Detection of SARS-CoV-2 antibodies is insufficient for the diagnosis of active or cured COVID-19

We assessed the performance of Abbott's SARS-CoV-2 IgG assay and the PanbioTM COVID-19 IgG/IgM rapid test device for the diagnosis of either active or cured COVID-19. Three cohorts of patients were chosen. Cohort 1, patients (n = 65) who attended the emergency department on March 30, 2020 with clinical suspicion of active COVID-19 (n = 56 with proven/probable COVID-19). Cohort 2, hospital workers (n = 92) who had either been (n = 40) or not (n = 52) diagnosed with proven/probable COVID-19 and were asymptomatic at the time of the sampling. Cohort 3, patients (n = 38) cared at the hospital before the start of the COVID-19 pandemic. Detection of serum antibodies was done using Abbott´s SARS-CoV-2 IgG assay and the PanbioTM COVID-19 IgG/IgM device. Both methods showed 98% agreement for IgG detection. No antibodies were detected in the 38 samples from hospitalized pre-COVID subjects. The diagnostic performance of IgGs detected by Abbott´s SARS-CoV-2 assay in Cohorts 1/2 was: sensitivity (60.7%/75%) and specificity (100%/84.6%). The diagnostic performance of IgM by PanbioTM COVID-19 in Cohorts 1/2 was: sensitivity (16%/17.5%) and specificity (100%/98.1%). We show that IgG detection alone is insufficient for the diagnosis of active or cured COVID-19. IgM detection has a limited diagnostic value.


Performance of Abbott´s SARS-CoV-2 IgG assay for the diagnosis of COVID-19. From the 38
samples from patients with either community-acquired pneumonia of unknown etiology or coronavirus infection other than SARS-CoV-2, none generated a positive result (index value below 0.2). IgG cross-reactivity with patients without COVID-19 was thus ruled out.
In Cohort 1, IgGs were detectable in samples from 34/65 patients; sensitivity, specificity, positive predictive value, and negative predictive value of IgG detection for the diagnosis of active COVID-19 were 60.7%, 100%, 100%, and 29.3%, respectively (Table 3). Serum samples from patients with proven/probable COVID-19 were collected at a median of seven days (IQR 5-10 days) from the onset of symptoms. There was no improvement in the sensitivity in serum samples taken from patients reporting disease onset > 7 days, nor by excluding probable cases. Twenty-two COVID-19-positive patients -most of which were proven cases (n = 20) -showed negative IgGs. We further compared patients with proven/probable COVID-19 with detectable or undetectable IgGs and found that patients with detectable IgGs had higher frequency of > 38 °C fever and/or pneumonia (P < 0.05). The remaining comparisons resulted in differences without statistical significance, although a trend towards higher frequency of dyspnoea in IgG-positive patients (P = 0.055; Table 1) was detected. Sensitivity of IgG detection was higher in patients with probable COVID-19 than in subjects with proven COVID-19 and it was unrelated to the number of days from the onset of symptoms. All patients not fulfilling COVID-19 criteria had negative serum IgGs.
In Cohort 2, IgGs were detectable in samples from 38/92 patients; sensitivity, specificity, positive predictive value, and negative predictive value of IgG detection for the diagnosis of cured COVID-19 (Cohort 2) were 75%, 84.6%, 79%, and 81.5%, respectively (Table 3). Median number of days for sample collection from the onset of symptoms in patients with proven/probable cured COVID-19 was 26 days (IQR 22-32 days). Ten patients with proven cured COVID-19 resulted in negative IgG and serum samples of 8 of the patients, taken one month later, remained negative. Anosmia was more frequent in patients with proven/probable cured COVID-19 and detectable IgGs than in subjects without detectable IgGs (P < 0.05) ( Table 2). No statistically significant differences were observed for the remaining comparisons. We detected IgGs (index values ranging between 2.08 and 6.75) in serum samples from eight patients not fulfilling COVID-19 criteria; four of them reported mild symptoms in the two months before sample collection: headache or anosmia (n = 3); cough, dyspnoea or ageusia (n = 2); myalgia, sore throat or rhinorrhoea (n = 1 each). Two women receiving prophylaxis with fosamprenavir were asymptomatic carriers who resulted PCR-positive and IgG-negative while participating in the study; one of them developed symptoms few days after the end of the study, suggesting an early detection COVID-19 case.
