key: cord-0829335-q4tka8vs authors: Pieri, Massimo; Nuccetelli, Marzia; Nicolai, Eleonora; Sarubbi, Serena; Grelli, Sandro; Bernardini, Sergio title: Clinical validation of a second generation anti‐SARS‐CoV‐2 IgG and IgM automated chemiluminescent immunoassay date: 2021-01-26 journal: J Med Virol DOI: 10.1002/jmv.26809 sha: 6d31451d4f14dc64bf0176663f3beb475a8366c4 doc_id: 829335 cord_uid: q4tka8vs Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection has proven to be extremely contagious and has spread rapidly all over the world. A key aspect in limiting the virus diffusion is to ensure early and accurate diagnosis. Serological assays could be an alternative in increasing testing capabilities, particularly when used as part of an algorithmic approach combined with molecular analysis. The aim of this study was to evaluate the diagnostic accuracy of a second generation chemiluminescent automated immunoassay able to detect anti‐SARS‐CoV‐2 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies. Data are carried out on healthy subjects and other infectious diseases pre‐pandemic sera, as controls, and on two different coronavirus disease 2019 hospitalized patient groups (early and late infection time). Data obtained have been analyzed in terms of precision, linearity, sensitivity and specificity. Specificities are: 100% for anti‐SARS‐CoV‐2 IgG and 98% for anti‐SARS‐CoV‐2 IgM, in all patient groups. Sensitivities are: 97%, 100%, and 98% for anti‐SARS‐CoV‐2 IgG and 87%, 83%, and 86% for anti‐SARS‐CoV‐2 IgM in the early infection, in the late infection and in the total patient group, respectively. The Mindray anti‐SARS‐CoV‐2 IgG and IgM assays demonstrated higher sensitivity and specificity, indicating that IgG and IgM simultaneous detection is useful even in the early phases of infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is extremely contagious and is still spreading around the world with minor local differences. Coronavirus disease is mild in most cases; in some people, usually elderly and with co-morbidities, it may progress to pneumonia, acute respiratory distress syndrome and multi organ dysfunction. As the global impact of this new epidemic is yet uncertain, 1, 2 a crucial aspect in containing the virus spread is based on ensuring an early and accurate diagnosis and adequate quarantine for infected subjects. Nucleic acid tests to detect SARS-CoV-2 RNA genome have been quickly developed and are now widely employed to diagnose COVID-19. 3 However, the real-time polymerase chain reaction (PCR) often fails as a result of suboptimal collection procedures, a low patient viral load due to early stage of the disease or suppressed by host immunity, or if the samples are analyzed at an advanced stage of infection. 4 Unfortunately, a false negative diagnosis allows patients to spread the infection, hindering efforts to contain the virus and producing serious consequences. 5 In such conditions, additional screening methods able to detect the infection despite low viral titers, can be extremely helpful to guarantee a prompt diagnosis of all infected patients. Specific serological assays are particularly required to understand the epidemiology of SARS-CoV-2, but often still need to be accurately validated. Even though, the development of specific immunoglobulin M (IgM) and immunoglobulin A antibodies after symptom onset may occur as quickly as genetic viral material can be detected in respiratory samples, their production time usually ranges from less than 5 days to more than 10 days, thus limiting the serologic tests suitability for diagnosis of acute infections. 6 Nevertheless COVID-19 patient diagnostic sensitivity may significantly increase when combining serological tests with molecular tests. 7 In particular, serological assessment is able to support a number of highly relevant clinical and epidemiological applications in detecting SARS-CoV-2 infection: they can be used to determine the infection prevalence, useful to accurately define the laboratory test positive predictive values; to recognize individuals with a strong antibody response, who might be convalescent plasma donors; to analyze the immune response in a dynamic qualitative and quantitative manner; to evaluate vaccine trials results and the therapeutic antibodies development in the near future. Moreover, validated serological assays are critical for tracking patient contact, identifying viral reservoir hosts and for epidemiological studies, which are primarily needed to help uncover disease burden, especially the rate of asymptomatic individuals, and to obtain better estimates of morbidity and mortality. 