key: cord-284841-flhfagp3
authors: Nicol, Thomas; Lefeuvre, Caroline; Serri, Orianne; Pivert, Adeline; Joubaud, Françoise; Ducancelle, Alexandra; Lunel-Fabiani, Françoise; Le Guillou-Guillemette, Hélène
title: 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)
date: 2020-06-15
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104511
sha: 
doc_id: 284841
cord_uid: flhfagp3

BACKGROUND: The emergence of new SARS-CoV-2 has promoted the development of new serological tests that could be complementary to RT-PCR. Nevertheless, the assessment of clinical performances of available tests is urgently required as their use has just been initiated for diagnose. OBJECTIVES: The aim of this study was to assess the performance of three immunoassays for the detection of SARS-CoV-2 antibodies. METHODS: Two automated immunoassays (Abbott SARS-CoV-2 CLIA IgG and Euroimmun Anti-SARS-CoV-2 ELISA IgG/IgA assays) and one lateral flow immunoassay (LFIA NG-Test® IgG-IgM COVID-19) were tested. 293 specimens were analyzed from patients with a positive RT-PCR response, from patients with symptoms consistent with COVID-19 but exhibiting a negative response to the RT-PCR detection test, and from control group specimens. Days since symptoms onset were collected from clinical information sheet associated with respiratory tract samples. RESULTS: Overall sensitivity for IgG was equivalent (around 80%) for CLIA, ELISA and LFIA. Sensitivity for IgG detection, >14 days after onset of symptoms, was 100.0% for all assays. Overall specificity for IgG was greater for CLIA and LFIA (more than 98%) compared to ELISA (95.8%). Specificity was significantly different between IgA ELISA (78.9%) and IgM LFIA (95.8%) (p < 0.05). The best agreement was observed between CLIA and LFIA assays (97%; k = 0.936). CONCLUSION: Excellent sensitivity for IgG detection was obtained >14 days after onset of symptoms for all immunoassays. Specificity was also excellent for IgG CLIA and IgG LFIA. Our study shows that NG-Test® is reliable and accurate for routine use in clinical laboratories.

A new acute respiratory syndrome named coronavirus disease 2019 has emerged from the region of Wuhan in China in December 2019. This infection, widespread all over the world, is caused by a novel Sarbecovirus designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), associated with severe morbidity and mortality [1] [2] [3] . The detection of viral RNA by real time reverse transcriptase-Polymerase chain reaction (RT-PCR) in respiratory tract samples is considered as the gold standard method for screening and diagnosis in the early phase of infection. However, sensitivity is variable depending on sample types, suitable sampling technique, the anatomic site, time of infection and viral load [4] [5] [6] .

Chest computed tomography (CT) may be helpful for the diagnosis, complementary to RT-PCR, but it remains unspecific [7] . Development of new serological tests [8, 9] , readily available and easier to perform compared to requirements of molecular assays in laboratories [10] , could be helpful as a complementary diagnostic tool and to increase the sensitivity of tests especially in patients with late complications i.e. severe pneumonia. Different assays have recently been commercialized: automated tests (enzyme-linked immunosorbent assays [ELISA] or chemiluminescence enzyme immunoassays [CLIA]) or rapid detection test (lateral flow immunoassays, LFIA). LFIA seems to be very attractive for large seroprevalence studies because these tests can be used easily as point of care tests or in the laboratory, with a result in less than 15 minutes. Serological tests can be used for symptomatic individuals for which RT-PCR testing was either not performed at the time of acute illness or for which nasopharyngeal swab result was found to be negative, and also for epidemiological studies (close contacts screening, screening of health care workers …) [11, 12] . However, the relevance of serological tests is highly related to their clinical performance, hence antibody (Ab) assays with good sensitivity and specificity are needed. Despite a growing number of available assays, related J o u r n a l P r e -p r o o f clinical performances are still scarce [13] [14] [15] [16] [17] or unknown and individual studies are usually inconclusive. Moreover, the quality and diagnostic performance of rapid tests have already been questioned in Spain and United Kingdom [18, 19] .

The aim of the study was to assess the clinical performance of CE marked assays available in Europe to detect SARS-CoV-2 antibodies: two automated immunoassays (Euroimmun and Abbott assays) targeting two different proteins and also one lateral flow immunoassay (NG Biotech). Then, 25 serum samples with a potential cross-reaction to the SARS-CoV-2 immunoassays were investigated (Table 1) . Samples from 10 pregnant women and 10 sera from patients with positive rheumatoid factor (RF) were also tested.

