key: cord-0792978-tsgj730x
authors: Serre-Miranda, C.; Nobrega, C.; Roque, S.; Canto-Gomes, J.; Silva, C. S.; Vieira, N.; Barreira-Silva, P.; Alves-Peixoto, P.; Cotter, J.; Reis, A.; Formigo, M.; Sarmento, H.; Pires, O.; Carvalho, A.; Petrovykh, D. Y.; Diéguez, L.; Sousa, J. C.; Sousa, N.; Capela, C.; Palha, J. A.; Cunha, P. G.; Correia-Neves, M.
title: Performance assessment of 11 commercial serological tests for SARS-CoV-2 on hospitalized COVID-19 patients
date: 2021-01-20
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.01.038
sha: 3b341643c40bb04bfa0227f64006fe56a9c573eb
doc_id: 792978
cord_uid: tsgj730x

Background Commercial availability of serological tests to evaluate immunoglobulins (Ig) towards severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has grown exponentially since the onset of disease outbreak. Their thorough validation is of extreme importance before using them as epidemiological tools to infer seroprevalence in specific populations, and as complementary diagnostic tools to molecular approaches (e.g. RT- qPCR). Methods Commercial serological tests from 11 suppliers were assayed side-by-side using 126 samples from SARS-CoV-2-infected inpatients, and 36 healthy and HIV-infected individuals. Results Most of the tests assayed have >95% specificity. For sensitivity calculation, samples were stratified by days since symptoms onset; sensitivity peaks at 16-21 days for IgM and IgA (maximum: 91.2%; Euroimmun); and, varying with the test, at 16-21 or at >21 days for IgG (maximum: 94.1%; Snibe). Data from semiquantitative tests show that patients with severe clinical outcome, in comparison with patients with a non-severe presentation, reveal lower levels of Ig against SARS-CoV-2 at <10 days since symptoms onset, and higher levels at >21 days. Conclusions This study highlights the sensitivity heterogeneity accompanied by an almost general high specificity of most tests and sets the basis for the usefulness of these tests as a complement for diagnosis and population seroprevalence studies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a large RNA virus from the Coronaviridae virus family that is currently globally spread causing coronavirus disease 2019 ([CSL STYLE ERROR: reference with no printed form.]; Lu et al. 2020) . Considering the absence of an effective vaccine or treatment for SARS-CoV-2 infection, early diagnosis of infection and isolation of infected individuals is critical to control the ongoing pandemic (Gudbjartsson et al. 2020b) . Most efforts for case detection involve collection of swab samples from the upper respiratory tract, and the amplification of viral nucleic acids sequences by RT-qPCR ). More recently, tests detecting SARS-CoV-2 proteins from swab samples were also developed. While viral detection is essential for active infection surveillance, detection of antibodies towards SARS-CoV-2 arise as interesting complementary surveillance tools. Following SARS-CoV-2 infection, most patients produce detectable immunoglobulins (Ig) against a set of viral antigens, particularly to the immunodominant nucleocapside (N) and spike (S) proteins Tang et al. 2020; Walls et al. 2020; Zhao et al. 2020) . Current evidence suggests that Ig produced against these antigens may confer protection to SARS-CoV-2 infection (Cao et al. 2020; Ju et al. 2020; Poh et al. 2020) . Nevertheless, there is still insufficient data on the timing of Ig production upon infection. Literature suggests that, considering the timing since symptoms onset, blood IgA and IgM can be detected after 6-8 days Montesinos et al. 2020; Okba et al. 2020; Padoan et al. 2020; Yongchen et al. 2020) , and IgG seroconversion seems to occur slightly later (at 9-10 days). However, many patients seem to seroconvert for both IgM and IgG simultaneously, peaking at around 21 days (Liu et al. 2020b; Sun et al. 2020; Yongchen et al. 2020; Zhang et al. 2020) . Moreover, it is yet debatable whether Ig levels varies with disease severity.