Index values of Abbott´s SARS-CoV-2 IgG assay are shown in Fig. 1 Table 2. Clinical characteristics of subjects with proven/probable cured COVID-19 (Cohort 2) and comparisons between patients with detectable and undetectable IgGs; NA, not applicable. Numbers in bold indicate differences reaching statistical significance (P < 0.05). *Chest X-rays were performed in 22 out of the 40 patients.

Patients with proven/probable cured COVID-19
Overall IgG detection in the 157 serum samples tested using both procedures showed high concordance: positive (n = 69) and negative (n = 85); three samples (1.9%) yielded discrepant positive results with Abbott´s SARS-CoV-2 IgG or negative results by the Panbio TM COVID-19 IgG/IgM rapid test device. Discrepancies were retested (CLIA index results ranging between 2.1 and 3.3), resulting in two patients from Cohort 2 who were not infected and one patient with proven COVID-19 from Cohort 1.

Discussion
Our study shows that serum IgG detection alone is insufficient for the diagnosis of active or cured COVID-19, with sensitivity values that range between 60 and 75%, respectively. Detection of IgM adds limited value to the performance of serological strategies. However, the coupled detection of SARS-CoV-2 RNA and IgG may improve the performance of any of the two procedures alone.
As PCR is insufficient for the diagnosis of active COVID-19, alternative procedures are essential. Non-PCR based procedures rely on the detection of either viral antigens or antibodies against SARS-CoV-2 17 . Antigen detection has shown sensitivity and specificity values ranging between 50 and 100%, respectively. Low viral loads and variability in a sample collection limit sensitivity 18 . IgG detection may improve and speed up the diagnosis of COVID-19 in symptomatic patients, particularly in negative SARS-CoV-2 PCR cases. It has been shown that in up to 15% of COVID-19-positive patients IgG antibodies are detectable during the first week following the onset of symptoms 8 , although reaching 100% of the patients required two additional weeks 8,12,13,19 . Here, we examined the performance of serology as an adjuvant for the diagnosis of COVID-19 using commercially available procedures. Detection of IgG antibodies using Abbott´s SARS-CoV-2 IgG assay (Architect analyser) and the Panbio TM COVID-19 IgG/IgM rapid test device resulted in high agreement (98%). To the best of our knowledge, to date there is only one study that has assessed Abbott´s SARS-CoV-2 IgG assay (Architect analyser) in samples from patients admitted to the hospital 16 . The performance of the test was particularly good in samples collected ≥ 10 days from the onset of the disease. Bryan et al. report 100% sensitivity in cases for which samples were taken ≥ 17 days from the onset of the disease. The results communicated by these authors are significantly higher than the ones reported in our study 16 . Here, we tested patients who attended the emergency department with acute and active clinical presentations (Cohort 1). Our serum samples were collected at a median of seven days from the onset of symptoms, mostly < 10 days. This may explain the high number of IgG-negative determinations in patients with proven COVID-19. Of note, there is a trend of positive samples that show high index values (> 2), particularly in probable COVID-19 cases (Fig. 1). Coupling IgG detection to PCR testing may have helped increase the number of COVID-19 cases from 49 (proven cases) to 56 (proven and probable cases).
