key: cord-0743503-1rhhmlux
authors: De Marinis, Y.; Sunnerhagen, T.; Bompada, P.; Blackberg, A.; Yang, R.; Svensson, J.; Ekstrom, O.; Eriksson, K.-F.; Hansson, O.; Groop, L.; Goncalves, I.; Rasmussen, M.
title: Serology assessment of antibody response to SARS-CoV-2 in patients with COVID-19 by rapid IgM/IgG antibody test
date: 2020-08-06
journal: nan
DOI: 10.1101/2020.08.05.20168815
sha: 83e8cbe4ee47f927b1214ae431a5e787e410f803
doc_id: 743503
cord_uid: 1rhhmlux

The coronavirus disease 2019 (COVID-19) pandemic has created a global health- and economic crisis. Lifting confinement restriction and resuming to normality depends greatly on COVID-19 immunity screening. Detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19 by serological methods is important to diagnose a current or resolved infection. In this study, we applied a rapid COVID-19 IgM/IgG antibody test and performed serology assessment of antibody response to SARS-CoV-2. In PCR-confirmed COVID-19 patients (n=45), the total antibody detection rate is 92% in hospitalized patients and 79% in non-hospitalized patients. We also studied antibody response in relation to time after symptom onset and disease severity, and observed an increase in antibody reactivity and distinct distribution patterns of IgM and IgG following disease progression. The total IgM and IgG detection is 63% in patients with < 2 weeks from disease onset; 85% in non-hospitalized patients with > 2 weeks disease duration; and 91% in hospitalized patients with > 2 weeks disease duration. We also compared different blood sample types and suggest a potentially higher sensitivity by serum/plasma comparing with whole blood measurement. To study the specificity of the test, we used 69 sera/plasma samples collected between 2016-2018 prior to the COVID-19 pandemic, and obtained a test specificity of 97%. In summary, our study provides a comprehensive validation of the rapid COVID-19 IgM/IgG serology test, and mapped antibody detection patterns in association with disease progress and hospitalization. Our study supports that the rapid COVID-19 IgM/IgG test may be applied to assess the COVID-19 status both at the individual and at a population level.

The coronavirus disease 2019 (COVID-19) is caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of June 02, 2020, it had caused a total of 6, 194 ,533 cases of infection and 376,320 deaths worldwide. 1 The COVID-19 pandemic has become a global crisis, and the outbreak has had a substantial impact on the world economy. Resuming from society lockdown to normality depends largely on population screening of COVID-19 immunity, and serology assessment of antibody response to SARS-CoV-2 by blood antibody test is a crucial aid to track the true spread of the pandemic.

In the current clinical practice, the reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used method for diagnosis of COVID-19. The test is performed on upper respiratory tract specimens including nasopharyngeal and throat swabs. Although PCR offers detection of viral RNA, the result may not necessarily reflect the presence of viable virus. 2 False-negative rates up to 50% may occur due to inappropriate sampling procedure or timing in relation to disease onset. 3, 4 Furthermore, PCR tests are also technically demanding and costly in operation. To compromise the limitation of the PCR test, COVID-19 infection can also be detected by serology assessment of antibody responses to SARS-CoV-2 infection. The antibody test captures the presence of antibodies both during and after the infection and is able to determine if a person has been infected with the virus, even without the development of symptoms. This is particularly important for individuals who present to the health care late after disease onset. 5 Blood antibody tests All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint may therefore complement the PCR test to accurately diagnose a current or resolved infection, map the extent of community spread of COVID-19, and profile the potential protection from future virus exposure by acquired immunity.

In this study, we assessed antibody responses to SARS-CoV-2 in patients with COVID-19 by a rapid COVID-19 IgM/IgG test. The evaluation was performed on patients from the acute phase of the disease; as well as hospitalized and recovered COVID-19 patients.

The specificity of the test was verified on COVID-19 negative blood samples collected prior to the pandemic. We also tested whether different blood sample types (serum/plasma/whole blood) provided the same results for this assay.

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Patients with RT-PCR verified COVID-19 were enrolled prospectively either in the convalescence phase (2-8 weeks after start of symptoms) or when they were acutely ill or/and hospitalized. The study was conducted at the Department for Infectious Diseases, Skåne University Hospital, Lund, Sweden. All patients gave their informed consent to participate in the study. Blood was drawn in serum tubes and analyzed directly or after coagulation over night at 8 0 C followed by centrifugation at 2 000 g for 10 minutes.

The MSAT is an ongoing study since 2016 that recruits healthy male volunteers for exercise testing at the Department of Clinical Sciences, Malmö, Lund University, Sweden.

All 39 MSAT serum samples included in this analysis were collected between 2016 and 2017.