The performance (sensitivity and specificity) of serological assays can be affected by many variables including: timing of assessment since symptoms/infection, course of COVID-19 (from asymptomatic to lethal) and, potentially, population and virus genetics (Osório and Correia-Neves 2020; Peeling et al. 2020) . It is thus unequivocally important to evaluate the performance of commercially available serological tests, comparing their performance side-by-side using J o u r n a l P r e -p r o o f samples from extremely well characterized patients with well define clinical presentation and distinct time-points upon symptoms onset, to be able to select the most adequate tests for defined populations.

We evaluated the performance of serological tests (3 semiquantitative and 8 qualitative) from 11 suppliers using plasma samples of hospitalized patients with COVID-19 from the Minho region, in the North of Portugal. These tests were chosen considering previous reports on their sensitivity, specificity, and availability.

Patients living in the Minho region of Portugal, followed-up as inpatients at Senhora da Oliveira Hospital (Guimarães) and Braga Hospital, diagnosed with COVID-19 (by RT-qPCR at a reference laboratory; at least two positive RT-qPCR results were obtained from each patient) were invited to participate in the study. This study was approved by the Ethics Committees of both participating Hospitals (Senhora da Oliveira Hospital: 25/2020; Braga Hospital: 37/2020). An explanation of the project was provided to those individuals, and the ones that agreed to participate signed an informed consent form. The informed consent was prepared according to the Declaration of Helsinki principles, the Oviedo Convention and according to the General Data Protection Regulation -Regulation (EU) 2016/679. Patients' blood samples were collected throughout hospitalization, at different timepoints following symptoms onset. The number of samples available from each participant varied depending on the duration of their hospitalization.

For sensitivity calculation, COVID-19 patients were stratified based on the number of days since symptoms onset as follows: <10 days; 10 to 15 days; 16 to 21 days; >21 days. Days since symptoms onset were calculated based on each patient's self-report of symptoms manifestation.

COVID-19 patients were further categorized according to the severity of their clinical presentation.

Patients given oxygen therapy above 10 L/min and/or needing mechanical ventilation (noninvasive or invasive) were considered as having a severe clinical presentation. All other patients J o u r n a l P r e -p r o o f (needing supplementary oxygen therapy below 10 L/min and not requiring mechanical ventilator support) were considered as having non-severe clinical presentation. SARS-CoV-2 non-infected controls were selected from banked human plasma samples from two studies at pre-COVID-19 pandemic (the first COVID-19 case in Portugal was reported in March 2 nd , 2020): i) a study with healthy individuals older than 55 years of age (samples collected between April 2019 and January 2020); ii) a study with HIV-infected patients on antiretroviral therapy (54 to 60 months; samples collected between January 2016 and August 2018) (Rb-Silva et al. 2019) . In both cases, matched samples were selected based on COVID-19 patients' sex and age. Control samples were collected, processed and preserved at -80 ºC using a similar protocol as the one used for samples from COVID-19 inpatients (bellow).

Data was handled anonymously. Individuals' sex, age and comorbidities are summarized in Table   1 .

From each patient, venous blood was collected into K2EDTA collecting tubes and processed on the same day: blood collecting tubes were centrifuged at 2000 xg for 15 min, at 20 ºC. Plasma was aliquoted into screw-cap tubes and frozen at -80ºC until tested.

Semiquantitative [enzyme linked immune-absorbent assays (ELISA) and chemiluminescence immunoassays (CLIA)] and qualitative assays [lateral flow immunoassays (LFIA)] from 11 different suppliers were tested according to manufacturer's instructions (Table 2) . At least two different tests were performed for each sample (Supplementary Table S1 and S2).

For each test, specificity was calculated as the percentage of negative tests among the pre-COVID-19 controls, and sensitivity as the percentage of positive tests among the SARS-CoV-2 confirmed cases. Sensitivity was calculated upon stratification in days since symptoms onset J o u r n a l P r e -p r o o f (<10, 10-15; 16-21 and >21 days) . Whenever the same patient was tested multiple times within the same time range during the course of the disease, the results of only one plasma sample was considered to calculate sensitivity (Supplementary Table S1 ). The 95% confidence intervals for sensitivity and specificity were calculated using the Wilson score with continuity correction method. Positive predictive values (PPV) and negative predictive values (NPV) were calculated for 3 prevalence scenarios, as described elsewhere (Tenny and Hoffman 2020) : 3% seroprevalence in a general population, which is the estimated prevalence for the Portuguese population (2020); 50% seroprevalence in a high risk sub-population; and 95% seroprevalence in hospitalized patients suspect for COVID-19. Tests' agreement was evaluated using Cohen's Kappa. For the semiquantitative tests, receiver-operator characteristic (ROC) curves were constructed and used to calculate the area under the curve (AUC) of the different serologic tests.

