key: cord-0989461-0bekb7ys
authors: Krempe, Friederike; Schöler, Lara; Katschinski, Benjamin; Herrmann, Anke; Anastasiou, Olympia E.; Elsner, Carina; Ross, R. Stefan; Scholz, Friedrich; Dittmer, Ulf; Miethe, Peter; Le-Trilling, Vu Thuy Khanh; Trilling, Mirko
title: A rapid test recognizing mucosal SARS-CoV-2-specific antibodies distinguishes prodromal from convalescent COVID-19
date: 2021-09-30
journal: iScience
DOI: 10.1016/j.isci.2021.103194
sha: 4fe304a37be15bf9d757de4ea37a9d7853f87a8c
doc_id: 989461
cord_uid: 0bekb7ys

The COVID-19 pandemic poses enormous challenges to global healthcare sectors. To prevent the overburden of medical systems, it is crucial to distinguish individuals approaching the most infectious early phase from those in the declining non-infectious phase. However, a large fraction of transmission events occur during pre- or asymptomatic phases. Especially in absence of symptoms, it is difficult to distinguish prodromal from late phases of infection just by RT-PCR since both phases are characterized by low viral loads and corresponding high Ct values (>30). We evaluated a new rapid test detecting IgG antibodies recognizing SARS-CoV-2 nucleocapsid protein using two commercial antibody assays and an in-house neutralization test before determining suitability for testing clinical swab material. Our analyses revealed the combination of the well-known RT-PCR and the new rapid antibody test using one single clinical nasopharyngeal swab specimen as fast, cost-effective, and reliable way to discriminate prodromal from subsiding phases of COVID-19.

Over 225 million people have acquired a laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and more than 4.64 million fatalities were associated with coronavirus infectious disease 19 (COVID-19) (Dong et al., 2020) . As indicated by surveillance studies (e.g., (Lau et al., 2020) ) and excess mortality calculations (e.g., (Vestergaard et al., 2020) ), the true numbers of infections and fatalities are certainly far higher, and the COVID-19 pandemic is far from being over. In December 2019, the SARS-CoV-2 outbreak was first recognized in the Hubei province in China . On January 31, 2020, the World Health Organization (WHO) declared the outbreak a Public Health Emergency of International Concern (PHEIC) and on March 11, 2020, the spread of SARS-CoV-2 fulfilled all criteria of a global pandemic.

SARS-CoV-2 and SARS-CoV-1 share several virological and clinical similarities, but the easy transmission supported by replication in the upper respiratory is a special feature of SARS-CoV-2 . Given the extent, pace, and severity of the COVID-19 pandemic, diagnostics departments around the globe struggle to provide sufficient test capacities, which often limit the possibilities to conduct a series of follow-up tests for individuals with suspected or proven SARS-CoV-2 infections. The most relevant methods for SARS-CoV-2 diagnosis are real-time reverse transcription-PCR (qRT-PCR) assays using nasopharyngeal swab specimens or other upper respiratory tract samples . Despite some doubts (Woloshin et al., 2020 , Arevalo-Rodriguez et al., 2020 , we found that the vast majority (>90%) of swab specimens are actually of sufficient quality and contain enough nucleic acids to enable the recognition of viruses such as SARS-CoV-2 (Klingen et al., 2020) . or if the qRT-PCR just recognizes viral remnants such as viral genome fragments and/or non-infectious immune complexes. From this, there is a continuous debate if such 'high-Ct' individuals can be discharged from hospitals and need to be quarantined. During the acute infection, SARS-CoV-2 genomes usually become detectable one week before symptom onset, peak during the first symptomatic week, decline afterwards, and eventually viral RNAs become undetectable (Sethuraman et al., 2020) . Therefore, high (>30) Ct values especially from non-recurring clinical sampling are particularly inconclusive. A very important question is whether a low viral burden, represented by a high Ct value, is indicative for an early phase of infection or if the infection is already subsiding (conclusively visualized in (Sethuraman et al., 2020) ). This is especially relevant if no or only very mild symptoms occur and when only single measurements without a longitudinal follow-up can be assessed. This question is important in terms of health policies, since health authorities should be enabled to optimally allocate their limited resources in terms of contact tracing and containment efforts, hospitals need to decide which patients can be discharged or transferred (e.g., to other wards, hospitals, or care facilities), and when medical staff is redeployable after infections. Obviously, the issue concerning the infectiousness also has relevant socio-economic implications regarding the question if people need to be quarantined or can continue to work.

