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.1Pallett SJC Rayment M Patel A et al.Point-of-care serological assays for delayed SARS-CoV-2 case identification among health-care workers in the UK: a prospective multicentre cohort study.Lancet Respir Med. 2020; 8: 885-894Summary Full Text Full Text PDF PubMed Scopus (84) Google Scholar 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.2Wang YS Shen H Sun SH et al.Analysis of false-positive associated with antibody tests for SARS-CoV in SLE patients.Shi Yan Sheng Wu Xue Bao. 2003; 36 (in Chinese).: 314-317PubMed Google Scholar Similarly, consideration has also been given to how age could affect viral load and the subsequent development of SARS-CoV-2 IgG antibodies,3Chen Y Li L SARS-CoV-2: virus dynamics and host response.Lancet Infect Dis. 2020; 20: 515-516Summary Full Text Full Text PDF PubMed Scopus (193) Google Scholar 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.4Public Health EnglandGuidance on shielding and protecting people who are clinically extremely vulnerable from COVID-19.https://www.gov.uk/government/publications/guidance-on-shielding-and-protecting-extremely-vulnerable-persons-from-covid-19/guidance-on-shielding-and-protecting-extremely-vulnerable-persons-from-covid-19Date: August 18, 2020Date accessed: August 26, 2020Google Scholar 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.5Flower B Brown JC Simmons B et al.Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey.Thorax. 2020; (published online Aug 12.)https://doi.org/10.1136/thoraxjnl-2020-215732Crossref PubMed Scopus (90) Google Scholar 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.6Pallett SJ Jones R Pallett MA et al.Characterising differential antibody response is integral to future SARS-CoV-2 serostudies.J Infect. 2020; (published online July 31.)http://doi.org/10.1016/j.jinf.2020.07.029Summary Full Text Full Text PDF PubMed Scopus (3) Google Scholar LSPM has consulted for bioMerieux (2013–20), DNAelectronics (2015–18), Dairy Crest (2017–18), Umovis Lab (2020), and Pfizer (2018–20), received speaker fees from Profile Pharma (2018), received research grants from the National Institute for Health Research (2013–20), CW+ Charity (2018–19), and Leo Pharma (2016), and received educational support from Eumedica (2016–18). NM has received speaker fees from Beyer (2016) and Pfizer (2019) and received educational support from Eumedica (2016) and Baxter (2017). RJ has received honoraria, speaker fees, travel support and research grant funding from Gilead, ViiV Healthcare, BMS, AbbVie, Janssen and Merck. SJCP has received a research grant from the Scientific Exploration Society with support from the Viscount Gough. EC has received speaker fees from bioMerieux (2019). All other authors declare no competing interests. Point-of-care serological assays for delayed SARS-CoV-2 case identification among health-care workers in the UK: a prospective multicentre cohort studyAlthough 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. Full-Text PDF Serological assays for delayed SARS-CoV-2 case identificationIn their Article, published in The Lancet Respiratory Medicine in July, 2020, on point-of-care serological assays for delayed severe acute respiratory coronavirus 2 (SARS-CoV-2) case identification among health-care workers in the UK, Pallett and colleagues1 evaluated two lateral flow serological assays. Full-Text PDF
Abstract Background: Accurately predicting patient outcomes in SARS-CoV-2 could aid patient management and allocation of healthcare resources. There are a variety of methods which can be used to develop prognostic models, ranging from logistic regression and survival analysis to more complex machine learning algorithms and deep learning. Despite several models having been created for SARS-CoV-2, most of these have been found to be highly susceptible to bias. We aimed to develop and compare two separate predictive models for death during admission with SARS-CoV-2. Method: Between March 1 - April 24, 2020, 398 patients were identified with laboratory confirmed SARS-CoV-2 in a London teaching hospital. Data from electronic health records were extracted and used to create two predictive models using: 1) a Cox regression model and 2) an artificial neural network (ANN). Model performance profiles were assessed by validation, discrimination, and calibration. Results: Both the Cox regression and ANN models achieved high accuracy (83.8%, 95% confidence interval (CI): 73.8 - 91.1 and 90.0%, 95% CI: 81.2 - 95.6, respectively). The area under the receiver operator curve (AUROC) for the ANN (92.6%, 95% CI: 91.1 - 94.1) was significantly greater than that of the Cox regression model (86.9%, 95% CI: 85.7 - 88.2), p=0.0136. Both models achieved acceptable calibration with Brier scores of 0.13 and 0.11 for the Cox model and ANN, respectively. Conclusion: We demonstrate an ANN which is non-inferior to a Cox regression model but with potential for further development such that it can learn as new data becomes available. Deep learning techniques are particularly suited to complex datasets with non-linear solutions, which make them appropriate for use in conditions with a paucity of prior knowledge. Accurate prognostic models for SARS-CoV-2 can provide benefits at the patient, departmental and organisational level.
