key: cord-0684653-7arc4y97 authors: Cacaci, Margherita; Menchinelli, Giulia; Ricci, Rosalba; De Maio, Flavio; Mariotti, Melinda; Torelli, Riccardo; Morandotti, Grazia Angela; Bugli, Francesca; Sanguinetti, Maurizio; Posteraro, Brunella title: Re-evaluating positive serum samples for SARS-CoV-2-specific IgA and IgG antibodies using an in-house serological assay date: 2021-01-02 journal: Clin Microbiol Infect DOI: 10.1016/j.cmi.2020.12.014 sha: 8271fac8ddde0abd2ee429d1cf7bd8f56790f21c doc_id: 684653 cord_uid: 7arc4y97 This letter takes a cue from a recently published study, which looked at diagnostic tests currently available for SARS-CoV-2 infection, to present and discuss the results of a re-evaluation of 122 positive serum samples from COVID-19 patients using an in-house serological assay for SARSCoV-2 antibodies detection. While serology targeting the SARS-CoV-2 S protein should be preferable, IgA testing is proposed in all situations for which serology is the solely practicable diagnostic strategy for SARSCoV-2 infection. We read the recent article by Caruana et al. exploring the current landscape of diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), and signalling interpretive issues of test results [1] . We were particularly interested in serological testing, which may fill the gap between negative results of RT-PCRdthe reference standard for SARS-CoV-2 diagnosis [2] dand clinical (and radiological) findings suggestive of COVID-19 [3, 4] . Like molecular testing [5] , targeting the SARS-CoV-2 spike (S) protein (or the subunit S1 thereof) rather than the nucleocapsid (N) protein with an ELISA to detect virus-specific antibodies in patient serum may be crucial for diagnostic yield [6] . Sensitivity of ELISAs based on the N or S protein varies depending on the infection timing [1] . Additionally, testing for only IgM and IgG [7e9] may be limited in samples taken around symptom onset [10] . In this context, individuals who present within the first week after symptom onset could benefit from IgA testing [11] . In a recent study [11] , the S1based IgA Euroimmun (Lübeck, Germany) assay revealed good sensitivity compared with an S (or S1) -based IgG Wantai test (Beijing, China) or Euroimmnun assays with individuals sampled at early infection times. Consistently, Caruana et al. experienced a 96% sensitivity with samples collected 15e30 days post infection, using an N-based ELISA (Epitope Diagnostics, San Diego, CA, USA) [1] . Finally, mild (non-hospitalized), moderate (hospitalized) or severe (admitted to the intensive care unit) illness may affect antibody responses in individuals with COVID-19 [8, 9] . Using in-house ELISA targeting the SARS-CoV-2 N protein [7] , we re-evaluated positive results from the Euroimmnun ELISA for SARS-CoV-2-specific IgA and IgG detection for 122 serum samples of individuals admitted to the emergency department of our institution for suspicion of COVID-19. The institutional ethics committee approved the study (no. 27015/20), and informed consent was obtained from all individuals. Except for 105 individuals with RT-PCR-confirmed SARS-CoV-2 infection, COVID-19 diagnosis in 17 RT-PCR-negative individuals was based on both abnormal radiological findings and positive serology results. Initially, reproducibility of in-house ELISA was assessed testing 30 serum samples from individuals with COVID-19 with different levels of IgA or IgG antibodies. We found that the coefficients of variation were 1.38%e 32.22% and 2.06%e21.05% for IgA and IgG, respectively, whereas intra-class correlation coefficients were 0.88 and 0.98 for IgA and IgG, respectively. As shown in Table 1 and depicted in Fig. 1 , all samples with positive IgA/IgG results by Euroimmnun ELISA included samples positive for IgA (n ¼ 119) and IgG (n ¼ 113); of these samples, 110 were positive for both IgA and IgG, nine for only IgA and three for only IgG. In parallel, samples with positive IgA/IgG results by inhouse ELISA included samples positive for IgA (n ¼ 98) and IgG (n ¼ 111); of these samples, 95 were positive for both IgA and IgG, 3 for only IgA and 16 for only IgG. The in-house assay detected 96/119 IgA-positive samples and 109/113 IgG-positive samples, corresponding to a positive per cent agreement of 80.7% (95% CI 72.4%e 87.3%) and 96.5% (95% CI 91.2%e99.0%), respectively. Discrepancies between the two assays mainly involved samples that tested negative for IgA by the in-house assay (Table 1) . These samples were from individuals with mild (11/30 samples) or moderate (12/ 62 samples) disease, as well as those collected within the first 5 days (9/30 samples) or after 40 days (9/56 samples) of admission. Although