All variables analysed had a non-normal distribution, as verified by Shapiro-Wilk tests.

Comparisons of the relative amounts of Ig at the various time ranges were performed using the Kruskal-Wallis test, followed by Dunn's post-hoc tests. Comparisons of the relative amount of Ig in the groups of patients with severe and non-severe clinical presentation were performed using the Mann-Whitney U-test. Comparison of demographic and clinical conditions percentages between groups of patients, with severe and non-severe clinical presentation, were performed using Fisher Exact Probability test.

Differences were considered significant when p<0.05. Statistical analyses were performed on GraphPad Prism version 8.4.3 (La Jolla, San Diego, CA, USA).

This study includes 89 inpatients infected with SARS-CoV-2 (diagnosed and re-confirmed during hospitalization by RT-qPCR). Plasma samples from 40 individuals were tested at different timepoint during hospitalization (Supplementary Table S1 ). Most participants are women (57%) J o u r n a l P r e -p r o o f with a median age of 71 years old (Table 1) . None of the COVID-19 patients are HIV-positive or have history of organ transplantation.

Serologic tests from 11 suppliers were assayed for their performance. Considering the combined results of IgM and IgG tests, specificity ranges, in most of the tests, from 95.8% to 100.0%, except for Cellex and Snibe (76.0% and 88.6%, respectively). Considering each Ig class independently, 4 out of 10 tests assessing IgG, and 5 out of 9 tests assessing IgA or IgM show 100.0% specificity (Table 3 and Supplementary Figure S1 ). From the thirty-six samples used as negative controls (collected at the pre-COVID-19 pandemic), 10 negative controls are positive for at least 1 test; 5 of which are positive in more than 1 test (Supplementary Table S2 ). As for the semiquantitative tests (Abbott, Euroimmun and Snibe), 2 of the negative control samples are positive in the Snibe IgG test but have a value close to the negative-to-positive cut-off value (1.124 and 1.652 AU/mL; cut-off 1.000 AU/mL; Supplementary Figure S2 and Supplementary Table S2) .

To analyse sensitivity, samples were stratified according to time since symptoms onset (<10, 10-15, 16-21 and >21 days). For each Ig, the lowest sensitivities are observed at <10 days since symptoms onset: the Leccurate IgG test presents the highest sensitivity (71.4%) and Snibe IgM the lowest (33.3%; Table 3 and Supplementary Figure S1 ). Detection of IgA or IgM reaches maximum sensitivity at 16-21 days in 7 of the 9 assays. The maximum sensitivity for IgG peaked at 16-21 days in half of the tests; the other half at >21 days (Abbott, Cellex, Innovita, Leccurate, and Render). As for Getein, that detects total Ig towards SARS-CoV-2, the peak detection is at 16-21 days. Considering the combined Ig classes, at >21 days, the Euroimmun test (IgA and IgG) provides the highest sensitivity (91.4%), followed by the Snibe test (IgM and IgG; 90.6%). The combined IgM and IgG Leccurate test provides the lowest sensitivity value (75.0%; Table 3 and Supplementary Figure S1 ).

Positive and negative predictive values (PPV and NPV, respectively), vary according to the estimated disease seroprevalence. For the assayed tests, three distinct population seroprevalence scenarios were tested, from low (3%; considered to be the prevalence in the general population, and the estimated to be found among the Portuguese population (2020)) to high (95%; considering the hospitalized settings). To estimate the adjusted PPV and NPV the overall sensitivity of each test was calculated (i.e. without stratification by says since symptoms onset); Abbot, Euroimmun, Snibe and Cellex present the highest overall sensitivity for the detection of IgG, with values ranging from 74.3 to 76.73% (Table 4 ). Detection of IgA by the Euroimmun test has an overall sensitivity of 84.9%, and Cellex and SD for IgM of 78.6% and 77.1%, respectively; all the other tests detecting IgM have overall sensitivities bellow 69.4%.