The CPIP is an ongoing cohort since 2006 that recruits patients undergoing carotid endarterectomy at Lund University Hospital to study atherosclerosis and inflammatory and immune markers. Blood samples were collected the day before surgery. For the current study, thirty serum samples collected between 2016 to 2018 were included. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint

The serology assessment of COVID-19 antibodies was performed using ZetaGene COVID-19 Antibody Test IgM/IgG (ZetaGene, Sweden; www.zetagene.com). The ZetaGene COVID-19 Antibody Test IgM/IgG is a lateral flow immunoassay intended for the qualitative detection and differentiation of IgM and IgG antibodies to SARS-CoV-2 in serum, plasma or whole blood samples from patients suspected of COVID-19 infection. The antibody assessment was conducted according to the manufacturer's instruction (www.zetagene.com). In brief, 10 µl of serum/plasma or whole blood was dispensed into the test sample well, followed by addition of 100 µl of diluent buffer provided in the kit.

The results were visualized between 15-20 minutes.

The study was performed in accordance with the Declaration of Helsinki. Patients in all study cohorts included gave informed consent to serum donation and study participation.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. 

The data is presented in n (% of total) in respective analysis, and 95% confidence intervals for proportions are calculated according to the Clopper-Pearson method.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. Table 1 ). Together, the overall positive percentage agreement (PPA) for COVID-19 antibody detection is 92% (95%CI: 62%-100%) for hospitalized patients, and 79% (95%CI: 61%-91%) for non-hospitalized patients.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint Previous studies revealed that COVID-19 seroconversion for detectible IgM and IgG begins from the second week of symptom onset. 6 To better understand the dynamics of IgM/IgG detection in response to time after onset and hospitalization, the cohort patients were grouped into (1) acute patients with < 2 weeks after onset of symptoms (n=8, Table   2 ); (2) convalescent patients with > 2 weeks after onset, non-hospitalized (n=26, Table 3 );

(3) convalescent patients with > 2 weeks after onset, hospitalized (n=11, Table 4 ).

In sera from 8 acute patients (7 non-hospitalized and 1 hospitalized) with less than 2 weeks after onset, five patients were tested IgG and IgM positive (63%), and 3 were negative to IgG or IgM (37%, Fig. 3 and 4, Table 2 ). The overall PPA for COVID-19 antibody detection in this subgroup is 63% (95%CI: 26%-90%).

In convalescent sera from 26 non-hospitalized patients with > 2 weeks after onset, eleven (42%) were positive for both IgG and IgM, one (4%) was only IgM positive, ten (38%) were only IgG positive, and 4 (15%) were IgG/IgM negative ( Fig. 3 and 4 , Table   3 ). The overall PPA for COVDI-19 antibody detection was 85% (95%CI: 65%-96%) for this subgroup.

In convalescent sera from 11 hospitalized patients with > 2 weeks after onset, seven (73%) patients were positive for both IgG and IgM, and 2 (18%) were IgG positive only.

There was also 1 (9%) patient with no detectible IgG or IgM ( Fig. 3 and 4 , Table 4 ). The overall PPA in this subgroup for COVDI-19 antibody detection was 91% (95%CI: 56%-100%).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. .

The distribution of antibody detection in the above three groups of patients is 63%, 46% and 73% for total IgM; 63%, 77% and 91% for total IgG; and 63%, 85% and 91% for total antibody (Fig. 3) .

Among all individuals, three patients (ID 2, 5 and 12) were tested at both < 2 weeks and > 2 weeks. Patients 2 and 5 were hospitalized at both samplings, and patient 12 was home quarantined. Patient 2 was IgG and IgM positive at first sampling, and converted to IgG positive IgM negative at the second sampling. Patient 5 was IgG and IgM positive at both time points. Patient 12 was antibody negative at < 2 weeks, and converted to IgG and IgM positive at > 2 weeks.

Serum and plasma antibody detection from the same individuals were compared in 8 patients, and no difference between the two measurements was observed. We also compared serum and whole blood readout from 19 patients. In 18 patients, serum and whole blood gave uniform antibody detection. However, in 1 patient, we observed that IgG positive was only detected in the serum but not in the whole blood.

For accurate interpretation of the rapid IgM/IgG serology assessment of COVID-19 sera, and to determine the specificity of this rapid test, we performed qualitative measurements All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint By combining both MSAT and CPIP cohorts, we obtained an overall NPA 97% (95%CI: 90%-100%) for total negative antibody detection.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. Recent studies reveal that the spike glycoprotein (S) of SARS-CoV-2 mediates receptor binding on host cells, membrane fusion and virus entry. Additionally, it is the main target for neutralizing antibodies. 7 Each monomer of trimeric S protein contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively, with the S1 region being the major immune-epitope for antibody binding. In this study, we employed a lateral flow test that detects antibodies binding to the receptor-binding domain (RBD) of S1 protein.

