key: cord-0844625-gryx88ac authors: Veyrenche, Nicolas; Bolloré, Karine; Pisoni, Amandine; Bedin, Anne‐Sophie; Mondain, Anne‐Marie; Ducos, Jacques; Segondy, Michel; Montes, Brigitte; Pastor, Patrick; Morquin, David; Makinson, Alain; Le Moing, Vincent; Van de Perre, Philippe; Foulongne, Vincent; Tuaillon, Edouard title: Diagnosis value of SARS‐CoV‐2 antigen/antibody combined testing using rapid diagnostic tests at hospital admission date: 2021-02-15 journal: J Med Virol DOI: 10.1002/jmv.26855 sha: 026a10de0526dcc6eb42272d08041acc05f0c14a doc_id: 844625 cord_uid: gryx88ac The implementation of rapid diagnostic tests (RDTs) may enhance the efficiency of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) testing, as RDTs are widely accessible and easy to use. The aim of this study was to evaluate the performance of a diagnosis strategy based on a combination of antigen and immunoglobulin M (IgM) or immunoglobulin G (IgG) serological RDTs. Plasma and nasopharyngeal samples were collected between 14 March and 11 April 2020 at hospital admission from 45 patients with reverse transcription polymerase chain reaction (RT‐PCR) confirmed COVID‐19 and 20 negative controls. SARS‐CoV‐2 antigen (Ag) was assessed in nasopharyngeal swabs using the Coris Respi‐Strip. For IgM/IgG detection, SureScreen Diagnostics and Szybio Biotech RDTs were used in addition to laboratory assays (Abbott Alinity i SARS‐CoV‐2 IgG and Theradiag COVID‐19 IgM enzyme‐linked immunosorbent assay). Using the Ag RDT, 13 out of 45 (29.0%) specimens tested positive, the sensitivity was 87.0% for cycle threshold (C (t)) values ≤25% and 0% for C (t) values greater than 25. IgG detection was associated with high C (t) values and the amount of time after the onset of symptoms. The profile of isolated IgM on RDTs was more frequently observed during the first and second week after the onset of symptoms. The combination of Ag and IgM/IgG RDTs enabled the detection of up to 84.0% of COVID‐19 confirmed cases at hospital admission. Antigen and antibody‐based RDTs showed suboptimal performances when used alone. However when used in combination, they are able to identify most COVID‐19 patients admitted in an emergency department. Reported for the first time in December 2019, coronavirus disease 2019 (COVID-19) has become a major public health concern worldwide. Currently, clinical management of COVID-19 is mainly based on the prevention of transmission, viral tests, and supportive care. Wide access to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing is one of the keys to protecting populations. To be efficient, diagnostic assays must be accessible in different settings, ranging from the hospital to the community level, and from low to high incomes countries. 1 Reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2 RNA in upper and lower respiratory tract specimens (nasopharyngeal swab, throat swab, and sputum) is the gold standard to confirm COVID-19. 2 RT-PCR tests have an overall sensitivity estimated around 70.0% in nasopharyngeal sampling 3 with a high specificity. RT-PCR is ideal for the diagnosis of COVID-19 during the first week after the onset of symptoms because the viral load is high during this period. Beyond Day 14, when the viral load is low or undetectable, the performance of RT-PCR diminishes. 4 In such situations, serological tests may help to confirm a COVID-19 diagnosis in individuals with a high clinical suspicion but who tested negative for SARS-CoV-2 RNA. Both nucleic acid tests and automated serological tests require sample collection, transportation, and laboratory analysis, leading to a delayed response and limiting the efficiency of SARS-CoV-2 testing strategies. Furthermore, insufficient access to nearby laboratory facilities is a major concern in intermediate and low-income countries. A diagnostic test is characterized not only by its analytical performance, mainly estimated by sensitivity (Se) and specificity (Sp) but also by its overall accessibility. 5 The implementation of rapid diagnostic tests (RDTs) in the diagnosis of COVID-19 could have significant benefits by enhancing the efficiency of large testing strategies. 6 RTDs are useful devices that facilitate testing outside of laboratory settings, a capability needed for hard to reach populations. 7 In addition, RTDs deliver results in a shorter amount of time than RT-PCR. This time saving is important for the identification, isolation and provision of appropriate clinical care to patients with COVID-19. RTDs also reduce overloads in emergency departments. 8 COVID-19 RDTs are based on the detection of either SARS-CoV-2 antigen in respiratory specimens or anti-SARS-CoV-2 antibodies in whole blood or plasma or serum. Experience with RDTs used to detect antigens from other respiratory viruses in respiratory samples suggests that the sensitivity of these tests is lower than that of nucleic acid tests, ranging from 34.0% to 80.0%. 9 Recent publications have confirmed that COVID-19 RDTs are considerably less sensitive than molecular tests, and may therefore generate false negative results. 10 Antigen detection is mainly dependant on the viral concentration, hence most specimens with high viral concentrations test positive for antigen. [11] [12] [13] In contrast to RDTs based on Ag detection, RDTs that detects anti-SARS-CoV-2 antibodies are widely available, and a very large number have been approved by the FDA and CE. Although weak or absent humoral responses have been reported, especially in mild and moderate forms of COVID-19, most patients develop an antibody response within the first two weeks of COVID-19. 