Antibody response in patients admitted to the hospital with suspected SARS-CoV-2 infection: results from a multicenter study across Spain

Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome produced by a novel coronavirus (SARS-CoV-2) that has spread globally and very quickly since its first appearance in Wuhan, China, in December 2019 [1]. SARS-CoV-2 is the seventh known coronavirus that infects humans; SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43, and 229E are associated with mild respiratory illness [2]. The virus has a genome size of 30 kilobases that encodes multiple structural and nonstructural proteins. The structural proteins include the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.

The diagnostic approach to SARS-CoV-2 includes the detection of viral RNA by real-time PCR (RT-PCR). Different factors could contribute to false negative results of RNA tests with RT-PCR, such as insufficient amount of virus at the site of sample collection, incorrect sample collection or being outside in the viral replication time window [3‐6]. Serological methods combined with PCR could be of help for increasing the sensitivity and accuracy of the diagnosis, especially in patients with negative RT-PCR results; serology may also help to identify asymptomatic and past infections. SARS-COV-2 serology undoubtedly helps to understand the immune status of the population and to evaluate viral spread [7]; hence, serology should be used for epidemiological studies to investigate the rate of asymptomatic infections and to better estimate morbidity and mortality [7]. Serological methods include binding and neutralization assays. Binding assays such as ELISAs are easily automatized, and they are very well adapted to a pandemic situation; neutralization assays require viral culture, and they must be performed in a facilities with higher biosecurity levels [8].

Preliminary studies have analyzed antibody responses against SARS-COV-2. Some authors [2, 9‐12] found that IgM was detected on day 7 and peaked on day 28, and IgG appeared by day 10 and peaked on day 49, while others [13] determined that seroconversion among 173 patients took place at median times of 12 (IgM), 14 (IgG), and 11 (neutralizing antibodies) days. The duration and nature of SARS-CoV-2 immunity is unknown. The timescale of protection is a critical determinant of the future impact of the pathogen. The presence or absence of protective immunity due to infection or vaccination (when available) will affect future transmission and illness severity and will allow the identification of individuals with protective immunity [2, 7, 10, 13, 14].

Additional studies are needed to characterize how anti-SARS-CoV-2 antibodies will change over prolonged periods of time. The present study presents data from a large series of samples covering both IgG and IgM + IgA responses to two of the main viral antigenic proteins (N and S).

The utility of serological tests to help in the diagnosis of COVID-19 was evaluated in a multicenter study with a large series of samples collected from 1236 patients in several Spanish hospitals during the peak of the pandemic. In our study, we observed high values of sensitivity, especially for the IgM + IgA test at early periods of time after hospitalization (61.7% presented a positive serology in the first 4 days of the diagnosis, and 90.5% after the first week). We believe that our findings support the use of serological assays as supplementary diagnostic tests for the clinical management of COVID-19. We also observed a decreased sensitivity of serology in older patients in samples collected early in the disease.

Two commercial kits were used to measure the IgG and IgM + IgA responses against two major antigenic components of the virus, the N and S proteins. To our knowledge, few commercial tests are simultaneously based on both proteins. Studies with good performances for assays based either on the N protein [15, 16], the S protein [7, 13, 17] or both antigens [9, 10, 14, 18] have been published. Another peculiarity of the assays used in the study is the inclusion of IgA together with IgM for early detection of the disease. Several studies have described a good sensitivity of this immunoglobulin class, better than that of IgG, although with a lower specificity [2, 7, 12, 18, 19]. The specificity of the test was established by the manufacturer of the kit on the basis of samples collected from prepandemic populations (98.8% CI 95% 95.52–99.68). The high levels of sensitivity reported in the present study, above those reported in other studies [17, 20, 21], may be related to the aforementioned special features of the assays used in the study: the use of two viral antigens and the inclusion of IgA.

The percentage of IgM + IgA-positive patients with IgG-negative levels was higher in samples collected at early stages of the disease, as can be expected for these immunoglobulin classes. However, we did not observe a decrease in IgM + IgA values, as has been shown by other authors [9], probably due to the low number of samples after hospital discharge included in the study. Positivity of the serological tests increases with time, reaching values close to 100% in samples taken 10 days or more after the first PCR result. Conversely, in studies carried out with MERS patients, 20% of the patients did not show a detectable serological response after more than 30 days of evolution [22]. For SARS, all patients developed antibodies [23], but 28% showed negative IgM after 60 days [24], and most patients had lost the antibody response by six years [25].

However, it is surprising that the large proportion of patients with positive IgG levels in samples were surveyed so close to the first PCR-positive results: 45.0% within the first 3 days. Some of the patients even debuted with an IgG-positive/IgM + IgA-negative pattern. This finding could be in agreement with previous contact with other coronaviruses showing antigenic similarities with the novel SARS-CoV-2, as has been found by authors studying immune cell responses [26], and it could also be explained by the fact that patients are attending the emergency room and need hospitalization, suggesting advanced disease. However, it is not in agreement with the very low levels of IgG against the spike protein and nucleoprotein found in the prepandemic population that translates to the very high specificity of the serological assays used in this and other studies [27].

Due to the large series of sera studied, serological results could be stratified according to the age of the patients. A decreased number of patients with positive serology was observed among the older patients for samples collected early in the disease. However, the proportion increased to near 100% positivity in all age groups for samples collected in the second week or later after the first positive PCR result. Some authors have suggested a relationship between this delayed response in elderly individuals, with less favorable disease evolution in this population group [28]. For the comparison of serological responses between men and women, no global significant differences were observed either in the proportion of positive results or in the immunoglobulin levels attained in each group. The low number of samples in some of the subgroups may have limited the statistical potency of the comparisons.

In summary, the high values of sensitivity attained in the present study from a relatively early period of time after hospitalization support the use of the evaluated serological assays as supplementary diagnostic tests for the clinical management of COVID-19. The large number of samples gives a particular strength to the evaluation compared with that of others previously published. However, the lack of clinical and evolutionary data of the patients, as well as having used time from positive PCR result rather than time from symptom onset, constitutes its major limitations.