Antibodies play an important immunological role protecting the host against virus dissemination and viral reinfections. Usual targets of immunoglobulin (Ig) M and IgG antibodies are for example the SARS-CoV-2-encoded nucleocapsid protein (N) and the receptor-binding domain (RBD) of the spike protein (S). SARS-CoV-2-specific IgM and IgG responses increase approximately 2 weeks post-infection and during the first week after symptom onset. The amount of IgM positivity peaks by week 5 and then usually becomes undetectable by week 13. SARS-CoV-2-specific IgG peaks at week 4 and stabilizes at intermediate levels during a six-month observation period after a contraction phase between week 6 and 14 , Wu et al., 2021 . Several studies showed that specific IgG antibodies are present in the blood or sera of persons who had been infected with SARS-CoV-2 (see e.g., (Amanat et al., 2020, Zhao et al., J o u r n a l P r e -p r o o f 2020, Premkumar et al., 2020) ). Interestingly, recent findings suggested that, in addition to IgA, also SARS-CoV-2-specific IgG seems to be present in saliva (Isho et al., 2020) . We anticipated that a positive result in a rapid antibody test (rAbT) in conjunction with a very high Ct value might be a simple, inexpensive, and rapid way to discriminate early prodromal and late subsiding phases of the SARS-CoV-2 infection and may be instrumental to support the decision making, e.g., concerning patient discharge and medical staff reemployment following SARS-CoV-2 infections. To this end, we first evaluated a rapid antibody test recognizing IgG molecules binding the nucleocapsid protein of SARS-CoV-2 applying clinical serum specimens. For comparison, two approved and certified antibody binding assays (manufactured by Diasorin and Euroimmun) and an in-house in-cell-ELISA-based neutralization test (Scholer et al., 2020) were conducted. In order to assess the presence of IgG antibodies by the rAbT, we examined nasopharyngeal swab samples obtained from uninfected people, newly infected individuals, and persons being SARS-CoV-2positive since more than two weeks. Our results demonstrated that it is feasible to differentiate prodromal from convalescent COVID-19 by analyzing one single clinical nasopharyngeal swab specimen.

We intended to apply a rAbT for the recognition of SARS-CoV-2-specific immune responses in mucosal samples such as clinical nasopharyngeal swab specimens. To this end, we first evaluated the overall performance of the rAbT using clinical serum specimens. For comparative purposes, we used the Euroimmun Anti-SARS-CoV-2 ELISA and the Diasorin Liaison® XL SARS-CoV-2 S1/S2 IgG CMIA. According to the manufacturer's instructions, cut-off ratios of >1.1 and >15 in the Euroimmun ELISA and the Diasorin CMIA, respectively, were considered as clear indications for the presence of SARS-CoV-2-specific IgG recognizing the S protein. Given that the Euroimmun ELISA was more frequently used in our diagnostics department at the time of the study, we applied the ELISA results to stratify and select serum samples for our analysis. We re-tested 22 ELISA-negative clinical serum specimens by CMIA and found 19 serum samples double-negative in both the ELISA and the CMIA test (ELISA ratio <0.8 and J o u r n a l P r e -p r o o f CMIA <12). When we performed the Senova rAbT, we obtained negative results for all 22 samples, indicating that the 3 CMIA-positive results were most likely false positives (Table 1 ). In light of the discrepancy between ELISA and CMIA, we included an additional antibody assay, the in-cell-ELISA-based neutralization test (icNT) (Scholer et al., 2020) , in our analysis to detect the presence of SARS-CoV-2-neutralizing antibodies (nAb). We examined 18 ELISA-positive serum specimens also by CMIA, icNT, and rAbT. In addition, we included two of the three ELISA-negative, but CMIApositive samples (see Table 1 ). Fifteen samples were double-positive in both IgG assays ( Table 2 ). The ELISA and the CMIA showed concordant results in 85% (34/40) of cases ( Table 1 & Table 2 ). The icNT analysis of the 20 clinical samples that were positive in at least one antibody assay revealed that all samples double-positive in both ELISA and CMIA were also positive in icNT, whereas all samples with discrepant antibody assay results were negative in icNT ( Table 2) . Notably, an evaluation of the 20 clinical specimens by rAbT yielded high consistency with the icNT results ( Table 2) .

