An LOD of {approx} 5.0 x 102 pfu/ml (1.0 x 106 genome copies/ml) in culture media is defined as acceptable by the World Health Organization. Fourteen of nineteen Ag-RDTs (ActiveXpress, Espline, Excalibur, Innova, Joysbio, Mologic, NowCheck, Orient, PanBio, RespiStrip, Roche, Standard-F, Standard-Q and Sure-Status) exceeded this performance criteria using direct culture supernatant applied to the Ag-RDT. Six Ag-RDT were not compatible with Amies media and a decreased sensitivity of 2 to 20-fold was observed for eleven tests on the stored dilutions at -80{degrees}C for 7 days. Here, we provide analytical sensitivity data to guide appropriate test and sample type selection for use and for future Ag-RDT evaluations.

Immunoglobulin-G Enzyme-Linked Immunosorbent Assay Predicts Neutralising Antibody Response in Convalescent SARS-CoV-2 Patients (preprint)

Severe acute respiratory coronavirus 2 (SARS-CoV-2) has spread globally since its emergence in 2019. Most SARS-CoV-2 infections generate immune responses leading to rising levels of immunoglobulins (Ig) M, A and G which can be detected using diagnostic tests including enzyme-linked immunosorbent assays (ELISA). Whilst implying previous SARS-CoV-2 infection, the detection of Ig by ELISA does not guarantee the presence of neutralising antibodies (NAb) that can prevent the virus infecting cells. Plaque reduction neutralisation tests (PRNT) detect NAb but are not amenable to mass testing as they take several days and require use of viable SARS-CoV-2 in high biocontainment laboratories. We evaluated the ability of IgG and IgM ELISAs targeting SARS-CoV-2 spike subunit 1 (S1) and nucleocapsid protein (NP) at predicting the presence and magnitude of NAb determined by PRNT. SARS-CoV-2 IgG ELISA correlated well with NAb and was highly sensitive (93.8% [95% CI 79.2–99.2]) and specific (88.9% [95% CI 51.8–99.7%]) at predicting the presence of NAb. There was not a strong correlation between IgM ELISA and PRNT result. IgG ELISA provides a useful, high throughput method of predicting the presence of neutralising antibodies, with higher ELISA results increasing the likelihood of having a greater NAb titre.

SARS-CoV-2 Transmission Risk from sports Equipment (STRIKE) (preprint)

OBJECTIVES: To investigate the potential of shared sporting equipment as transmission vectors of SARS-CoV-2 during the reintroduction of sports such as soccer, rugby, cricket, tennis, golf and gymnastics. SETTING: Laboratory based live SARS-CoV-2 virus study. INTERVENTIONS: Ten different types of sporting equipment were inoculated with 40l droplets containing clinically relevant high and low concentrations of live SARS-CoV-2 virus. Materials were then swabbed at time points relevant to sports (1, 5, 15, 30, 90 minutes). The amount of live SARS-CoV-2 recovered at each time point was enumerated using viral plaque assays, and viral decay and half-life was estimated through fitting linear models to log transformed data from each material. MAIN OUTCOME MEASURE: The primary outcome measure was quantification of retrievable SARS-CoV-2 virus from each piece of equipment at pre-determined time points. RESULTS: At one minute, SARS-CoV-2 virus was recovered in only seven of the ten types of equipment with the low dose inoculum, one at five minutes and none at 15 minutes. Retrievable virus dropped significantly for all materials tested using the high dose inoculum with mean recovery of virus falling to 0.74% at 1 minute, 0.39% at 15 minutes and 0.003% at 90 minutes. Viral recovery, predicted decay, and half-life varied between materials with porous surfaces limiting virus transmission. CONCLUSIONS: This study shows that there is an exponential reduction in SARS-CoV-2 recoverable from a range of sports equipment after a short time period, and virus is less transferrable from materials such as a tennis ball, red cricket ball and cricket glove. Given this rapid loss of viral load and the fact that transmission requires a significant inoculum to be transferred from equipment to the mucous membranes of another individual it seems unlikely that sports equipment is a major cause for transmission of SARS-CoV-2. These findings have important policy implications in the context of the pandemic and may promote other infection control measures in sports to reduce the risk of SARS-CoV-2 transmission and urge sports equipment manufacturers to identify surfaces that may or may not be likely to retain transferable virus.