Regarding the combined detection of IgG and IgA or IgM, semiquantitative tests present the highest overall sensitivity (Euroimmun 86.5%; Snibe 80.3%); all the qualitative tests have an overall sensitivity lower than 78.6% (Table 4 ). The estimated NPV in a low seroprevalence scenario (3%) vary from 98.6% to 99.6%, dropping drastically in a high seroprevalence scenario (95%) to 10.1% to 27.5%. The estimated PPV reached 100% for some tests, irrespectively of the prevalence scenario. Additionally, estimated PPV is >98.4% in high prevalence scenarios, >76.6% in intermediated prevalence scenarios (50%) and >9.2% in low prevalence scenarios.

Euroimmun, Innovita, Leccurate, Liming, Medomics, Render and SD have PPV of 100%, independently of the Ig detected; in the combined tests only Liming and Render have 100% PPV (Table 4) .

Regarding test's agreement, the two tests with the overall highest sensitivity (Euroimmun and Snibe; Table 4 ) show a moderate agreement index (Cohens' Kappa = 0.723; Table 5 ). Euroimmun has the best agreement with SD (Cohens' Kappa = 0.827), and Snibe correlates the best with Getein (Cohens' Kappa = 0.874). For semiquantitative tests, most of the samples from COVID-

Analysis of the ROC curves of the semiquantitative tests reveals that Euroimmun is the test that best distinguishes SARS-CoV-2 non-infected controls from the SARS-CoV-2 confirmed cases, both for IgA and IgG [AUC (IgG) = 0.911; AUC (IgA) = 0.935; Figure 1 ].

To further understand the immune response profile to SARS-CoV-2 Ig levels were associated with the clinical presentation: COVID-19 inpatients were classified as having severe or non-severe clinical presentation based on the need, or not, of oxygen therapy above or below 10L/min, respectively, as specified in the Methods section. When comparing COVID-19 patients with severe or non-severe clinical presentation, no differences were observed on their clinical and demographic characteristics (Table 1 ; i.e. gender, hypertension, diabetes, obesity, neoplasia, autoimmune disease, smoking habits, and administration of corticosteroids or other immunosuppressive drugs; as assessed by Fisher Exact Probability test).

Independently of clinical presentation, all Ig measured in the semiquantitative tests are detected at early days since symptoms onset (<10 days). IgM and IgG present a plateau in their relative amount from 16 to 20 days since symptoms onset onwards; a peak in the relative amount of IgA is observed at 16 to 21 days since symptoms onset (Supplementary Figure S3) . When comparing COVID-19 inpatients with severe and non-severe clinical presentation, IgG relative amounts tend to be lower in the severe group at <10 days since symptoms onset, reaching statistical significance for Abbot and Euroimmun [Abbott: U = 7.5, p = 0.0006; Euroimmun IgG: U = 23.5, p = 0.0045; Figure 2 ]. Accordingly, Euroimmun IgA and Snibe IgM also present lower relative amounts in the severe group at <10 days since symptoms onset when compared with the non- J o u r n a l P r e -p r o o f DISCUSSION Serological testing to SARS-CoV-2 infection is a rapid, inexpensive, and easy-to-perform diagnostic approach complementary to RT-qPCR, but also an essential tool to access the presence and profile of Ig towards SARS-CoV-2, both individually and at the population level. The present study compares the performance of serological tests towards SARS-CoV-2 using well characterized COVID-19 inpatients at various moments of the disease course, and with diverse clinical presentation severity. Many studies exploring the production of Ig against SARS-CoV-2 have been reported, but few compare several tests between them using the same set of plasma samples. Comparison of tests performance, as presented here, is of relevance since it depends on inherent population genetic variations, on time during the course of disease, and on disease severity.