The outcome of rAbT assessment was even more concordant with icNT than the results of ELISA and CMIA, indicating that the Senova rAbT has a very good diagnostic performance.

Our aim was to develop a simple method enabling us to distinguish the early prodromal phase from the late declining phase in individuals whose clinical swab specimens exhibited high Ct values in PCR-based SARS-CoV-2 diagnostics. To begin with, we tested if IgA and/or IgG recognizing S or N of SARS-CoV-2 were present in a small number of nasopharyngeal swab specimens of convalescent individuals applying commercially available sandwich ELISAs recognizing IgA and IgG binding to N and S.

The sample size was rather small, but the results suggested that N-specific IgG is present rather frequently in nasopharyngeal swabs (data not shown). Given abovedescribed performance of the rAbT and the consistency between rAbT and icNT ( Table   2) , we tested if we can apply mucosal nasopharyngeal swab material to the rAbT to recognize individuals who had already raised a specific immune response against SARS-CoV-2. To this end, we selected 45 clinical nasopharyngeal swab specimens with positive RT-PCR result based on which the initial SARS-CoV-2 infection was diagnosed, indicating that these samples correspond to early infection events. In addition, we included 45 SARS-CoV-2 RT-PCR-positive swab specimens derived from J o u r n a l P r e -p r o o f 34 individuals who had acquired SARS-CoV-2 at least two weeks earlier. Both the group of newly infected and the group of persons infected for at least 2 weeks comprised samples with Ct values exceeding 30, although the mean values of these two groups differ significantly (Fig. 1A) . This demonstrates that the individual Ct values decline during the course of infection. However, the individual Ct values are insufficient to unambiguously define that a person has progressed to post-infectious period. This also applies to the group of individuals in the late stage of infection: There was no correlation between Ct value and time after the first positive RT-PCR result (Fig. 1B) .

When we determined the suitability of the rAbT for testing clinical nasopharyngeal swab material, we included as control samples swab specimens derived from 42 SARS-CoV-2-uninfected individuals, defined by negative SARS-CoV-2 RT-PCR results and the absence of indications of previous COVID-19 episodes. All of the 42 uninfected individuals showed negative rAbT results ( Fig. 2A) . Furthermore, we applied the rAbT to above-described 45 RT-PCR-positive swab specimens of newly infected individuals. All of these 45 specimens were negative in rAbT ( Fig. 2A) ,

suggesting that these donors did not had time to develop a SARS-CoV-2-specific IgG response. Conversely, when we tested the 45 SARS-CoV-2 RT-PCR-positive swab specimens derived from individuals infected since 2-8 weeks, 33 specimens clearly showed N-specific antibodies recognizable by the rAbT (Fig. 2A) . Six samples showed results, which were evaluated as intermediate rAbT-positive because the test line was visible with a weak appearance, and six samples were rAbT-negative ( Fig. 2A) . These findings reveal that most individuals in the late phase of infection exhibited IgG antibodies to SARS-CoV-2 N which can be detected in clinical nasopharyngeal swab specimens by rAbT. Accordingly, classification into infectious and non-infectious individuals based on a combination of high Ct value (e.g., >30) and positive rAbT appear reliable, whereas assessment based only on Ct values has the potential to be misleading.

The 9 newly infected individuals who had a Ct value above 30 were most likely in their prodromal phase, as indicated by the absence of N-specific antibodies, and might have proceeded to higher viral loads briefly after. In the group of individuals infected for 2-8 weeks, 14 swab specimens had a Ct value under 30. Here, the rAbT was positive in 71.4% of cases (10/14), in 14.3% (2/14) the rAbT was negative or intermediate positive (Fig. 2B) . When we grouped the samples according to the rAbT result, there was no significant difference between the groups in terms of post-infection time (as indicated J o u r n a l P r e -p r o o f by days since the first positive RT-PCR), although a tendency to a shorter postinfection period was observed for the rAbT-negatives (Fig. 2C) . Of these 6 rAbTnegative specimens, 3 samples belonged to individuals who were tested more than once. In these 3 cases, additional clinical swab samples were obtained 2-7 days after the rAbT-negative sampling, all of which tested positive in rAbT ( Table 3) , suggesting that the rAbT-negative sample collection was conducted just before antibodies developed. Of the 3 remaining rAbT-negative specimens, 2 individuals were known to have underlying conditions ( Table 3) Taken together, our analysis revealed the combination of positive rAbT in conjunction with low but positive RT-PCR (e.g., Ct >30) conducted on the basis of a single clinical nasopharyngeal swab specimen as fast, cost-effective, and reliable way to discriminate prodromal from subsiding phases of COVID-19 and to distinguish increasing from decreasing trends of SARS-CoV-2 virus burdens.