Evaluation of the accuracy, ease of use and limit of detection of novel, rapid, antigen-detecting point-of-care diagnostics for SARS-CoV-2 (preprint)

Abstract Background: Reliable point-of-care (POC) diagnostics not requiring laboratory infrastructure could be a game changer in the COVID-19 pandemic, particularly in the Global South. We assessed performance, limit of detection (LOD) and ease-of-use of three antigen-detecting, rapid POC diagnostics (Ag-RDT) for SARS-CoV-2. Methods: This prospective, multi-centre diagnostic accuracy study, recruited participants suspected to have SARS-CoV2 in Germany and UK. Paired nasopharyngeal swabs (NP) or NP and/or oropharyngeal swabs (OP) were collected from participants (one for clinical real-time reverse transcription polymerase chain reaction (RT-PCR) and one for Ag-RDT testing). Performance of each of three Ag-RDTs was compared to RT-PCR overall, and according to predefined subcategories e.g. cycle threshold (CT)-value, days from symptom onset, etc. In addition, limited verification of analytical limit-of-detection (LOD) was determined. To understand the usability of each Ag-RDT a System Usability Scale (SUS) questionnaire and ease-of-use assessment were performed. Results: Between April 17th and August 25th, 2020, 2417 participants were enrolled, with 70 (3.0%) testing positive by RT-PCR. The best-performing test (SD Biosensor, Inc. STANDARD Q) was 76.6% [95% Confidence Interval (CI) 62.8-86.4] sensitive and 99.3% [CI 98.6-99.6] specific. A sub-analysis showed all samples with RT-PCR CT-values <25 were detectable by STANDARD Q. The test was considered easy-to-use (SUS 86/100) and suitable for POC. Bioeasy and Coris showed specificity of 93.1% [CI 91.0%-94.8%] and 95.8% [CI 93.4%-97.4%], respectively, not meeting the predefined target of [≥]98%. Conclusion: There is large variability in performance of Ag-RDT tests with one test showing promise. Given the usability at POC, these tests are likely to have impact despite imperfect sensitivity; however further research and modelling are needed.

Self-sampling of capillary blood for serological testing of SARS-CoV-2 by COVID-19 IgG ELISA (preprint)

Serological testing is emerging as a powerful tool to progress our understanding of COVID-19 exposure, transmission and immune response. Large-scale testing is limited by the need for in-person blood collection by staff trained in venepuncture. Capillary blood self-sampling and postage to laboratories for analysis could provide a reliable alternative. Two-hundred and nine matched venous and capillary blood samples were obtained from thirty nine participants and analysed using a COVID-19 IgG ELISA to detect antibodies against SARS-CoV-2. Thirty seven out of thirty eight participants were able to self-collect an adequate sample of capillary blood ([≥]50 l). Using plasma from venous blood collected in lithium heparin as the reference standard, matched capillary blood samples, collected in lithium heparin-treated tubes and on filter paper as dried blood spots, achieved a Cohen's kappa coefficient of >0.88 (near-perfect agreement). Storage of capillary blood at room temperature for up to 7 days post sampling did not affect concordance. Our results indicate that capillary blood self-sampling is a reliable and feasible alternative to venepuncture for serological assessment in COVID-19.

Wide variabilities identified among spike proteins of SARS Cov2 globally-dominant variant identified (preprint)

SARS Cov2 is a newly emerged virus causing pandemic with fatality and co-morbidity. The greatest limitations emerged is the lack of effective treatment and vaccination due to frequent mutations and reassortment of the virus, leading to evolvement of different strains. We identified a wide variability in the whole genome sequences as well as spike protein variants (responsible for binding with ACE2 receptor) of SARS Cov2 identified globally. Structural variations of spike proteins identified from representative countries from all the continents, seven of them have revealed genetically similar, may be regarded as the dominant type. Novel non-synonymous mutations as S247R, R408I, G612D, A930V and deletion detected at amino acid position 144 . RMSD values ranging from 4.45 to 2.25 for the dominant variant spike1 with other spike proteins. This study is informative for future vaccine research and drug development with the dominant type.

