key: cord-0779934-a8sr6od5
authors: Van Elslande, Jan; Houben, Els; Depypere, Melissa; Brackenier, Anouk; Desmet, Stefanie; André, Emmanuel; Van Ranst, Marc; Lagrou, Katrien; Vermeersch, Pieter
title: Diagnostic performance of 7 rapid IgG/IgM antibody tests and the Euroimmun IgA/IgG ELISA in COVID-19 patients
date: 2020-05-28
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2020.05.023
sha: 4d94e0394cf52d90d86ed534f84bf3d103815c5d
doc_id: 779934
cord_uid: a8sr6od5

OBJECTIVES: To evaluate the diagnostic performance of 7 rapid IgG/IgM tests and the Euroimmun IgA/IgG ELISA for antibodies against SARS-CoV-2 in COVID-19 patients. METHODS: Specificity was evaluated in 103 samples collected before January 2020. Sensitivity and time to seropositivity was evaluated in samples from 94 patients with COVID-19 confirmed with PCR on nasopharyngeal swab. RESULTS: Specificity [confidence interval] of lateral flow assays (LFA) was ≥ 91.3% [84.0-95.5] for IgM, ≥ 90.3% [82.9-94.8] for IgG, and ≥ 85.4% [77.2-91.1] for the combination IgM OR IgG. Specificity of the ELISA was 96.1% [90.1-98.8] for IgG and only 73.8% [64.5-81.4] for IgA. Sensitivity 14-25 days after onset of symptoms was ≥ 92.1% [78.5-98.0] to 100% [95.7-100] for IgG LFA compared to 89.5% [75.3-96.4] for IgG ELISA. Positivity of IgM OR IgG for LFA resulted in a decrease in specificity compared to IgG alone without a gain in diagnostic performance except for VivaDiag. The results for IgM varied significantly between the LFA with an average overall agreement of only 70% compared to 89% for IgG. The average dynamic trend to seropositivity for IgM was not shorter than for IgG. At time of admission to the hospital, the sensitivity of LFA was <60%. CONCLUSIONS: Sensitivity for the detection IgG antibodies 14-25 days after onset of symptoms was ≥ 92.1% for all 7 LFA compared to 89.5% for the IgG ELISA. The results for IgM varied significantly and including IgM antibodies in addition to IgG for the interpretation of LFA did not improve the diagnostic performance.

The coronavirus SARS-CoV-2 is the cause of coronavirus disease 2019 (COVID-19), 2 an acute respiratory syndrome that was first identified at the end of 2019 in Wuhan 3 China, and evolved into a pandemic. The current gold standard for the diagnosis of 4 COVID-19 is the detection of viral RNA in respiratory tract samples [1] . The 5 sensitivity of nucleic acid amplification techniques is, however, lower than 100%. 6 False-negatives can occur, especially when using nasopharyngeal swabs (positivity 7 rate estimated at 54%-74%) due to difficulty in sampling and in patients with low viral 8 loads, especially in patients who present at day 8 or later, and mild cases [1] . 9 Detection of antibodies has been proposed as an additional diagnostic tool which 10 could help for the diagnosis of patients suspected of COVID-19 which have a 11 negative PCR result, or in whom no respiratory sample for PCR was taken at the 12 time of acute illness (e.g. due to lack of adequate resources during an outbreak). 13 Seroconversion for SARS-CoV-2 is estimated to occur 7-14 days after onset of 14 symptoms when the sensitivity of the PCR decreases [3, 4] . Detection of antibodies 15 could be useful in patients in whom a past asymptomatic, atypical or mild infection is 16 suspected. Antibody tests can provide epidemiologic information about the number 17 of affected individuals and guide control measures taken by governments [2, 5, 6] . 18 There are currently two main ways of investigating these antibodies: by enzyme- 19 linked immunosorbent assay (ELISA) and by lateral flow assay (LFA). End of March 20 2020 the first ELISA, the Euroimmun IgA and IgG ELISA, received CE marking. 21 Although ELISA is a long-established method for antibody detection, disadvantages 22 include a longer turn around time, need for a laboratory environment and more labor 23 cost needed to produce a result. LFA on the other hand, are medical diagnostic tests 24 which can be used at the point of care or in the laboratory and typically give a 25 response in less than 15 minutes. 26 In the first quarter of 2020 more than 100 so called "rapid tests" for the detection of 27 IgM/IgG antibodies were marketed. There are, however, important concerns about 28 the quality and diagnostic performance of rapid tests for SARS-CoV-2. End of March, 29 the Spanish government said they had returned a shipment rapid antigen LFA after 30 they were found to be unreliable [7] and beginning of April, the British government 31 reported problems with the performance of antibody LFA [8] . As a result of these 32 problems, doctors and regulators throughout the world started to look with suspicion 1 at rapid tests for COVID-19. 