The test offers qualitative assessment and indicates the presence or absence of SARS-CoV-2 antibodies. The test has been previously evaluated on 38 COVID-19 hospitalized patients and 228 healthy controls during Wuhan COVID-19 outbreak in China, and achieved a sensitivity and a specificity of 95% and 97% (www.zetagene.com). In the current study, All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint we observed that 92% of hospitalized patients have detectable antibodies, which is similar to the clinical validation in China (unpublished). The negative test results may be explained by a combination of test sensitivity, the complexity of antibody development biology, as well as the heterogeneity in antibody titres among individuals.

A previous study revealed that IgM kinetics is notably different between COVID-19 ICU and non-ICU patients. In patients with mild symptoms, IgM levels start to decline after 2 weeks while IgG continues to increase, while in patients with severe symptoms, IgM levels remains static or increases even after 4 weeks. 9 In our study, IgM was detected in 49% of non-hospitalized convalescent patients, and 75% in hospitalized patients (Fig.   1 ). This observation suggest that IgM may be present in the blood for a longer period in hospitalized patients. Furthermore, it has been shown that high titers of IgG antibodies positively correlate with neutralizing antibodies. 10 In our study we also observed higher prevalence of IgG detection in non-hospitalized vs. hospitalized convalescent patients (30% vs. 17%, Fig. 1 ). Furthermore, we mapped a clear increase in the antibody response from early disease onset (< 2 weeks, 63%) to a later time point (>2 weeks, 85% and 91%, Fig. 3 and 4) , which is in line with the antibody development profile following COVID-19 progression and an earlier investigation in Sweden. 8 A previous study on COVID-19 recovered patients showed that there is a heterogeneity in antibody titers among the recovered patients, and around 15% of recovered patients had very low antibody titers or even levels below the threshold of detection. 11 This observation is also in line with our study on recovered patients, where we observed that 85% of non-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint hospitalized recovered patients and 91% of hospitalized recovered patients had detectable COVID-19 antibodies (Fig. 3) .

We compared antibody detection in whole blood, serum and plasma. In one patient out of 19 patients, IgG positivity was only detected in serum but not in whole blood. Also, upon comparison of the sensitivity in serum versus plasma, we did not observe any difference in the 8 patients where both plasma and serum were analyzed. Albeit suggestive, clinical assessment of COVID-19 antibody reactivity performed on serum/plasma is likely to be more accurate than that of whole blood. Further evaluation is needed to compare pinprick blood, whole blood and serum/plasma sensitivity.

The choice of negative control cohorts also warrants explanation. The COVID-19 pandemic was reported to have reached Sweden on 31 January 2020 with the first clinically confirmed case. 12 The epidemic outbreak of COVID-19 in Sweden started on 26 February, with confirmed community transmission on 9 March in the Stockholm region. However, according to the Public Health Agency of Sweden, it is likely that there were individual cases of COVID-19 in Sweden as early as November 2019. Therefore, we chose to use cohorts with blood samples collected before 2019 as the negative controls of the test. We tested both a healthy cohort and a diseased cohort with cardiovascular morbidity, and both cohorts displayed uniform specificity.

In summary, our study provides a comprehensive validation of the rapid COVID-19

IgM/IgG serology assessment. Also, the antibody detection patterns in association with disease progress and hospitalization was mapped, which provides a potential reference for All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint accurate clinical antibody assessment interpretation. The rapid COVID-19 IgM/IgG test may be a valuable application for COVID-19 clinical diagnosis, and can be applied as a powerful tool to assess the COVID-19 status at both individual and population level.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint and whole-body metabolism -implications primarily for human health, but also for fundamental evolutionary questions. The overarching goal is to tailor, for the individual, the most beneficial exercise program to counteract genetic predisposition to metabolic disease and type 2 diabetes. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint . 3 The distribution of antibody detection in response to time from onset and hospitalization. Total IgM, IgG and total antibody detection percentage (%) are presented for PCR-confirmed COVID-19 patients divided into the following groups: (1) acute patients with < 2 weeks (w) after onset (n=8); (2) convalescent patients with > 2 weeks after onset, non-hospitalized (n=26); (3) convalescent patients with > 2 weeks after onset, hospitalized (n=11). Different groups are presented in respective colors as indicated in the figure.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint . 4 The dynamics of COVID-19 IgM/IgG detection in response to time from onset and hospitalization. Qualitative detection of IgM and IgG to SARS-CoV-2 is mapped in COVID-19 cohort patients from the following groups: (1) acute patients with < 2 weeks (w) after onset (n=8); (2) convalescent patients with > 2 weeks after onset, non-hospitalized (n=26); (3) convalescent patients with > 2 weeks after onset, hospitalized (n=11). IgM and IgG detection percentage (%) is presented in respective color as indicated in the figure.

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168815 doi: medRxiv preprint Positive percentage agreement (PPA) = 10/11 (91%, 95%CI: 56%-100%) perpetuity.

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World Health Organization

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Detection of SARS-CoV-2 in Different Types of Clinical Specimens

False positives in reverse transcription PCR testing for SARS-CoV-2

Interpreting Diagnostic Tests for SARS-CoV-2

Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for assessment of past exposure to SARS-CoV-2

Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study