14-16 Serologic assays also can be useful in conjunction with molecular assays for the clinical assessment of persons who present themselves for testing long after the onset of symptoms. While RDTs might constitute a simple screening method, they have shown limitations in the early phase of acute infections due to the time required for an antibody response. Consequently, COVID-19 diagnosis based on immunoglobulin M (IgM) and immunoglobulin G (IgG) detection is often delayed to the second phase of the disease, when some opportunities for therapy and prevention of SARS-CoV-2 transmission already have been lost. Due to the overall performance of the tests, World Health Organization (WHO) and FDA do not recommend the use of Ag or antibody-detecting RDTs as the sole basis for the diagnosis of infection, but are encouraging research studies to establish their usefulness. Diagnostic algorithms based on Ag plus antibodies detection using RDTs need to be compared to the molecular techniques which currently are the gold standard for COVID-19 diagnosis. 7 The aim of this study was to evaluate the performance of a combination of antigen and serological RDTs to diagnose COVID-19 in hospitalized patients who tested positive for SARS-CoV-2 RNA using RT-PCR. ClinicalTrials. gov Identifier: NCT04347850). All tests were performed in the laboratory of Virology. IgM directed against SARS-CoV-2 protein S were detected using the ELISA COVID-19 THERA02 IgM assays (Theradiag). The IgM positive cut-off is ratio ≥1. All tests were performed according to the manufacturer's instructions. The experimental data were summarized by number and percentage for categorical variable, that is, positive and negative results. The COVID-19 confirmed patients were grouped in three categories according to RT-PCR values: C t ≤ 25, 25 < C t < 35, and C t ≥ 35. Quantitative variables with non-normal distribution (C t values, IgM ratio or IgG ratio) were compared between the different groups using Mann-Whitney U test. Because the distribution was nonnormal and total of the discordant pairs was too low, Exact binomial's test was used to compare the performance of diagnostic tests. The median and interquartile range (IQR) were used for analysis distribution of C t values because this variable follows a non-normal distribution. Analyses were performed using GraphPad Prism 8.0 (GraphPad Prism Software Inc.). (Tables S1A,B ). Seventeen samples were IgM positive but IgG negative, and among these, IgM were also detected by ELISA in 11 samples ( Figure S2A ). All samples that tested positive for IgG using both the Sur-eScreen and the Szybio RDTs also tested positive for IgM. A good overall agreement between Surescreen and Szybio RDTs was recorded regardless the time since onset of symptom (Table S1B ). The presence of anti-SARS-CoV-2 IgM and IgG was also assessed using laboratory assays (SARS-CoV-2 IgG Alinity and Theradiag IgM ELISA) on plasma samples collected at admission in the emergency department (Tables S1A,B). A total of 24 out of 45 COVID-19 confirmed patients tested positive for IgG using the Abbott assay, resulting in a sensitivity (95% CI) of 53.3% (38.8-67.9) (Tables S1A,B). All but one patient tested two weeks after the onset of symptoms tested positive for IgG using the Abbott assay ( Figure S3A ). IgG signal to cut-off values were weakly correlated with the amount of time after the onset of symptoms (R 2 = 0.3139, Figure S3A ). IgM directed against the S protein were detected in 24 specimens using the Theradiag ELISA, sensitivity (95% CI): 53.3% (38.8-67.9) (Tables S1A,B). Theradiag ELISA IgM ratios were not correlated with the amount of time after the onset of symptoms (R 2 = 0.0413) (data not shown). IgM were detected by ELISA in 11 out of 17 samples tests positive using the rapid tests ( Figure S2A ). CT values were compared with the amount of time following the onset of symptoms. The two parameters were weakly correlated (R 2 = 0.3151, Figure S3B ). The Ag RDT had a sensitivity (95% CI) of 87.0% (70.0-100.0) for C t values ≤25% and 0% for C t values greater than 25 ( p < .0001; Figure 1 ). RT-PCR C t values were higher in patients who tested positive using the SureScreen IgM compared to those who tested negative (Tables S1A). A good overall agreement between Surescreen and Szybio RDTs was recorded regardless the RT-PCR C t values (Table S1A ). The proportion of IgG positive samples rises according to the C t values and the time from the onset of COVID-19 symptoms ( Figure 3 and Table S1A ,B). (Figure 4 and Table S4A ). The combination of RDTs based on Ag plus IgM detection significantly improved the identification of COVID-19 cases at hospital admission compared to the Coris Ag RDT alone (p < .0001) or the IgM/IgG RDTs alone (p = .0113 and p = .0142, respectively, Figure 4 and Table S4A ). In this study, we observed that a majority of nasopharyngeal samples Ag RDT that targets the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen does not cross-react with seasonal coronaviruses. 