We established and validated a rAbT to assess the presence of SARS-CoV-2 Nspecific IgG antibodies in clinical serum samples and nasopharyngeal swab specimens. Using SARS-CoV-2-positive and -negative serum samples, we first demonstrated that the Senova rAbT correlates very well with the approved and certified antibody binding assays Euroimmun Anti-SARS-CoV-2 ELISA and Diasorin Liaison® XL SARS-CoV-2 S1/S2 IgG CMIA (Amanat et al., 2020 , Premkumar et al., 2020 . In 100% of cases, the rAbT showed consistent results with the ELISA and the CMIA double-positive serum samples and the icNT analyses. All samples only single-positive in the ELISA or the CMIA were rAbT-and icNT-negative. Thus, the result of the rAbT assessment matched even better with the icNT than the results of J o u r n a l P r e -p r o o f ELISA and CMIA. Therefore, we concluded that the IgG antibody levels required for a positive rAbT result seem to correlate very well with the neutralization capacities and that the Senova rAbT has a very good diagnostic performance.

Based on recent findings suggesting the presence of SARS-CoV-2-specific IgG at the nasopharyngeal mucosa (e.g., (Isho et al., 2020) ), we suspected that clinical swab specimens used for RT-PCR-based SARS-CoV-2 diagnostics might also contain sufficient N-specific IgG to be suitable for antibody tests. Application of the rAbT faithfully discriminated samples derived from individuals who were in the prodromal early phase of COVID-19 from samples derived from late phases of SARS-CoV-2 infections when humoral immune response already raised N-specific IgG recognizable by the rAbT. Indeed, we found that a high frequency of nasopharyngeal swab samples derived from donors who were shedding low levels of SARS-CoV-2 genomes at least two weeks post primary infection contain rAbT-reactive N-specific IgG, while the specimens either derived from uninfected individuals or individuals who were infected very recently remained rAbT-negative. Thus, rAbT positivity can provide additional information and add a layer of safety concerning the decision if individuals with low virus doses (Ct >30) are currently in the increasing or the decreasing phase concerning viral burden. Obviously, this discrimination is particularly relevant during presymptomatic phases of infection and in the case of asymptomatic courses when symptoms cannot be used as a reference. The latter is in fact also important given that a considerable fraction of people acquire a SARS-CoV-2 infection without experiencing symptoms , Wu et al., 2021 . Additionally, modeling studies suggest that approximately half of all infections originate from individuals without symptoms (Johansson et al., 2021) . Thus, pre-and asymptomatic infections are an urgent issue which need to be correctly diagnosed. studies agree that IgG antibodies to SARS-CoV-2 spike and RBD antigens are detected in the blood in more than 90% of cases by 10-11 days post-symptom onset (e.g., (Amanat et al., 2020 , Long et al., 2020 ). Thus, rather similar results were evident here. The small difference may be explained by the assumption that IgG antibody responses may be detected a bit earlier or may be more abundant in the blood compared to the nasopharyngeal mucosa.

In conclusion, our study provides evidence that the Senova rapid SARS-CoV-2 IgG antibody test is an easy and cost-effective way to diagnose IgG antibodies in the nasopharyngeal swab specimens of individuals during the late phase of infection. The entire rAbT can be processed in less than 20 minutes. The fact that the identical clinical nasopharyngeal swab specimens can be used for the RT-PCR as well as the rAbT is clearly an advantage. It saves time of clinicians, laboratory staff and patients, since no further sampling is necessary. In contrast to automated diagnostic IgG tests or ELISA that require plate readers, the rapid antibody test does not require equipment and can be processed under a laminar airflow workbench. Thus, undiluted and non-inactivated specimens can be examined safely. Additionally, the test is inexpensive (in our case, approx. 5 € per test), ready-to-use and easy to handle. Taken together, the rAbT may have a diagnostic value and may support clinical decision-making.