N-based serological correlates of protection from SARS- were not reported because these results were beyond the comparison purposes between in-house and Euroimmun assays. Fig. 1 . Agreement of results for 122 serum samples obtained with the Euroimmun and the in-house ELISA tests. Unlike the commercial Euroimmun assay, the in-house assay for IgA and IgG detection was developed based on the use of a recombinant nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as described elsewhere [7] . For both assays, the antibody levels are shown expressed as spectrometrically measured values divided by the cut-off (S/CO), as are the percentage between-assay agreement values calculated for IgA and IgG antibodies, respectively. The cut-offs for IgA (0.08 and 1.10) and IgG (0.45 and 1.10) antibodies in both assays are marked with vertical blue (inhouse assay) or green (Euroimmun assay) lines. The Cohen's k values indicate fair (range 0.21e0.40) or substantial (range 0.61e0.80) agreement for IgA and IgG results, respectively. Among five samples that tested positive with the in-house assay but negative with the Euroimmnun assay, two were positive for IgA antibodies and three for IgG antibodies, respectively. CoV-2 infection are not fully understood [12] , similar to us, other investigators emphasized the role of anti-SARS-CoV-2 IgA in the current serodiagnostic arsenal for SARS-CoV-2 [13, 14] , especially in the early phase of infection [15] . We also determined the specificity of N-based serological testing using sera from 85 healthy blood donors or from 15 individuals with non-SARS-CoV-2 respiratory infection and we found that no sera were positive with the N-specific IgA (and IgG) assay. Furthermore, we observed that IgG antibodies detected in two individuals who tested positivedone with the in-house ELISA only and one with both the in-house and Euroimmun ELISAsdwere able to neutralize the Vero E6 cell-cultured SARS-CoV-2 (titres were 1 : 80 in both individuals). Likewise, IgA antibodies detected in two other individuals who tested positivedone with the in-house ELISA only and one with both in-house and Euroimmun ELISAsdwere able to neutralize the Vero E6 cell-cultured SARS-CoV-2 (titres were 1 : 20 and 1 : 640, respectively). Although these observations are consistent with recently published data [16, 17] , for reasons of comparability, we did not include data regarding the detection of IgM antibodies by the assay. In conclusion, we suggest that serology targeting the SARS-CoV-2 S protein, such as the Euroimmun ELISA, should be preferable. We recorded the highest sample positivity rates with S-based testing for IgA antibodies in individuals tested early or in individuals with mild COVID-19 (not requiring hospitalization) on admission (Table 1) . Hence, we propose considering IgA testing in all situations where serology is the solely practicable diagnostic strategy for SARS-CoV-2 infection. Future studies will help to decide on the deployment of serological assays for specific contexts in COVID-19 diagnostics. The authors declare that they have no conflicts of interest. No external funding was received for this study. Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance. World Health Organization Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis Diagnostic testing for severe acute respiratory syndrome-related coronavirus 2: a narrative review Laboratory diagnosis of COVID-19: current issues and challenges Novel antibody epitopes dominate the antigenicity of spike glycoprotein in SARS-CoV-2 compared to SARS-CoV Profiling early humoral response to diagnose novel coronavirus disease (COVID-19) Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Antibody responses to SARS-CoV-2 in patients with COVID-19 Antibody tests for identification of current and past infection with SARS-CoV An evaluation of COVID-19 serological assays informs future diagnostics and exposure assessment Serodiagnostics for severe acute respiratory syndrome-related coronavirus 2: a narrative review Anti-SARS-Cov-2 IgA in current scenario of IgM and IgG rapid test: a new alternative for the diagnostic of covid-19 Kinetics of SARS-CoV-2 specific antibodies (IgM, IgA, IgG) in non-hospitalized patients four months following infection SARS-CoV-2 infection serology validation of different methods: usefulness of IgA in the early phase of infection A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations A comprehensive, longitudinal analysis of humoral responses specific to four recombinant antigens of SARS-CoV-2 in severe and non-severe COVID-19 patients We wish to thank Franziska Lohmeyer for her English language assistance.