2 The aim of this study was to critically evaluate the diagnostic performance of 7 rapid 3 LFA tests for professional use only to detect SARS-Cov-2 antibodies as well as the 4 Euroimmun IgA/IgG ELISA. We determined the specificity, the sensitivity and the 5 time to seropositivity of IgM and IgG. 6 1 This study was performed at the University Hospital Leuven and approved by the 2 local ethics committee (protocol number S63897). To assess specificity, we selected 3 samples from 103 patients collected before January 2020 as negative controls. 4 These included (i) a disease control group of 49 consecutive patients with a 5 respiratory infection who had a PCR test for respiratory pathogens in the period 6 September to November 2019. The serum samples were collected day 1 to day 40 7 after the PCR test. Table 1 ). All 11 samples were stored at -20°C until use. 12 For analysis of sensitivity and dynamic trend to seropositivity, a total of 167 samples 13 of 94 patients who presented with a clinical suspicion of COVID-19 in March and 14 April 2020 at the University Hospitals Leuven and were diagnosed with COVID-19. 15 Only patients positive for SARS-CoV-2 with RT-PCR on nasopharyngeal swabs 16 (UTM®, Copan, Italy) and for whom residual samples were available were included. 17 RT-PCR was performed using an in-house method complying with the WHO 18 guidelines [9] . Two patients that were initially considered for the study were excluded 19 because of treatment with rituximab for a B-cell malignancy. Table 2 ) and data analysis. We calculated the positive likelihood ratio (LR+: sensitivity/(1-specificity)) as a measure of the diagnostic performance of a 1 test. the low specificity of the IgA ELISA, this assay was not further tested. Multi-G IgM 10 and Prima IgG were the only assays with more than 1 false-positive result in the 14 11 samples from non-SARS-CoV-2 coronaviruses (2 and 3, respectively) 12 (Supplementary Table 3 ). 13 14 Sensitivity and dynamic trend to seropositivity 15 The sensitivity of LFA (IgM, IgG, IgM OR IgG, and IgM AND IgG) and the IgG ELISA 16 was <50% during the first week after onset of symtoms (day 0-6) except for the 17 Prima IgM OR IgG ( to 89% for IgG (Table 2) . 32 The average dynamic trend to seropositivity for IgM antibodies was not shorter than 33 for IgG antibodies (Figure 1 & Supplementary Figure 1 ). The dynamic trend to seropositivity for IgG followed the same pattern for all 7 LFA and the Euroimmun IgG 1 assay, but the trends for the different LFA varied strongly for IgM. LFA, although the difference did not reach statistical significance. This can be 10 attributed to a slower time to seroconversion for the ELISA (Figure 1 ). Between day 11 3 and day 17 after onset of symptoms, nine patients tested negative with the 12 Euroimmun IgG ELISA but positive with all 7 LFA. The 6 samples tested day 18-25 13 were positive for IgG with all assays including Euroimmun IgG ELISA. 14 15 Diagnostic performance of LFA at the time of admission to the hospital 16 In the 63 diagnostic samples, sensitivity ranged from 7.9 [3. The sensitivity of the 7 LFA included in our study for IgG was at least as good as the 2 first CE marked IgG ELISA during the first 3 weeks after onset of symptoms with a 3 faster seroconversion for IgG LFA. Seropositivity was >92% with all 7 IgG LFA 14-25 4 days after onset of symptoms. The specificity for IgG was more than 97% for 5 of the 5 7 LFA which can be considered very good given the challenging nature of the control 6 samples used in our evaluation.The performance of the IgM LFA, however, varied 7 greatly and the average dynamic trend to seropositivity was not shorter than for IgG. 8 For the LFA, including IgM also did not improve the diagnostic performance. The low 9 specificity of the IgA ELISA has since been confirmed by the manufacturer who now 10 recommends not to use the IgA ELISA for screening of asymptomatic persons. 11 Initial reports suggested that IgM antibodies against SARS-Cov-2 might appear 12 earlier than IgGs and that measuring both IgM and IgG would improve the diagnosis 13 of SARS-Cov-2 infection [1, 10] . To et al., however, found that more patients had 14 earlier seroconversion for IgG than for IgM. In addition, they also found a 100% 15 seroconversion for IgG antibodies, but not for IgM, 14 days after onset of symptoms 16 in 16 patients for whom serial serum samples were available [3] . Recently, Long et 17 al. reported 100% seroconversion for IgG after 19 days [11] . Our results confirm 18 these observations in a group of more than 80 patients and suggest that the antibody 19 response to SARS-CoV-2 might be comparable to the response to SARS-CoV-1 20 where the three antibodiy classes IgA, IgG and IgM seroconverted simultaneously, 21 or even 1 day earlier for IgG [12] . 