In our study, we analyzed 132 nasopharyngeal swab specimens derived from 42 uninfected, 45 newly infected, and 45 individuals infected since 2-8 weeks. Although our cohort showed clear differences among the groups, the number of samples is rather limited for a clinical cohort.

Since neutralizing antibodies are not determined by the rAbT, we would like to emphasize that we do not claim that rAbT-positive individuals are immune to SARS-CoV-2. Instead, our test discriminates prodromal from late phases of infection. We are convinced that a very high CT value in combination with a positive rapid antibody test reduces the likelihood that the person sheds infectious SARS-CoV-2.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mirko Trilling (mirko.trilling@uk-essen.de).

No new materials have been generated. All reagents are commercially available and all protocols are either presented here or are publicly available e.g., in previously published open-access papers. Technical questions should be directed and will be answered by the Lead Contact, Mirko Trilling (mirko.trilling@uk-essen.de).

The published article includes all data sets generated or analyzed in this study. This study did not generate new algorithms.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Vero E6 cells (ATCC CRL-1586) were cultivated in high glucose Dulbecco`s minimal essential medium (DMEM [Gibco 41966-029]), supplemented with 10% (v/v) FCS, penicillin, and streptomycin at 37°C in an atmosphere of 5% CO2. SARS-CoV-2 was isolated from a patient sample using Vero E6 and confirmed by SARS-CoV-2 diagnostic qRT-PCR. Viral titers were determined by TCID50 titration.

The collection of serum and swab specimens was part of the routine diagnostic procedure.

The assessment of test samples for the improvement of diagnostic procedures and the virus isolation have been approved by the ethics committee of the medical faculty of the University of .

The Euroimmun Anti-SARS-CoV-2 ELISA was conducted in accordance to the recommendations of the manufacturer (Euroimmun, Lübeck, Germany). If the ratio exceeded 1.1, the test was considered to be positive. Results below 0.8 were judged to be negative. Borderline samples (ELISA ratio between 0.8 and 1.1) were excluded.

The Diasorin Liaison® XL SARS-CoV-2 S1/S2 IgG CMIA was conducted in accordance to the recommendations of the manufacturer (Diasorin, Saluggia, Italy).

RLU were converted to arbitrary units (AU/ml) based on a standardized master curve.

If the value was >15 AU/ml, the test was considered to be clearly positive. Values below 12 AU/ml indicated the absence of recognizable SARS-CoV-2-specific IgG antibodies binding to S1/S2.

For direct SARS-CoV-2 detection, the RealStar® SARS-CoV-2 RT-PCR kit (Altona, Hamburg, Germany), which targets the SARS-CoV-2 genes S and E, was used.

The SARS-CoV-2 icNT was recently described in an open access journal including a detailed laboratory protocol (Scholer et al., 2020) . For the analysis of neutralizing antibodies, serum samples were inactivated at 56°C for 30 min. Briefly, defined doses of SARS-CoV-2 were incubated with different serum dilutions for 1 h at 37°C prior to Vero E6 infection in a 96-well plate. At 16 to24 h p. i., cells were fixed with 4% (w/v) paraformaldehyde/PBS. Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. The primary antibody was added and incubated for 2 h at room temperature or overnight at 4°C. Peroxidase-labelled secondary antibody was incubated for 1 to2 h. Washing steps were performed with 0.05% (v/v) Tween-20/PBS. Tetramethylbenzidin (TMB) substrate was added to visualize the enzyme reaction. The reaction was stopped with 0.5 M HCl. Subsequently, the absorbance was measured using a microplate multireader (Mithras2 LB 943; Berthold Technologies, Bad Wildbad, Germany). An anti-N mAb (ABIN6952435) and POD-coupled secondary antibodies (Dianova) were used.

To perform the rapid test, 10 µl of the virus transport medium (VTM) derived from the clinical nasopharyngeal swab specimens was pipetted onto the IgG antibody test strip.

Subsequently, 2 drops of the buffer from the kit were added to the strip. The test was 

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