Regardless test methodology (ELISA, CLIA, LFIA), we observe here, as reported previously, that test sensitivity is dependent on time since symptoms onset. In addition, the combined detection of IgG and IgA/IgM against SARS-CoV-2 leads, in most cases, to a better performance than measurements of a single Ig class, regardless of the time range evaluated. These observations were previously reported by others, and are in agreement with the establishment of the immune response, where IgA, IgM and IgG have different dynamics throughout disease progression (Alter and Seder 2020; Guo et al. 2020; Liu et al. 2020b; Montesinos et al. 2020; Okba et al. 2020; Padoan et al. 2020; Sun et al. 2020; Yongchen et al. 2020; Zhang et al. 2020) . This dynamic nature of tests sensitivity needs to be considered when interpreting performance. Inconsistent reporting by manufactures, or lack of details about the timepoints used to establish tests performance, caused confusion about the expected sensitivity. This has contributed to the recent decision by the US Food and Drug Administration to remove some tests from the Emergency Use Authorization.

Previous reports concluded that N-protein/peptide-based tests present better sensitivities than the ones based on S-protein/peptide. This difference may result from an earlier immune response towards the N-protein/peptide in comparison to the one towards the S-protein/peptide, or related to the higher specificity of Ig towards the N-protein/peptide (27) (28) (29) ]. It is not possible to confront J o u r n a l P r e -p r o o f these previous results with ours, as 4 of the assayed tests displayed no information regarding the target antigen, 4 target both proteins, 2 target only the N-protein/peptide alone, and 1 target only the S-protein.

Regarding seroconversion rate, it is striking to notice that Ig towards SARS-CoV-2 are detectable at very early days since symptoms onset (at 3 to 5 days either for IgM, IgA or IgG; e.g. Patients 012, 033 and 070; Supplementary Table S1), though for others, detection using the same tests occurs only much later (>21 days; e.g. Patients 004, 037 and 064; Supplementary Table S1).

Several factors might account for these observations. Antibody production kinetics during SARS-CoV-2 infection is not yet fully elucidated, nor are the factors responsible for differences in patients' response (Chen et al. 2020; Gudbjartsson et al. 2020a; Liu et al. 2020b; Wang et al. 2020; Zhao et al. 2020) . Identifying the variable responsible for distinct profiles of seroconversion is necessary to be able to fully understand false-negative results.

It is important to recall that the time since symptoms onset is self-reported, which might introduce variability taking into consideration the diversity of clinical manifestation of COVID-19 and of symptoms perception by each individual. However, we consider that it introduces less variability than time since disease diagnosis by RT-qPCR, which is performed for some patients before symptoms onset and for others several days after.

Despite the variability in the overall sensitivity among the assayed tests, the PPV in COVID-19 hospitalized patients when assuming a 50% seroprevalence, or higher, is above 87.5%, with exception of Cellex. However, serological tests usage as a seroprevalence and surveillance tool when the estimated prevalence is low (e.g. 3%, which is the estimated seroprevalence for the Portuguese population (2020)), is more challenging due the observed variation. Even though, some of the tests are promising in low prevalence scenarios. Regarding the semiquantitative tests, which can only be used in a laboratory setup, Euroimmun present the best PPV (mainly IgG), as expected due their high specificity. Considering the rapid diagnostic tests, the combo IgG and IgM from Liming and Render present a 100% PPV. The high NPV in low prevalence scenarios using these serological tests reinforce their use in the general population, if the objective is to detect the presence of SARS CoV-2 antibodies in seroprevalence studies. However, for J o u r n a l P r e -p r o o f population seroprevalence studies during a pandemic phase, a highly specific test is required to assure an acceptable PPV in a low seroprevalence population, as well as a reasonable sensitivity.

Euroimmun IgG (ELISA) and the Liming IgG and IgM combo (LFIA) meet the criteria. They both have 100% PPV and an overall specificity close to 75% on a 3% seroprevalence scenario, meaning that all the positive tests will be specific for SARS CoV-2 antibodies though, they will detect only 75% of the subjects with Ig towards SARS-CoV-2 (Table 4 ).

