key: cord-0872512-ze4uq8kd
authors: Infantino, Maria; Manfredi, Mariangela; Grossi, Valentina; Lari, Barbara; Fabbri, Sergio; Benucci, Maurizio; Fortini, Alberto; Damiani, Arianna; Mobilia, Emanuela Maria; Panciroli, Marta; Pancani, Silvia; Pesce, Giampaola
title: Closing the Serological Gap in the Diagnostic Testing for COVID‐19: The Value of Anti‐SARS‐CoV‐2 IgA Antibodies
date: 2020-08-13
journal: J Med Virol
DOI: 10.1002/jmv.26422
sha: a157d7ee80dfb3bf516a8e5e4d65416ede0d46dd
doc_id: 872512
cord_uid: ze4uq8kd

BACKGROUND: During COVID‐19 pandemic, the early diagnosis of patients is a priority. Serological assays, in particular IgM and IgG anti‐SARS‐CoV‐2, have today several applications but the interpretation of their results remain an open challenge. Given the emerging role of the IgA isotype in the COVID‐19 diagnostics, we aimed to identify the SARS‐CoV‐2 IgA antibodies in a COVID‐19 population seronegative for IgM. METHODS: A total of 30 patients hospitalized in San Giovanni di Dio Hospital (Florence, Italy) for COVID‐19, seronegative for IgM antibodies, have been studied for anti‐SARS‐CoV‐2 antibodies. They all had a positive oro/nasopharyngeal swab reverse transcription polymerase chain reaction result. Assays used were a chemiluminescent assay measuring SARS‐CoV‐2 specific IgM and IgG (S+N) and an ELISA, measuring specific IgG (S1) and IgA antibodies against SARS‐CoV‐2. RESULTS: Among the 30 patients, eight were positive for IgA, seven were positive for IgG (N+S) and two for IgG (S1), at first point (5‐7 days from the onset of symptoms). The IgA antibodies mean values at the second (9‐13 days) and third (21‐25 days) time points were even more than twice as high as IgG assays. The agreement between the two IgG assays was moderate (Cohen's K = 0.59; SE = 0.13). CONCLUSIONS: The inclusion of the IgA antibodies determination among serological tests of the COVID‐19 diagnostic is recommended. IgA antibodies may help to close the serological gap of the COVID‐19. Variations among anti‐SARS‐CoV‐2 IgG assays should be considered in the interpretation of results. This article is protected by copyright. All rights reserved.

At the end of December 2019, twenty-seven cases of pneumonia of unknown etiology were identified in Wuhan, China [1] . The causative agent was identified by the Chinese Center for Disease Control and Prevention in January 2020 directly from bronchoalveolar-lavage fluid samples and was then named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The disease associated to it was referred as coronavirus disease 2019 . Recently the World Health Organization (WHO) provided an interim guidance to laboratories, showing the strategic use of diagnostic tests in different transmission scenarios of the COVID-19 outbreak, including how to justify their use when prioritizing patients due to the lack of proper facilities. The WHO document specifies the conditions necessary to consider a case laboratory-confirmed by nucleic acid amplification tests (NAAT) for areas with not known or established SARS-CoV-2 circulation [2] .

It has been shown that the viral RNA can be detected from nasal and pharyngeal swabs, bronchoalveolar lavage, and blood plasma using real-time reverse-transcription polymerase chain reaction (RT-PCR) [3] [4] [5] . The molecular diagnostic testing is particularly useful for the diagnosis and triage of patients, monitoring the spread of the disease, identifying strains and mutations, with the next-generation sequencing, and assessing the current infection status. These tests provide the message whether the infection is This article is protected by copyright. All rights reserved.

active or not and can often be of great utility early in the course of infection as they are able to confirm the viral presence up to 2 days before the onset of symptoms [6] . Given that antibodies may not be detectable until 6-7 days after the symptom onset, molecular tests can accelerate the diagnostic window by up to 9 days. In cases where the NAAT are negative, the serological evaluation, both of the acute phase and the convalescence phase, may support and complete a diagnosis [7] . Serological assays are capable of verifying the immune response to SARS-CoV-2, the identification of seroconversion, and finally the characterization of the virus course [8] . The production of specific antibodies, in particular IgM, IgA, and IgG anti-SARS-CoV-2, should be used as an additional and non-invasive method, together with NAAT, in the COVID-19 diagnosis. It's increasingly evident the role of the serological testing also for the disease surveillance, therapeutics, return-to-work screening tests, and vaccine applications. For all the above reasons, antibody tests commercially available are continuously increasing, with a wide variability of kits and test protocols [7] . antibodies (IgG, IgM, and IgA) increased to detectable levels at the third week of illness [9] . On the contrary, an earlier study, in 2003, showed that the three isotypes began to be measurable at the second week [10] .