Variation of SARS-CoV-2 viral loads by sample type, disease severity and time: a systematic review (preprint)

Background. To describe whether SARS-CoV-2 viral loads (VLs) and cycle thresholds (CTs) vary by sample type, disease severity and symptoms duration. Methods. Systematic searches were conducted in MEDLINE, EMBASE, BioRxiv and MedRxiv. Studies reporting individual SARS-CoV-2 VLs and/or CT values from biological samples. Paired reviewers independently screened potentially eligible articles. CT values and VLs distributions were described by sample type, disease severity and time from symptom onset. Differences between groups were examined using Kruskal-Wallis and Dunn's tests (post-hoc test). The risk of bias was assessed using the Joanna Briggs Critical Appraisal Tools. Results. 14 studies reported CT values, 8 VLs and 2 CTs and VLs, resulting in 432 VL and 873 CT data points. VLs were higher in saliva and sputum (medians 4.7x108 and 6.5x104 genomes per ml, respectively) than in nasopharyngeal and oropharyngeal swabs (medians 1.7x102 and 4.8x103). Combined naso/oropharyngeal swabs had lower CT values (i.e. higher VLs) than single site samples (p=<0.0001). CT values were also lower in asymptomatic individuals and patients with severe COVID-19 (median CT 30 for both) than among patients with moderate and mild symptoms (31.4 and 31.3, respectively). Stool samples were reported positive for a longer period than other specimens. Conclusion. VLs are higher in saliva and sputum and in individuals who are asymptomatic of with severe COVID-19. Diagnostic testing strategies should consider that VLs vary by sample type, disease severity and time since symptoms onset.

High SARS-CoV-2 seroprevalence in Health Care Workers but relatively low numbers of deaths in urban Malawi (preprint)

Background In low-income countries, like Malawi, important public health measures including social distancing or a lockdown, have been challenging to implement owing to socioeconomic constraints, leading to predictions that the COVID-19 pandemic would progress rapidly. However, due to limited capacity to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, there are no reliable estimates of the true burden of infection and death. We, therefore, conducted a SARS-CoV-2 serosurvey amongst health care workers (HCW) in Blantyre city to estimate the cumulative incidence of SARS-CoV-2 infection in urban Malawi. Methods Five hundred otherwise asymptomatic HCWs were recruited from Blantyre City (Malawi) from 22nd May 2020 to 19th June 2020 and serum samples were collected all participants. A commercial ELISA was used to measure SARS-CoV-2 IgG antibodies in serum. We run local negative samples (2018 - 2019) to verify the specificity of the assay. To estimate the seroprevalence of SARS CoV-2 antibodies, we adjusted the proportion of positive results based on local specificity of the assay. Results Eighty-four participants tested positive for SARS-CoV-2 antibodies. The HCW with a positive SARS-CoV-2 antibody result came from different parts of the city. The adjusted seroprevalence of SARS-CoV-2 antibodies was 12.3% [CI 9.0-15.7]. Using age-stratified infection fatality estimates reported from elsewhere, we found that at the observed adjusted seroprevalence, the number of predicted deaths was 8 times the number of reported deaths. Conclusion The high seroprevalence of SARS-CoV-2 antibodies among HCW and the discrepancy in the predicted versus reported deaths, suggests that there was early exposure but slow progression of COVID-19 epidemic in urban Malawi. This highlights the urgent need for development of locally parameterised mathematical models to more accurately predict the trajectory of the epidemic in sub-Saharan Africa for better evidence-based policy decisions and public health response planning.

Saliva offers a sensitive, specific and non-invasive alternative to upper respiratory swabs for SARS-CoV-2 diagnosis. (preprint)

RT-qPCR utilising upper respiratory swabs are the diagnostic gold standard for SARS-CoV-2 despite reported low sensitivity and limited scale up due to global shortages. Saliva is a non-invasive, equipment independent alternative to swabs. We collected 145 paired saliva and nasal/throat (NT) swabs at diagnosis (day 0) and repeated on day 2 and day 7 dependent on inpatient care and day 28 for study follow up. Laboratory cultured virus was used to determine the analytical sensitivity of spiked saliva and swabs containing amies preservation media. Self-collected saliva samples were found to be consistent, and in some cases superior when compared to healthcare worker collected NT swabs from COVID-19 suspected participants. We report for the first time the analytical limit of detection of 10-2 and 100 pfu/ml for saliva and swabs respectively. Saliva is a easily self-collected, highly sensitive specimen for the detection of SARS-CoV-2.