key: cord-0838579-lsb1s8y7
authors: Isabel, Montesinos; Damien, Gruson; Benoit, Kabamba; Hafid, Dahma; Sigi, Van den Wijngaert; Soleimani, Reza; Vincenzo, Carbone; Olivier, Vandenberg; Beatrice, Gulbis; Fleur, Wolff; Hector, Rodriguez-Villalobos
title: Evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-SARS-CoV-2 antibodies
date: 2020-05-05
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104413
sha: 57626a1add7bdbd1e608a80904a6bf12b540561b
doc_id: 838579
cord_uid: lsb1s8y7

Abstract Introduction Several SARS-CoV-2 immunoassays have been developed recently. The purpose of this study was to assess the performance of five immunoassays for the detection of SARS-CoV-2 antibodies. Methods Two quantitative automated immunoassays (MaglumiTM 2019-n-Cov IgG and IgM and Euroimmun Anti-SARS-CoV-2 IgG and IgA assays) and three lateral flow rapid tests were performed. This retrospective study included 200 residual sera from patients and healthy volunteers. Case serum samples (n = 128) were obtained from COVID-19 patients confirmed by RT-qPCR and CT-scan. Days since onset of symptoms was collected from their medical records. Control non-SARS-CoV-2 samples (n = 72) were obtained from anonymous stored residual serum samples. Results MaglumiTM IgG/IgM tests showed overall less sensitivity than Euroimmun IgG/IgA test (84.4% versus 64.3%). Both tests showed similar specificities of IgG at 99% and 100%, respectively. The two tests showed similar specificity for IgG at 99% and 100%, respectively. The results from the lateral flow assays were easily interpretable with unambiguous coloured reading bands. The overall sensitivity of the three tests was similar (around 70%) without any significant differences. The sensitivity of the three lateral flow assays and also of the serological quantitative assays increased during the second week after symptom onset and all reached similar values (91% to 94%) after 14 days. Conclusion This study shows accurate and equivalent performance of the five serological antibody assays (ELISA, CLIA and three lateral flow tests) in detecting SARS-CoV-2 antibodies 14 days after the onset of COVID-19 symptoms. This is compatible with their application in specific clinical contexts and in determining epidemiological strategies for the COVID-19 pandemic.

Since the emergence of the novel respiratory virus SARS-CoV-2 during December 2019 in the region of Wuhan (China), the virus has spread rapidly all over the world causing a pandemic coronavirus disease (COVID-19) [1] .

Adequate diagnosis of SARS-CoV-2 infection is essential for prompt therapeutic management of patients, control of the epidemic and the establishment of infection control measures. Even though RT-qPCR is considered the reference method for screening and diagnosis, the sensitivity of this method may vary depending on the quality and origin of the sample, the time of infection and the viral load [2, 3] .

On 30 January 2020, the World Health Organization (WHO) declared the COVID-19 outbreak to be a Public Health Emergency of International Concern, and shortly thereafter called for research on in-vitro diagnostics for use at the community level [4] . In response, several serological tests, including the enzyme-linked immunosorbent assay (ELISA), the chemiluminescence immunoassay (CLIA) and lateral flow rapid testing, are now under development or have already entered the market. The Foundation for Innovative New Diagnostics (FIND) (https://www.finddx.org/) lists over 150 rapid COVID-19 antibody tests that are "Communauté Européenne" (CE) marked [5] .

The detection of IgG, IgM and IgA antibodies against the SARS-CoV-2 can play a complementary role to the RT-qPCR test in the diagnosis of COVID-19 and in assessing the immune status of individuals. Moreover, serological data will enable the gathering of important epidemiological information, providing more realistic data on the spread of the epidemic, and on morbidity and mortality. In addition, the detection of antibodies against SARS-CoV-2 will play a key role in determining appropriate lockdown exit strategies and in vaccine development [2] [3] [4] [5] [6] [7] .

However data concerning the performance of these assays are scarce [8, 9] . The purpose of this study was therefore to assess the performance of CE marked assays available in Belgium-three lateral flow rapid tests and two quantitative automated immunoassays-for the detection of SARS-CoV-2 IgG, IgM and IgA antibodies.

This retrospective study included 200 residual sera from patients and healthy volunteers from Laboratoire Hospitalier Universitaire de Bruxelles -Universitair Laboratorium Brussel (LHUB-ULB) and the Microbiology 

Statistical analysis was performed with SPPS software. A receiver operator characteristic (ROC) curve was constructed and used for comparisons of the area under the curve (AUC) of the ROC curves. The Cohen Kappa index was calculated for agreement between all analysed assays. A p value < 0.05 was considered statistically significant. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for each serological test.