Since the outbreak of the pandemic COVID-19 in 2020, numerous research groups have become stubborn about the importance of kinetic of antibodies and about the immunological memory. However, the time required by serological tests is not negligible and the interpretation of the results is not always clear [11] [12] [13] . The group of Okba N. et al. demonstrated that most PCR-confirmed SARS-CoV-2 infected patients were seroconverted by 2 weeks after the onset of the infection. Using a commercial ELISA method, they found that IgA antibodies showed higher sensitivity than IgG, but lower specificity [14] . IgA and IgG assays could both be used for serologic diagnosis, but IgG was longer lived, as previously shown, after infection with SARS-associated coronavirus and was thus preferred for studies regarding the sero-surveillance [15] .

The group of Padoan A. et al. described the kinetics of IgM and IgG to SARS-CoV-2 using a chemiluminescent (CLIA) assay, showing a rapid increase of both IgM and IgG after 6-7 days from the onset of first symptoms. IgG presented 100 % and IgM 88% sensitivity on day 12 [16] . In a subsequent study, coming from the same group, the characteristics of the kinetics of IgA antibodies in comparison to IgM were deepened from the COVID-19 onset.

IgA response appeared and grew earlier, peaked at the third week, and maintained a response stronger and more persistent compared to the IgM one [17] .

Lippi et al. studied the antibodies profile comparing anti-SARS-CoV-2 ELISA tests (IgA and IgG) to CLIA tests (IgG and IgM). In patients with symptoms onset ≤5 days the rate of positive antibodies was very low, whilst in those with symptoms onset between 5-10 days the rate of positive antibodies ranged between 15.4% and 53.8%. Notably, in patients with symptoms onset between 11 and 21 days, the rate of positive antibodies was 100% except for IgM antibodies, which was positive only in 60% of patients [18] .

With the viewpoint upcoming from Sethuraman N. et al. [19] , the two SARS-CoV-2 diagnostic screening tests most represented, such as RT-PCR and IgM and IgG ELISA, were analyzed in their variation over the time. They found that the total antibodies levels started to increase from the second week of symptoms onset. Although IgM and IgG antibodies were positive even as early as the fourth day after the first symptom onset, higher levels occurred in the second and third week. IgM and IgG seroconversion appeared in all patients between the third and the fourth week of clinical illness onset. IgM antibodies began to decline and reached lower levels by week 5 and almost disappeared by week 7, whereas IgG persisted beyond 7 weeks [20] . ELISAbased IgM and IgG antibody tests had greater than 95% specificity for diagnosis of COVID-19, presenting a high sensitivity when used at the same time [21] . diagnostics. The main benefits of the anti-SARS-CoV-2 IgA antibodies are due to their early detection and to their high sensitivity [17, 22] . For these reasons, this study aims to assess the temporal profile of specific anti-SARS-CoV-2 antibodies, searching for the IgA isotypes in a COVID-19 population seronegative for IgM, to investigate how it could close the serological gap.

To study the kinetics of SARS-CoV-2 specific antibodies, two different assays were used: CLIA for IgM and IgG detection, and ELISA for IgG and IgA detection. This article is protected by copyright. All rights reserved.

For both IgG (S1) and IgA tests, the cut-off recommended from manufacturers is ≥1.1: hence sample with IgG and IgA value ≥ 1.1 ratio are considered positive (reactive).

This study enrolled a total of 30 patients (mean age 64±19 years; 20 men and 

Statistical analysis was performed with SPSS 25.0 software (SPSS, Chicago, IL, USA).

Wilks normality test (p ≤ 05). All data met the normality requirements for parametric statistics and were thus summarized as mean ± SD.

The agreement among methods was calculated by Cohen's Kappa agreement.