Our results highlight that Ig levels, on a hospitalized COVID-19 population, are associated with disease severity. In fact, our data suggests a delay in Ig detection in patients with worse disease presentation; at <10 days since symptoms onset they present lower relative amounts of Ig (IgA, IgM and IgG) when compared to patients with non-severe clinical presentation. To our knowledge, this was not previously reported. In fact, some of the studies did not find any difference in the Ig production between patients with distinct clinical presentations at the initial phase of the disease.

As others previously suggested (Chen et al. 2020; Gudbjartsson et al. 2020a; Liu et al. 2020b; Wang et al. 2020; Zhao et al. 2020 ), we also observe that patients with severe clinical presentation show higher relative amounts of Ig at latter periods since symptoms onset (>21 days).

Interestingly a recent study showed that the higher amounts of Ig in patients with severe clinical presentation are associated with higher neutralizing capability ). The association of Ig production with disease severity is of outmost relevance in clinical settings and needs to be further explored in a larger cohort of patients evaluated longitudinally, since the very beginning of symptoms manifestation perception, and throughout longer time-periods.

Our results are based on measurements performed in a specific population of hospitalized COVID-19 patients. Asymptomatic and non-hospitalized COVID-19 patients were not analysed and, for this reason, extrapolation of these data to other COVID-19 settings or to general population should be cautious. Still, it provides the basis for an informed selection of a serological test to be assayed and applied in a greater population setting to evaluate the potential value of each test as a complement for COVID-19 diagnosis, and to understand the dynamics of Ig production upon infection with SARS-CoV-2 or upon vaccination. 

Getein kits were provided free of charge by the manufacturer. No other conflicts of interest reported. Table 1 . Clinical and demographic characterization of the cohort. Table 2 . Assayed commercial tests to detect immunoglobulins specific for SARS-CoV-2 infection. Table 3 . Sensitivity and specificity of the assayed tests to detect immunoglobulins specific for SARS-CoV-2 infection. Sensitivity was evaluated in COVID-19 patients upon stratification by days since symptoms onset. Each colour gradient refers to higher (darker) towards lower (lighter) values of specificity (yellow) or sensitivity (green). Table 4 . Negative and positive predictive values (PPV and NPV, respectively) of assayed commercial tests to detect immunoglobulins specific for SARS-CoV-2 infection. PPV and NPV are adjusted for different population prevalence scenarios (3, 50 and 95%), as described in the "Data analysis" section of material and methods. Each colour gradient refers to higher (darker) towards lower (lighter) values of NPV (blue) or PPV (grey). Table 5 . Cohen's kappa coefficient for the strength of agreement between assayed tests to detect immunoglobulins specific for SARS-CoV-2 infection. Colour gradient refers to higher (darker) towards lower (lighter) values Cohen's kappa coefficient (purple). Obesity, n (%) 2 13 (14.6) 4 (12.5) 9 (15.8) n/a n/a Neoplasia, n (%) 8 (9.0) 4 (12.5) 4 (7.0) n/a n/a Autoimmune disease, n (%) 5 (5.6) 2 (6.3) 3 (5.3) n/a n/a Active smokers, n (%) 2 (2.2) 0 (0.0) 2 (3.5) n/a n/a Corticosteroids (during hospitalization), n(%) 6 (6.7) 3 (9.4) 3 (5.3) n/a n/a

Other immunosuppressive drugs, n (%)

Prior to hospitalization 5 (5.6) 3 (9.4) 2 (3.5) n/a n/a During hospitalization 1 (1.1) 1 (3.1) 0 (0.0) n/a n/a Getein Abbott Abbott Abbott Getein J o u r n a l P r e -p r o o f

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The authors gratefully acknowledge Drs. AC Braga and R Menezes (University of Minho) for discussions of the statistical properties of the data, Dr. Qi Huan (Nanjing Tembusu New Material Research Institute) for assistance in obtaining the Medomics kits, Dr. Lei Wu (INL) for assistance in obtaining the Getein kits and Hao Tu (Overseas Business, Getein) for providing the Getein kits for this project free of charge. We are thankful to all study participants.