The study was approved by the Institutional Board of the Angers University Hospital.

J o u r n a l P r e -p r o o f

The Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA assays (Euroimmun, Lüebeck, Germany) were performed according to the manufacturer's guidelines on the DS2® system, an automated microplate technology (Dynex Technologies GmbH, Denkendorf, Germany). The microplate wells are coated with recombinant S1 structural protein and the assay detects anti-SARS-CoV-2 IgG and IgA against the viral spike protein (Sp).

The Abbott SARS-CoV-2 IgG (Abbott, Ireland) was performed according to the manufacturer's instructions on the automated Abbott ARCHITECT i2000SR Instrument

The assay is a CLIA for qualitative detection of IgG antibodies against the SARS-CoV-2 nucleoprotein (Np) in serum or plasma.

NG-Test® IgG-IgM COVID-19 (NG Biotech Laboratoires, Guipry-Messac, France) is an immune colloidal technique intended for the qualitative detection of IgG and IgM antibodies against the SARS-CoV-2 nucleoprotein in serum or plasma. 10 μL of specimen, were added onto the sample loading area followed by 2 drops of sample dilution solution. The results were read and interpreted 15 min after testing.

All statistical analyses were performed using IBM® SPSS® 15.0 Statistics software (Statistical Package for Social Sciences, IBM Corp., Chicago, IL). To assess the sensitivity and specificity, J o u r n a l P r e -p r o o f we choose the RT-PCR method as gold standard. Time from onset symptoms was used to determine sensitivity and specificity. Grey zone was considered positive for the statistical analyses. A p value <0.05 was considered statistically significant. The Cohen's Kappa value was determined for agreement between assays.

Sensitivities and specificities obtained with three immunoassays are summarized in Table 2 Table 3 summarized overall agreement and agreement relative to the time of symptoms onset between three immunoassays. Overall, excellent agreement was observed between the three assays. The best agreement was observed between CLIA and LFIA (97%; Cohen kappa index of 0.936). Even for the first week of symptoms onset, an excellent agreement was observed between ELISA and LFIA assays (95%; k=0.810). However, poor agreement was observed between ELISA and CLIA (89%; k=0.687). Overall agreement between IgG/IgA ELISA and IgG/IgM LFIA was excellent (96%; k=0.914).

The IgA, IgM and IgG Ab kinetics were studied using specimens from seven patients (positive RT-PCR) with serial results and interesting kinetics ( Figure 2) . Then, five patients presented an earlier IgG seroconversion using CLIA compared to ELISA, the first week of symptom onset.

Among these patients, we observed in three patients an IgM line with LFIA and IgA ELISA was positive for four patients.

Using LFIA, results were more easily interpretable for IgG line than for IgM line. IgM line was difficult for reading, notably for seven sera.

J o u r n a l P r e -p r o o f Discussion A strong clinical performance of assays in diagnosis and management of COVID-19 is essential to quickly contain the COVID outbreak worldwide. Therefore, the development of serological assays, routinely used in clinical laboratories to determine recent infection or previous contact with viruses, is a good option complementary to RT-PCR method [21] . On May, 2020, the French Health Authority (Haute Autorité de Santé) and Infectious Diseases Society of America recommended that patients with symptoms consistent with COVID-19 but having a positive result for SARS-CoV-2 by RT-PCR may be diagnosed by serological tests [22, 23] . Various immunoassays are available on the European market [24, 25] and subjected to European regulations with the mandatory CE marked for sales. Nevertheless, the European Commission, in its April 2020 recommendations, allowed exceptionally the marketing of tests that do not have the CE marked, in the interest of public health [22] .

Here, we evaluated three different CE marked commercial immunoassays for detection of SARS-CoV-2 antibodies in human serum and plasma. ELISA assay was performed on a semiautomated microplate technology requiring high handling and with a limited capacity of tests per day (90 tests per 4h). In contrast, CLIA assay is a fully automated random-access test and that can perform over 4,000 tests per 24h. These two assays are used in clinical laboratories, unlike LFIA, which can be used as a point of care test or in clinical laboratories and provides a result within 15 min.