Kappa was considered moderate from 0.41 to 0.60, good from 0.61 to 0.80, and excellent from 0.81 to 1. P value < 0.05 was considered as significant. This article is protected by copyright. All rights reserved.

days from the onset of symptoms), eight were positive for IgA, seven were positive for IgG (N+S) and two for IgG (S1). The distribution of positive sera by each isotype is shown through the Venn diagram reported in Figure 1 . Table 2 ), as well as for IgG antibodies.

The agreement between the two assays, CLIA and ELISA, for measuring IgG antibodies, using N+S and S1 antigens respectively, was moderate (Cohen's K = 0.59; SE = 0.13).

Our results showed that anti-SARS-CoV-2 IgA rise rapidly and reach concentrations markedly higher (over 18-fold the cut off) than those observed where are locally produced to inhibit bacterial and viral barrier adhesion and invasion. Moreover, IgA are the only immunoglobulin capable to penetrate epithelial cells to neutralize intracellular viruses [24] . Notably, SARS-CoV2 is able to damage the respiratory mucosal barrier, entering in alveolar cell by ACE2 receptor, and respiratory symptoms are between the main complaints in infected patients. Also, the gastrointestinal mucosa can be affected, with diarrhea descripted as quite frequent during the first phase of the disease [25] .

Thus, it can be hypotized that high circulant IgA levels represent a mirror of this mucosal infiltration and of the reactive immune activation. It has to be clarified how, in COVID-19, the dual capacity of IgA antibodies, antiinflammatory on one side and pro-inflammatory on the other, is modulated and whether the proinflammatory capacity of IgA, eventually resulting not only in innate immunity but also in T-cell (notably, T helper-1 cell) activation, can affect IgG production and long-term immunity.

Our study focuses on patients seronegative for IgM anti-SARS-CoV-2 and highlights the early positivity for IgA and IgG antibodies. Other studies in the literature report data relating to COVID-19 patients who demonstrated a previous seroconversion for IgG compared to IgM [26] but, it is not clear, whether this evidence is attributable exclusively to a lower sensitivity of IgM tests compared to that for IgG.

As regards the IgG antibody response, two tests were used with different methods and different SARS-CoV-2 antigenic targets. In all the cases studied longitudinally, the IgG (S1) antibodies appeared later compared to the IgG (N+S), reaching 93.3% positivity at time T3 compared to 100% IgG positivity (N + S). In our total results, the agreement between the two assays, CLIA for IgG (N+S) and ELISA for IgG (S1), was moderate (Cohen's K = 0.59) at variance with the data published by Padoan et al. [17] , who reported a better agreement (Cohen's K = 0.83). Such difference may likely be due to a difference in timing: in our study the first point was very early (5-7 days), whereas in Padoan's study the late stage occurred much later (up to 16 weeks).

However, these observations focus our attention, together with consolidated literature data, on the ability of a number of viruses to induce a prompt antibody production by B lymphocytes, regardless of the activation of T lymphocytes. Such antibody production is due to repetitive viral epitopes capable of inducing cytokine production/release by innate immunity cells.

These antibodies are usually short-lived IgG. However, the cooperation of T cells is considered necessary for the development of sustained, long-lasting antiviral antibody responses, thanks to the formation of plasma cells and memory B cells [27] [28] [29] . The responses of CD4 T cells have been shown to be mainly directed towards protein spikes, and it is known that the production of neutralizing antibodies by plasma cells requires interactions with CD4 T cells specific for the same B lymphocyte activation protein [30] . On the basis of these literature data, it could be hypothesized that the production of IgG Accepted Article (N+S) antibodies, earlier than the appearance of IgG (S1) antibodies, would be attributable to T-cell-independent antibody production. IgG (S1) antibodies could represent the neutralizing and important humoral component in the development of immunity.

Prospective data with a long-term follow-up of antibody response in COVID-19 patients and Neutralization Test (PRNT) could elucidate the possible relationship between IgA response and sustained IgG response, likely protective.

Contributors MI, SF, MM and GP participated in the design of the study, and drafted the manuscript. SP supported data analysis and revised the manuscript.

VG, BL, MB, AD, AF, EMM, and MP participated in the design and revised the manuscript.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure 1 . Venn diagram of SARS-CoV-2 specific antibodies in a population seronegative for IgM antibodies. IgG was tested with two different tests: ELISA (S1 antigen) and CLIA (N+S antigens) This article is protected by copyright. All rights reserved. 

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