11 Hence, the specificity of the assay appears close to 100% in all published studies. [11] [12] [13] Thanks to its high specificity and positive predictive value, a positive result using the Coris RDT would make it possible to avoid or delay the RT-PCR test. 11 The two serological RDTs evaluated in this study also showed a high specificity but a variable sensitivity according to antibody isotype, time from onset of symptoms and RT-PCR C t values. In contrast of the Coris RDT, the sensitivity of IgM/IgG RDTs improve when days after onset of symptom increase. The performances of the RDTs to detect IgG was lower than that of the Abbott SARS-CoV-2 IgG assay. While these RDTs had a lower capacity to detect low IgG concentrations compared to the chemiluminescence immunoassay, both the SureScreen RDT and the Szybio RDT had a good capacity to detect IgM ( Figure S5A ,B antigen rapid tests among symptomatic cases and contacts of confirmed cases. 32 Our results showed that testing SARS-CoV-2 antigen plus antibodies using RDTs would improve the rate of COVID-19 confirmation compared to COVID-19 testing using antigen RDT alone. The effectiveness of antibody RDTs is obvious when the delay after onset of symptom is over seven days but request a capillary or venous blood collection. Furthermore, the cost of antigen plus antibodies COVID-19 RDTs is below 10 euros, compare to 25-30 euros for a random access RT-PCR test in our hospital. Our study has some limitations. We used an antigen rapid test that has lower performances than more recent antigen tests. The study was performed in a laboratory settings whereas rapid tests are especially useful when used as point of care tests. Finally, we did not include pauci-symptomatic or asymptomatic SARS-CoV-2 infections. A synthetic representation of variation over time in biological markers for COVID-19 diagnosis is proposed in Figure 5 . In conclusion, our results show that the Coris Ag immunochromatographic assay has insufficient sensitivity for the di- The authors declare that there are no conflict of interests or personal relationships that could have appeared to influence the work reported in this paper. critically reviewed the manuscript. Edouard Tuaillon has conceived the study, discussed the results and wrote the manuscript. All authors contributed to the article and approved the submitted version. The data that support the findings of this study are available from the corresponding author upon reasonable request. This study received an institutional ethics committee approval (CPP Ile de France III, n°2020-A00935−34; ClinicalTrials. gov Identifier: NCT04347850). Nicolas Veyrenche https://orcid.org/0000-0001-5948-5039 Audio interview: new research on possible treatments for Covid-19 RT-PCR) C t value less than 25 and antigen detection in nasopharyngeal specimens characterize the first week after the onset of symptoms when the risk of SARS-CoV-2 transmission is at its maximum. The second week of COVID-19 infection is the period when the absence of detectable Ag and IgM/IgG is the most probable. The eclipse phase of antigen/IgM/IgG combined RDTs is most likely observable during this time period. A low level or the absence of SARS-CoV-2 RNA alongside IgG and IgM detection is observed two weeks after the onset of symptoms in most patients. COVID-19, coronavirus disease 2019; C t , cycle threshold; IgG, immunoglobulin G; IgM, immunoglobulin M; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 2 False negative tests for SARS-CoV-2 infection-challenges and implications Virological assessment of hospitalized patients with COVID-2019 Dried blood spot tests for the diagnosis and therapeutic monitoring of HIV and viral hepatitis B and C. Front Microbiol EasyCOV: LAMP based rapid detection of SARS-CoV-2 in saliva Advice on the use of point-of-care immunodiagnostic tests for COVID-19: scientific brief COVID-19 infection: the perspectives on immune responses Rapid tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review and meta-analysis Clinical evaluation of selfcollected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19 Development and potential usefulness of the COVID-19 Ag Respi-strip diagnostic assay in a pandemic context Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis Evaluation of a rapid diagnostic assay for detection of SARS-CoV-2 antigen in nasopharyngeal swabs Multiplex detection and dynamics of IgG antibodies to SARS-CoV2 and the highly pathogenic human coronaviruses SARS-CoV and MERS-CoV Detection of SARS-CoV-2 antibodies using commercial assays and seroconversion patterns in hospitalized patients Cochrane Infectious Diseases Group. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database System Rev Clinical management of COVID-19 Cochrane Infectious Diseases Group. Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database System Rev Immunologic testing for SARS-CoV-2 infection from the antigen perspective Predicting infectious SARS-CoV-2 from diagnostic samples Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards SARS-CoV-2 antibody responses do not predict COVID-19 disease severity Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Antibody detection and dynamic characteristics in patients with coronavirus disease 2019 Profile of specific antibodies to SARS-CoV-2: the first report Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech) Four point-of-care lateral flow immunoassays for diagnosis of COVID-19 and for assessing dynamics of antibody responses to SARS-CoV-2 Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes Diagnostic performance of seven rapid IgG/IgM antibody tests and the Euroimmun IgA/IgG ELISA in COVID-19 patients Dynamics and significance of the antibody response to SARS-CoV-2 infection Comission recommendation of 18.11.2020: on the use of rapid antigen tests for the diagnosis of SARS-CoV-2 infection