Abstract Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Case identification is currently made by real-time polymerase chain reaction (PCR) during the acute phase and largely restricted to healthcare laboratories. Serological assays are emerging but independent validation is urgently required to assess their utility.We evaluated five different point-of-care (POC) SARS-CoV-2 antibody test kits against PCR, finding concordance across the assays ( n= 15). We subsequently tested 200 patients using the OrientGene COVID-19 IgG/IgM Rapid Test Cassette and find a sensitivity of 74% in the early infection period (day 5-9 post symptom onset), with 100% sensitivity not seen until day 13. Specificity was 96%, but in validating the serological tests uncovered potential false-negatives from PCR testing late-presenting cases. A positive predictive value (PPV) of 37% in the general population precludes any use for general screening. Where a case definition is applied however, the PPV is substantially improved (95·4%), supporting use of serology testing in carefully targeted populations. Larger studies in specific patient cohorts, including those with mild infection are urgently required to inform on the applicability of POC serological assays to help control the spread of SARS-CoV-2 and improve case finding of patients that may experience late complications.
Background: Neutrophils are key players in the immune and aid in the defense against microorganisms. Neutrophil extracellular traps (NETs) are extracellular DNA complexes, which are released during NETosis, a programmed form of cell death. Although NETs are crucial in the fight against infectious agents, an overabundance of neutrophils has been implicated in many inflammatory lung conditions. Our aim is to determine whether an overabundance of NETosis is associated with clinical deterioration of patients with COVID-19. Methods: Circulating polymorphonuclear cells (neutrophils) were isolated from human peripheral blood of 20 human subjects with COVID-19. Neutrophils were seeded in 96-well plates and treated with 0, 2.5 nM, 25 nM, and 250 nM of phorbol 12-myrisate 13-acetate (PMA) or 12 uM nigericin for 2 hours to stimulate NET production via canonical and noncanonical pathways, respectively. Following incubation, wells were treated with micrococcal nuclease, supernatants were collected from each well, and extracellular DNA content to quantify NETosis was detected by fluorescent plate reader. We calculated acute physiology and chronic health evaluation (APACHE-II) scores for every human subject. These were calculated at the same time point at which the neutrophils were collected. They were then compared to the degree of NETosis and absolute neutrophil count (ANC). These were analyzed using a simple linear regression model. We also categorized participants based on APACHE-II scores (APACHE-II 15) and compared them to rates of NETosis using a bar graph. Results: APACHE II is a widely used ICU mortality prediction score that is used to risk-stratify patients. We found that participants with higher APACHE-II scores had higher rates of NETosis, both at 0 nM PMA and when stimulated with nigericin (figure 1a-b). This suggests that higher rates of NETosis correlate with increased disease severity. Additionally, we found a positive correlation between ANC and NETosis (Figure 1c-1d), suggesting that ANC itself is a reliable marker of NETosis and disease severity. Conclusion: NETosis is an important player in immune system defense but has also been implicated in various inflammatory lung conditions. We found that in patients with COVID-19, there was a positive correlation between worsening disease state, measure by APACHE II scores, and increased NETosis. This suggests that over-activation of neutrophils may play a role in disease progression. We also found a positive correlation between NETosis and ANC, indicating that the degree of circulating neutrophils is a reliable marker of the functional state of neutrophils, as well as disease severity.