8 Furthermore, epidemiological studies can help reveal the extent of virus spread in specific settings, such as households and communities, which could assist in guiding control measures. Based on these considerations, the aim of this study was to evaluate the diagnostic accuracy of a second generation chemiluminescent automated immunoassay able to detect anti-SARS-CoV-2 immunoglobulin G (IgG) and IgM antibodies, carried out on healthy subjects and other infectious diseases pre-pandemic sera, as controls, and on two different COVID-19 patient groups (early infection time and late infection time), hospitalized at "Tor Vergata" University COVID-Hospital of Rome. The second generation CL-series SARS-CoV-2 IgG and IgM assays are EP15-A3 protocol. 9 The precision results obtained were compared to those claimed by the manufacturer. The study was performed according to "Tor Vergata" University The CV precision data are shown in Table 1 in comparison with those declared by the company, according to the procedure suggested in CLSI protocol. Results for low and high CV % values were satisfactory and with better outcomes than those obtained by the manufacturer (IgG low: 3.84% vs. 4.26%; IgG high: 3.12% vs. 3.85%; IgM high: 3.02% vs. 3.18%), except for anti-SARS-CoV-2 IgM low precision value, resulted moderately higher than those declared (2.71% vs. 2.62%). Specificities and sensitivities were calculated using receiver operating characteristic (ROC) curves. Results reporting the analytical parameters for each test (area under curve [AUC], sensitivity and specificity) are shown in Figure 2 and summarized in Table 2 . T A B L E 1 Coefficients of variation (CV %) precision data We also reported in Table 2 No cross-reactivity with antibodies from other pre-pandemic infectious disease sera (such as HBV and HCV) has been found. Cross-reactivity studies were carried out on HBV and HCV sera as pre-pandemic sera from other respiratory virus infections were not available. Serological assays represents an alternative in increasing COVID-19 testing capabilities, particularly when used as part of an algorithmic approach combined with molecular testing. 10, 11 In fact, specific antibodies detection (IgM and IgG to SARS-CoV-2 spike protein) is useful in infected but asymptomatic subjects, to confirm SARS-CoV-2 infection in PCR-negative COVID-19 patients and in COVID-19 patients examined many weeks after the disease onset or in those with a low viral load. 12, 13 In this regard, the Infectious Diseases Society of America recommends that patients with clinical symptoms consistent with COVID-19 but negative for SARS-CoV-2 molecular tests, may be diagnosed by serological examination. 14 In addition, anti-SARS-CoV-2 serology is also a valuable tool for assessing the adapted immunity status of patients, thus providing not only an important complement to RNA testing for specific pathogenic diagnoses. 15, 16 We have carried out a study to investigate the second generation | 2527 A review of coronavirus disease-2019 (COVID-19) Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia COVID-19 testing: the threat of false-negative results Transmission of 2019-nCoV infection from an asymptomatic contact in Germany Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19 An evolving approach to the laboratory assessment of COVID-19 User evaluation of precision of quantitative measurement procedure; approved guideline. Clinical and Laboratory Standards Institute (CLSI) SARS-CoV-2 testing Combined anti-SARS-CoV-2 IgA, IgG, and IgM detection as a better strategy to prevent second infection spreading waves SARS-CoV-2 infection serology validation of different methods: usefulness of IgA in the early phase of infection SARS-CoV-2 infection serology: a useful tool to overcome lockdown? Clinical performance of two SARS-CoV-2 serologic assays Serology characteristics of SARS-CoV-2 infection since the exposure and post symptoms onset. medRxiv Serological approaches for COVID-19: epidemiologic perspective on surveillance and control Comparison of different serological assays for SARS-CoV-2 in real life Comparison of the diagnostic sensitivity of SARS-CoV-2 nucleoprotein and glycoprotein-based antibody tests Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies Clinical validation of a second generation anti-SARS-CoV-2 IgG and IgM automated chemiluminescent immunoassay The authors would like to thank all the Clinical Biochemistry Laboratory staff of Tor Vergata Hospital, for their support; Mindray (Shenzhen Mindray Bio-Medical Electronics Co., Shenzen, China; distributed in Italy by Medical Systems S.P.A., Genova, Italy) for kindly having provided kits and instrument for this study. The authors declare that there are no conflict of interests. https://orcid.org/0000-0002-3463-0268