Performance of Euroimmun assay has been evaluated in some studies [13, 14, [26] [27] [28] [29] , showing sensitivity for IgG between 85% and 95% >14 days after symptoms onset and specificity between 95 and 100%. Few studies reported clinical performance of Abbott assay [14, 16, 26, 28] . Sensitivity for IgG was between 94% and 100% more than 14 days post symptom onset and specificity between 99 and 100%. In our study, we showed a sensitivity for IgG of J o u r n a l P r e -p r o o f 100% for CLIA Abbott and ELISA Euroimmun assays >14 days after symptoms onset and an overall specificity for IgG of 78.3% and 81.8% with ELISA and CLIA respectively. We carried out a large cross-reactivity study and more false positives results were observed using ELISA than CLIA as previously described [14] .

Recently, many CE marked LFIA became available. Two studies showed that sensitivity and specificity were similar to those of Euroimmun assay [13, 29] .However, to our knowledge, only one study compared clinical performance between CLIA Abbott and LFIA [30] and no study described diagnostic performance of NG-Test®. Here, we observed an excellent agreement for IgG between CLIA and LFIA 15 days after onset symptoms (k=0.810), and an excellent sensitivity and specificity for both assays. LFIA advantages are the ability to reach larger population groups, when used in point-of-care, and to evaluate the herd immunity without saturating the capacity of laboratories. However, these devices must be used with caution.

Trained staff or automated reader devices are needed for good interpretation of result.

Traceability of results may be at fault in case of use at the point-of-care and results may not be reported to the health authorities for seroprevalence studies.

To evaluate sensitivity, some manufacturers or authors used the time from positive RT-PCR rather than the time from symptom onset. However, there is a risk of misestimating sensitivity as some patients presented late after the onset of symptoms with disease progression at time of the first PCR testing. Then, sensitivity and specificity must be interpreted with caution. The use of RT-PCR as gold standard may decrease the real number of patients infected by SARS-CoV-2 due to false negative results.

In our study, we observed false positive results with IgA ELISA and few with IgM LFIA. No false positive with IgM LFIA were observed with for RF specimens whereas interferences were described with some other immunoassays [31] . Elslande et al pointed out that the ELISA IgA should not be used for the screening of asymptomatic persons. It might be better not to measure IgM or IgA since it may result in a significant number of false-positive results without improving diagnostic performance. [29] . It would appear here that IgM detection with the LFIA provides a gain in diagnostic performance.

Developed immunoassays target either the Sp or the Np of SARS-CoV-2 [32] , involving different immune Ab responses. However, related studies are controversial. Some studies described that early antibody response was targeted against Np and then Sp inducing an earlier positivity of the tests targeting Np [14, 33] . By contrast, another study revealed that the Spbased ELISA was more sensitive than the Np-based one in the detection of IgM [34] . Here, we did not observe any significant difference between sensitivity of IgA ELISA and IgM LFIA In conclusion, our study showed equivalent clinical performance for IgG of three immunoassays (ELISA, CLIA and LFIA) >14 days after symptoms onset. The three assays had, as expected, a poor sensitivity during first days of symptom onset. Therefore, serological tests can be useful to confirm past COVID-19, to do epidemiologic studies 15 days after symptoms J o u r n a l P r e -p r o o f onset [36] or to identify people who could return to the workplace, even if its use is still widely discussed [37] . For asymptomatic patients with RT-PCR negative, a higher threshold must be used [16] . A lower threshold (8-14 days) should be used for symptomatic patients >7 days with negative RT-PCR and clinical presentation consistent with COVID-19. Currently, it is not clear whether IgG antibodies are protective against reinfection [38] . Finally, even if the LFIA is reliable on serum or plasma, studies should be conducted to evaluate the performance on fingerstick; a process commonly used for seroprevalence studies.

This research did not receive any specific grant from funding agencies. NG-Test® IgG-IgM COVID-19 rapid test cassettes (NG Biotech Laboratoires) were kindly provided by the manufacturer.

The authors declare that they have no conflict of interest. 

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9%) Se: 59.4 (42.3-74.5%) Se: 28.1 (42.3-74.5%)

The authors thank the laboratory technicians who helped us.The authors thank Thomas Le Guillou for proofreading the English manuscript.J o u r n a l P r e -p r o o f