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      Serological assays for delayed SARS-CoV-2 case identification – Author's reply

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          Abstract

          We read with interest the insightful comments put forward by Kay Weng Choy, raising important considerations for clinicians planning to use point-of-care serological assays for delayed case identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in response to those presented in our Article. 1 We agree that along with evaluating assays for cross-reactivity of IgM and IgG with common infectious diseases, further benefit could be derived by assessing the potential of assay performance in those with autoimmune disease and immunodeficiency. Indeed, previously reported work in SARS-CoV-1 would suggest the potential for cross-reactivity of autoantibodies for SARS-CoV-2 IgG. 2 Similarly, consideration has also been given to how age could affect viral load and the subsequent development of SARS-CoV-2 IgG antibodies, 3 which is a focus of our ongoing work. Our study was designed specifically to evaluate the use of point-of-care assays for frontline health-care workers directly involved in the clinical care of patients with SARS-CoV-2 infection; therefore, it was not possible to evaluate any difference in detection of SARS-CoV-2 IgG in young or older people. At a strategic level, health-care workers with autoimmune disease or known immunodeficiency were required to be actively shielding during the study period and so were unable to take part. 4 An evaluation of the potential effect of immunodeficiency on assay performance was beyond the scope of our study; however, we strongly agree that this is an important issue for future studies where consideration can be given to testing in different populations. Additionally, Kay Weng Choy correctly highlights that whole blood is likely to be the primary sample type at point-of-care and, therefore, evaluation of diagnostic performance is warranted for whole blood and serum samples. Further research involving our group has been reported in August, 2020, comparing not only serum with whole blood samples in the laboratory, but also with finger-prick testing across a number of different point-of-care assays. 5 Observed test sensitivity was broadly similar; however, the reported variation in assay performance across these three methods highlights the need for robust evaluation of individual kits (as they become available) in specific populations. This variation is particularly relevant if consideration is being given to use with finger-prick blood. In our study, individuals were recruited on a single occasion and no repeat testing was considered in the performance evaluation. Finally, Kay Weng Choy highlights the value of orthogonal testing algorithms, advocating for a second test, each with unique assay design characteristics with the aim of improving the positive predictive value. Indeed, within our own institutions we have developed a testing algorithm that uses an anti-nucleocapsid and anti-spike protein immunoassay. This algorithm has potential to increase diagnostic yield but it is worth noting that insufficient antibody target data provided by a considerable number of manufacturers could provide additional challenges to the design of similar testing programmes. 6

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          SARS-CoV-2: virus dynamics and host response

          Since December, 2019, coronavirus disease 2019 (COVID-19) has affected more than 100 000 patients globally. 1 COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has a case-fatality rate of 2·3%, with higher rates among elderly patients and patients with comorbidities. 2 Person-to-person transmission is efficient, with multiple clusters reported. Clinically, patients with COVID-19 present with respiratory symptoms, which is very similar to the presentation of other respiratory virus infections. Radiologically, COVID-19 is characterised by multifocal ground-glass opacities, even for patients with mild disease. 3 Knowledge of virus dynamics and host response are essential for formulating strategies for antiviral treatment, vaccination, and epidemiological control of COVID-19. However, a systematic study on these aspects has not been done. In The Lancet Infectious Diseases, Kelvin To and colleagues 4 report the viral load and antibody profiles of a cohort of 23 patients admitted to hospital with COVID-19. In these patients, the viral load peaked during the first week of illness then gradually declined over the second week. Viral load was also shown to correlate with age. Furthermore, both IgG and IgM antibodies started to increase on around day 10 after symptom onset, and most patients had seroconversion within the first 3 weeks. Finally, the IgG and IgM antibody level against the SARS-CoV-2 internal nucleoprotein and the surface spike receptor binding domain correlated with neutralising activity. These findings have several practical implications. First, the high viral load during the early phase of illness suggests that patients could be most infectious during this period, and it might account for the high transmissibility of SARS-CoV-2. Furthermore, the high viral load on presentation suggests that SARS-CoV-2 could be susceptible to emergence of antiviral resistance. Second, age was associated with viral load in this study, which could explain the high degree of severe disease in older patients with SARS-CoV-2.5, 6 The high viral load in elderly patients is associated not only with low immunity but also with high expression of the ACE2 receptor (the cell-entry receptor for SARS-CoV-2) in older adults. 7 The timing of antibody seroconversion is crucial for determining the optimum timepoints for collecting serum specimens for antibody testing for diagnosis. Furthermore, this information is important for immunologists to choose the best timepoints for obtaining peripheral blood B cells for development of therapeutic monoclonal antibodies. 8 The major strength of the study by To and colleagues is the systematic analysis of the serial viral load and antibody profile for up to 4 weeks, which provides insights into viral and host interactions during the acute and convalescent phases. Another notable aspect is that self-collected posterior oropharyngeal saliva samples were used, instead of nasopharyngeal specimens, for viral load monitoring. Collection of nasopharyngeal specimens is an invasive procedure, and it is uncomfortable for the patient and poses an infection risk to health-care workers. Self-collected saliva is much more acceptable to patients and is safer for health-care workers. This study clearly shows the feasibility of using saliva for viral load monitoring. The information provided by To and colleagues is solid scientific evidence on COVID-19 for clinicians and scientists. Nonetheless, many questions are still outstanding on the viral characteristics and host response during infection. SARS-CoV-2 has been detected in faeces, blood, and urine samples,9, 10 and it is important to ascertain viral load dynamics in such samples, for prevention and control of the pandemic. Furthermore, the relation between viral load and disease severity needs to be further clarified. Studies with a larger sample size are needed to understand how different factors can affect viral load or antibody response. For example, immunocompromised patients might have higher viral load, prolonged viral shedding, and impaired antibody response. Future studies in the paediatric population are vital, because children seem to have much milder disease than in adults. Finally, a more detailed understanding of the innate and adaptive immune response against SARS-CoV-2 is important for understanding the pathogenesis and for designing vaccines. © 2020 Flickr-Ben (busy) 2020 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
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            Point-of-care serological assays for delayed SARS-CoV-2 case identification among health-care workers in the UK: a prospective multicentre cohort study