Sensitivity and specificity obtained with quantitative (ELISA and CLIA) serological assays are summarized in table 1. Overall the ELISA assay showed higher sensitivity than the CLIA (84% versus 64%, respectively). In J o u r n a l P r e -p r o o f contrast, the specificity of CLIA IgM (100%) was greater than that observed for ELISA IgA (86%). Both tests showed similar specificities of IgG at 99% for CLIA and 100% for ELISA. As shown in table 2, during the first week after the onset of symptoms, the ELISA IgA analysis was significantly more sensitive than the CLIA IgM (p < 0.001). Although not-statistically significant, higher sensitivity was also observed for the ELISA IgG Results for the lateral flow assays were easily interpretable with unambiguous coloured reading IgG lines. The colour intensity in the line regions correlated with the concentration of SARS-Cov-2 antibodies. In some cases, IgM line presented slight difficulty for reading in all commercial lateral flow tests. As predicted by the manufacturer, the results were obtained after ten minutes or less in all cases. All the tests performed in this study provided valid results. Overall sensitivity was similar (around 70%) without any significant differences between the three tests (Table 1) . However, the sensitivity for IgM was significant lower (p < 0.001) with LabOn Time as compared to the QuickZen and Avioq assays. The Avioq lateral flow test showed three false positive results of concomitant IgM and IgG reactivity in three samples from a previous epidemic period without any known confounding factor. In contrast, no false positive results were observed among the pool of 31 sera containing antibodies with potential cross reactivity. Globally, no significant differences were observed between the specificity values of the three tests with a positive predictive value of 100% for QuickZen and LabOn Time and 97.7% for the Avioq assays. As shown in Table 2 , similarly to the serological quantitative assays (ELISA and J o u r n a l P r e -p r o o f CLIA ), the sensitivity of the three lateral flow assays increased during the second week after the onset of symptoms and achieved similar values (91% to 94%) after 14 days. Table 3 shows the percentage of agreement between the five immunoassays. Overall, the best agreement was observed between the CLIA and LabOn Time assays (91%; Cohen Kappa index of 0.819) and between the two lateral flow assays LabOn Time and Avioq (91.5 %; Cohen Kappa index of 0.829). The absence of agreement between the five serological assays observed during the first week following symptom onset lessened 14 days after symptom onset, with all assays achieving 97% to 100% agreement.

Serological testing is a complementary test in COVID-19 diagnosis and a strategic vehicle in the second phase of the pandemic, necessary for epidemiological study and lockdown exit programmes. Immunoassays could provide identification of non-contagious and potentially protected individuals to support progressive de-confinement strategies in the process of gradually restoring safe economic and social activity [10, 11] . Different types of serological tests are available on the market and could be applied to the massive testing challenge the world is currently facing.

Fully automated CLIA and ELISA assays allow the quantitative determination of antibodies against SARS-CoV-2 by clinical laboratories with increased screening capacity. In this study, the ELISA IgG/IgA was tested on a fully automated microtiter plate analyser. The workflow for ELISA tests depends on the analyser used: in our laboratory, the capacity was 90 tests per 3-4 hours. In contrast, the CLIA IgG/IgM assays are fully automated random-access tests. This technology allows up to 180 tests per hour, depending on the platform used, with the results delivered in about 40 minutes. The performance of Maglumi IgG/IgM tests has been successfully evaluated by Padoan et al., showing the reliability of these immunoassays for assessing the immunological response in the sera of COVID-19 patients. These tests show that it takes at least 12 days to reach 100% sensitivity for IgG and a 88% positive rate for IgM [12] . In another study that compared the Maglumi IgG/IgM and Euroimmun IgG/IgA, 100% sensitivity was observed with Maglumi IgG 10 days following onset of symptoms. These authors reported a lower sensitivity for the Maglumi IgM (60%) compared to the Euroimmun IgA (100%) [13] . In the current study, the Maglumi IgG/IgM tests also showed overall lower sensitivity than the Euroimmun IgG/IgA test (64.3% vs 84.4%), but in contrast, their specificity increased to 100%. The mean days since symptom onset in the population tested for this study was 11.46 days (median of 10 days). This can explain the lower sensitivity of the Maglumi IgG/IgM tests in our study as compared to those in the literature. Obka et al. [8] also described higher sensitivity observed J o u r n a l P r e -p r o o f in the Euroimmun IgA test than in the Euroimmun IgG test (83.6% vs 61.7%). This was particularly evident in the two first weeks after symptom onset, but with less specificity for the former test (86.1% vs 98.6%).