Background Black, Asian and minority ethnic (BAME) populations are emerging as a vulnerable group in the severe acute respiratory syndrome coronavirus disease (SARS-CoV-2) pandemic. We investigated the relationship between ethnicity and health outcomes in SARS-CoV-2. Methods and findings We conducted a retrospective, observational analysis of SARS-CoV-2 patients across two London teaching hospitals during March 1 –April 30, 2020. Routinely collected clinical data were extracted and analysed for 645 patients who met the study inclusion criteria. Within this hospitalised cohort, the BAME population were younger relative to the white population (61.70 years, 95% CI 59.70–63.73 versus 69.3 years, 95% CI 67.17–71.43, p<0.001). When adjusted for age, sex and comorbidity, ethnicity was not a predictor for ICU admission. The mean age at death was lower in the BAME population compared to the white population (71.44 years, 95% CI 69.90–72.90 versus, 77.40 years, 95% CI 76.1–78.70 respectively, p<0.001). When adjusted for age, sex and comorbidities, Asian patients had higher odds of death (OR 1.99: 95% CI 1.22–3.25, p<0.006). Conclusions BAME patients were more likely to be admitted younger, and to die at a younger age with SARS-CoV-2. Within the BAME cohort, Asian patients were more likely to die but despite this, there was no difference in rates of admission to ICU. The reasons for these disparities are not fully understood and need to be addressed. Investigating ethnicity as a clinical risk factor remains a high public health priority. Studies that consider ethnicity as part of the wider socio-cultural determinant of health are urgently needed.
The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak is a public health emergency and the case fatality rate in the United Kingdom is significant. Although there appear to be several early predictors of outcome, there are no currently validated prognostic models or scoring systems applicable specifically to patients with confirmed SARS-CoV-2.
Abstract Background Older adults, particularly in long-term care facilities (LTCF), remain at considerable risk from SARS-CoV-2. Data on the protective effect and mechanisms of hybrid immunity are skewed towards young adults precluding targeted vaccination strategies. Methods A single-centre longitudinal seroprevalence vaccine response study was conducted with 280 LCTF participants (median 82 yrs, IQR 76-88 yrs; 95.4% male). Screening by SARS-CoV-2 polymerase chain reaction with weekly asymptomatic/symptomatic testing (March 2020-October 2021) and serology pre-/post-two-dose Pfizer-BioNTech BNT162b2 vaccination for (i) anti-nucleocapsid, (ii) quantified anti-receptor binding domain (RBD) antibodies at three time-intervals, (iii) pseudovirus neutralisation, and (iv) inhibition by anti-RBD competitive ELISA were conducted. Neutralisation activity: antibody titre relationship was assessed via beta linear-log regression and RBD antibody-binding inhibition: post-vaccine infection relationship by Wilcoxon rank sum test. Results Here we show neutralising antibody titres are 9.2-fold (95% CI 5.8–14.5) higher associated with hybrid immunity ( p < 0.00001); +7.5-fold (95% CI 4.6-12.1) with asymptomatic infection; +20.3-fold, 95% (CI 9.7-42.5) with symptomatic infection. A strong association is observed between antibody titre: neutralising activity ( p < 0.00001) and rising anti-RBD antibody titre: RBD antibody-binding inhibition ( p < 0.001), although 18/169 (10.7%) participants with high anti-RBD titre (>100BAU/ml), show inhibition <75%. Higher RBD antibody-binding inhibition values are associated with hybrid immunity and reduced likelihood of infection ( p = 0.003). Conclusions Hybrid immunity in older adults was associated with considerably higher antibody titres, neutralisation and inhibition capacity. Instances of high anti-RBD titre with lower inhibition suggests antibody quantity and quality as independent potential correlates of protection, highlighting added value of measuring inhibition over antibody titre alone to inform vaccine strategy.
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