            Summary Background Health-care workers constitute a high-risk population for acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Capacity for acute diagnosis via PCR testing was limited for individuals with mild to moderate SARS-CoV-2 infection in the early phase of the COVID-19 pandemic and a substantial proportion of health-care workers with suspected infection were not tested. We aimed to investigate the performance of point-of-care and laboratory serology assays and their utility in late case identification, and to estimate SARS-CoV-2 seroprevalence. Methods We did a prospective multicentre cohort study between April 8 and June 12, 2020, in two phases. Symptomatic health-care workers with mild to moderate symptoms were eligible to participate 14 days after onset of COVID-19 symptoms, as per the Public Health England (PHE) case definition. Health-care workers were recruited to the asymptomatic cohort if they had not developed PHE-defined COVID-19 symptoms since Dec 1, 2019. In phase 1, two point-of-care lateral flow serological assays, the Onsite CTK Biotech COVID-19 split IgG/IgM Rapid Test (CTK Bitotech, Poway, CA, USA) and the Encode SARS-CoV-2 split IgM/IgG One Step Rapid Test Device (Zhuhai Encode Medical Engineering, Zhuhai, China), were evaluated for performance against a laboratory immunoassay (EDI Novel Coronavirus COVID-19 IgG ELISA kit [Epitope Diagnostics, San Diego, CA, USA]) in 300 samples from health-care workers and 100 pre-COVID-19 negative control samples. In phase 2 (n=6440), serosurveillance was done among 1299 (93·4%) of 1391 health-care workers reporting symptoms, and in a subset of asymptomatic health-care workers (405 [8·0%] of 5049). Findings There was variation in test performance between the lateral flow serological assays; however, the Encode assay displayed reasonable IgG sensitivity (127 of 136; 93·4% [95% CI 87·8–96·9]) and specificity (99 of 100; 99·0% [94·6–100·0]) among PCR-proven cases and good agreement (282 of 300; 94·0% [91·3–96·7]) with the laboratory immunoassay. By contrast, the Onsite assay had reduced sensitivity (120 of 136; 88·2% [95% CI 81·6–93·1]) and specificity (94 of 100; 94·0% [87·4–97·8]) and agreement (254 of 300; 84·7% [80·6–88·7]). Five (7%) of 70 PCR-positive cases were negative across all assays. Late changes in lateral flow serological assay bands were recorded in 74 (9·3%) of 800 cassettes (35 [8·8%] of 400 Encode assays; 39 [9·8%] of 400 Onsite assays), but only seven (all Onsite assays) of these changes were concordant with the laboratory immunoassay. In phase 2, seroprevalence among the workforce was estimated to be 10·6% (95% CI 7·6–13·6) in asymptomatic health-care workers and 44·7% (42·0–47·4) in symptomatic health-care workers. Seroprevalence across the entire workforce was estimated at 18·0% (95% CI 17·0–18·9). Interpretation Although a good positive predictive value was observed with both lateral flow serological assays and ELISA, this agreement only occurred if the pre-test probability was modified by a strict clinical case definition. Late development of lateral flow serological assay bands would preclude postal strategies and potentially home testing. Identification of false-negative results among health-care workers across all assays suggest caution in interpretation of IgG results at this stage; for now, testing is perhaps best delivered in a clinical setting, supported by government advice about physical distancing. Funding None.
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              Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey

              Background Accurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required. Methods Sensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by reverse transcription PCR and were ≥21 days from symptom onset. In phase I, we evaluated five LFIAs in clinic (with finger prick) and laboratory (with blood and sera) in comparison to (1) PCR-confirmed infection and (2) presence of SARS-CoV-2 antibodies on two ‘in-house’ ELISAs. Specificity analysis was performed on 500 prepandemic sera. In phase II, six additional LFIAs were assessed with serum. Findings 95% (95% CI 92.2% to 97.3%) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8 out of 11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21% to 92% versus PCR-confirmed cases and from 22% to 96% versus composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2%–99.8%). Interpretation LFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI 97.1% to 99.4%)), moderate sensitivity (84.4% with finger prick (95% CI 70.5% to 93.5%)) and moderate concordance, suitable for seroprevalence surveys.
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                Author and article information

                Journal
                Lancet Respir Med
                Lancet Respir Med
                The Lancet. Respiratory Medicine
                Elsevier Ltd.
                2213-2600
                2213-2619
                14 September 2020
                14 September 2020
                Affiliations
                [a ]Centre of Defence Pathology, Royal Centre for Defence Medicine, Queen Elizabeth Hospital Birmingham, Birmingham B15 2WB, UK
                [b ]Chelsea and Westminster Hospital NHS Foundation Trust, London, UK
                [c ]NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance, Imperial College London, London, UK
                [d ]North West London Pathology, London, UK
                Article
                S2213-2600(20)30406-9
                10.1016/S2213-2600(20)30406-9
                7489942
                32941851
                bb77e48d-bd11-4044-8777-f2c0804eb03e
                © 2020 Elsevier Ltd. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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