22 Combining the results of IgG LFA and IgM LFA did not improve the diagnostic 23 performance, questioning the rationale for measuring IgM anti-SARS-CoV-2 24 antibodies. The fact that the specificity of 2 of the 7 LFA was <90% for IgM OR IgG 25 (either one positive) could explain concerns that have been raised regarding the 26 specificity of LFA. Concerns regarding sensitivity of LFA might be attributable to the 27 fact that these assays have been used in the emergency department. Zhao et al. 28 claimed that antibody detection (using ELISA) could be used as a diagnostic test 29 complementary to PCR, even in patients presenting in the first week since onset of 30 symptoms [13] . Antibody testing with LFA at the time of admission could also be 31 useful in resource-limited countries where PCR is not readily available. The 32 diagnostic performance at the time of admission in our study was, however, not very 33 good when both sensitivity and specificity, expressed as LR+, were taken into account. The 2 LFA IgM OR IgG with a LR+ ≥ 10 at the time of admission had a 1 sensitivity of only 30.2% and 31.7%. 2 The low sensitivity at time of admission in our study is not surprising given that the 3 median time of admission in our study was 7 days after onset of symptoms and 4 seroconversion typically occurs 7-14 days after onset of symptoms [3] . Our results 5 also confirm a recent report by Cassaniti et al. who did not recommend the use of a 6 SARS-Cov-2 IgM/IgG LFA for detection of COVID-19 in patients presenting at the 7 emergency department, stating a sensitivity of <20% in this patient population [14] . 8 The discussion about whether or not IgM/IgG LFA should be used in the emergency 9 department raises the question about the intended use of IgM/IgG LFA for the 10 detection of antibodies against SARS-CoV-2. Despite that all 7 of the tested assays 11 had a CE mark, none of the assays included information about the intended clinical 12 use other than that the assays are for the detection of antibodies against SARS- 13 CoV-2. Such a vague intended use, which might have contributed to the current 14 discussion about the diagnostic performance of LFA, will no longer be accepted for 15 CE marked after May 2022 when the IVD regulation 2017/746 enters into force. One 16 of the new requirements of the IVD regulation is that manufacturers will be required 17 to document the clinical evidence and the clinical benefit. 18 This study is to our knowledge the first peer-reviewed study that compared the limitation is that the samples used to evaluate specificity were challenging, and that 31 specificity in a routine laboratory setting will most likely be higher. A third limitation is 32 that we did not study the antibody response in asymptomatic persons. 33 The main expected use of antibody testing in the coming months is to confirm past 1 COVID-19 in patients, to determine (herd) immunity and epidemiologic studies [15] . 2 Our results suggest that detection of IgG antibodies can be very useful if performed 3 at least 18 days after onset of symptoms or, in asymptomatic persons, after the end 4 of an outbreak. There is currently no clear evidence that measuring IgA or IgM is 5 useful. Our results even suggest that it might be better not to measure IgM or IgA 6 since this could result in a significant number of false-positive results without a 7 significant gain in diagnostic performance. A number of important questions remain 8 regarding the use of antibody testing for epidemiologic purposes. Can someone 9 have a colonization with SARS-CoV-2 without developing IgG antibodies? In this 10 case, would this person be protected against reinfection? Finally, it is also still not 11 clear whether IgG antibodies are protective against reinfection [16] . 12 13 Conclusions 14 We found that the sensitivity for the detection of IgG antibodies 14-25 days after 15 onset of symptoms was > 92% for all 7 LFA compared to 89.5% for the IgG ELISA. 16 Five LFA even had a sensitivity and specificity of ≥ 94.7%. The average time to Author contributions PV devised the study, collected data and drafted the manuscript, JVE collected data 1 and drafted the manuscript, all other authors aided in collecting data and critically 2 reviewed the manuscript. 3 4 Conflicts of interest 5 Pieter Vermeersch reports personal fees from Roche, outside the submitted work. 6 Katrien Lagrou reports personal fees and non-financial support from Pfizer, personal 7 fees and non-financial support from MSD, personal fees from SMB Laboratoires, 8 personal fees from Gilead, and personal fees from FUJIFILM Wako, outside the 9 submitted work. The other authors state no conflicts of interests. 

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