Recently, many commercial lateral flow assays have been developed and CE-labelled. In this study, three of these were also evaluated. The results showed that their global sensitivity, specificity, NPV and PPV were equivalent to the ELISA IgG/IgA or the CLIA IgG/IgM tests. In addition, excellent concordance between the five immunoassays tested in this study was observed 14 days after the onset of symptoms. Similar results were reported by Li et al., showing good sensitivity (88.6%) and specificity (90.6%) with a rapid test in a large cohort of patient samples in China. They observed similar performances with serum, plasma and fingerstick blood [14] . Rapid serological tests can be performed in the laboratory or used as point-of-care tests (POCT). The latter will provide accurate results within 10 to 15 minutes with equivalent sensitivity and specificity as the quantitative automated immunoassays, particularly two weeks after onset of symptoms. The opportunity to test outside of the clinical laboratory by lateral flow assay makes it possible to reach larger population groups without saturating the capacity of the laboratories.

POCT may play an important role in large-scale testing in order to evaluate herd immunity against SARS-CoV-2.

However, mistakes in the interpretation of results in situations that are not under the control of trained staff must be taken into consideration. For this reason, the development of automated reader devices could help to reduce errors and increase sensitivity. In addition, such a device could support the transmission of the results to a public health institution to provide real-time information about seroprevalence at the population level.

While still awaiting the results of large seroprevalence studies in the community, Wu et al. report a detection rate of around 10% of SARS-CoV-2 IgG in asymptomatic subjects from a single-centre investigation [15] . The causal relationship between humoral response and illness severity is still unclear. Zhao et al. revealed a strong positive correlation between clinical severity and antibody titres two weeks after illness onset [16] . Yongchen et al. highlight the complementary role of immunoassays to RT-qPCR in the diagnosis of COVID-19, particularly in critical patients with a negative RT-qPCR. They observed seronegative asymptomatic patients four weeks after positive RT-qPCR results [17] . In the current study, two asymptomatic patients tested seronegative by all the immunoassays 20 days after a positive RT-qPCR result. Further studies in asymptomatic patients are necessary to understand better the humoral responses in this population and to understand the role of the real herd immunity in determining lockdown exit strategies.

This study has some limitations. First, this is a retrospective study performed with residual samples and the lack of fresh serum could impact the accuracy of the results. Second, no reliable gold standard for serological tests is currently available for comparative studies, and little literature exists concerning a comparison of immunoassay J o u r n a l P r e -p r o o f methods for SARS-CoV-2 detection. Furthermore, the criteria for assessing the time of illness onset were recovered from medical records and may contain imprecisions due to subjectivity in the perception of symptoms and timing.

In conclusion, this study shows the accurate and equivalent performance of five serological antibody assays Tables   Table 1 Analytical 

World Health Organization

Laboratory diagnosis of emerging human coronavirus infections -the state of the art

In vitro diagnostic assays for COVID-19: recent advances and emerging trends

World Health Organization. COVID 19 Public Health Emergency of International Concern (PHEIC): Global research and innovation forum: towards a research roadmap

Significance of serology testing to assist timely diagnosis of SARS-CoV-2 infections: implication from a family cluster

The Laboratory Diagnosis of COVID-19 Infection: Current Issues and Challenges

Severe acute respiratory syndrome Coronavirus 2-specific antibody responses in Coronavirus disease 2019 patients

Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients

Preparing for a responsible lockdown exit strategy

Laboratory testing of SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV): Current status, challenges, and countermeasures

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics

Assessment of immune response to SARS-CoV-2 with fully automated MAGLUMI 2019-nCoV IgG and IgM chemiluminescence immunoassays

Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2infection diagnosis

Serological Tests Facilitate Identification of Asymptomatic SARS-CoV-2 Infection in Wuhan

Antibody Responses to SARS-CoV-2 in Patients of Novel Coronavirus Disease

Different Longitudinal Patterns of Nucleic Acid and Serology Testing Results Based on Disease Severity of COVID-19 Patients Emerg Microbes Infect

Table 2 Analytical sensitivities for SARS-Cov-2 serological test depending on the onset of COVID-19 symptoms

We wish to thank the personnel of the LHUB-ULB and CUSL laboratories for its daily technical assistance. 

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.J o u r n a l P r e -p r o o f