Another added value of IgG detection is to unearth undetected and cured COVID-19 cases in order to assess the proportion of the population that is protected (serological status) and understand the real burden of the disease. At this point, serology may be the only method to track down cured infected patients. Here, we confirm the observations by Bryan et al., who reported no IgG detection in samples taken from patients during periods before the COVID pandemic using Architect Abbott´s SARS-CoV-2 IgG 16 . Furthermore, we show the same lack of cross-reactivity in the Panbio TM COVID-19 IgG/IgM rapid test device. We chose patients with cured proven Table 3. Sensitivity, specificity, positive predictive value, and negative predictive value of IgG detection in serum samples for the diagnosis of active and cured COVID-19. PPV, positive predictive value; NPV, negative predictive value; NA, not applicable. www.nature.com/scientificreports/ COVID-19 (Cohort 2) to study the performance of Architect Abbott´s SARS-CoV-2 IgG, but the rate of falsenegative serologic results increased 25%. This rate was surprisingly high given that serum samples were taken at a median of 26 days from the onset of symptoms. This may indicate the existence of a group of outpatients for whom the role of serology testing is lower than for hospitalised and sicker subjects. Patients in Cohort 2 may be representative of the general population who passed COVID-19 and did not attend the hospital. We found that CLIA index values in serum samples taken ≥ 3 weeks after symptoms onset from patients in Cohort 1 were invariably high. Values in samples from patients in Cohort 2 were wider and some of them high as well. It should be noted that the number of samples taken from the second week of symptoms onset on in patients from Cohort 2 was higher compared to patients from Cohort 1, what could explain the differences.
Considering the data presented in this study, we conclude that IgG detection is insufficient to assess the current percentage of subjects with cured infections. Other authors have also failed to detect IgGs in 15%-20% of COVID-19-positive patients 7,9,11,14 . Sensitivity of IgGs antibody detection against either the SARS-CoV-2 nucleoprotein or the spike protein receptor-binding domain could make the difference 10 . Since Architect Abbott´s SARS-CoV-2 IgG detects anti-nucleoprotein antibodies, we cannot rule out the presence of antibodies against other viral epitopes.
We compared study patients grouped by IgG detection results aiming to discover potential predictors of IgGnegative patients with COVID-19 in both cohorts. We failed to find such predictors, which illustrates the difficulties to determine which patients will not have detectable IgGs. Whether the absence of detectable IgG antibodies in patients with cured infection means a lack of protection to prevent a new infection remains to be defined.
Our study is subjected to limitations. IgM detection using the Architect analyser was not available at the moment of the study. Moreover, it was not possible to collect additional serum samples from patients in Cohort 1 in order to check if false-negative determinations may have turned positive over time.
In conclusion, IgG detection alone is insufficient for the diagnosis of active and cured COVID-19. IgM has marginal diagnostic value. Coupled detection of SARS-CoV-2 RNA and IgG may improve the performance of any of the two procedures alone. The Panbio TM COVID-19 IgG/IgM rapid test device is a good point-of-care alternative to Abbott´s SARS-CoV-2 IgG.   Clinical data and analysis. The following demographic and clinical data were collected: age, sex, presence of symptoms compatible with COVID-19 (fever > 38 °C, headache, cough, asthenia, myalgia, sore throat, runny nose, dyspnoea, anosmia, dysgeusia, abdominal pain, and diarrhoea), date of symptom onset, hospital admission, radiological images, immunosuppression, and outcome (favourable or death during admission). Clinical data were collected in a pre-established protocol that was either retrospectively filled out retrieving the information from electronic charts for Cohort 1 patients or filled out at sample collection for Cohort 2 patients. Categorical variables were described and compared using Chi-square or Fisher´s exact tests and continuous variables were compared using the t-student and the Mann-Whitney U tests (IBM SPSS Statistics, version 26; Armonk, NY, USA). Samples from patients in Cohort 3 were used to rule out antibody detection cross-reactivity. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated separately for Cohorts 1 and 2. Independent analyses were carried out considering or excluding probable cases at different stages of the disease (less or more than seven days from clinical disease onset) for patients in Cohort 1; no further analyses of diagnostic performances were done for subjects in Cohort 2 given that only one patient had probable COVID-19 and all samples were taken > 7 days after the onset of the symptoms. We compared the results generated by Abbott´s SARS-CoV-2 IgG assay and the Panbio TM COVID-19 IgG/IgM rapid test device. Representation of index values of IgG´s detected by Abbott´s SARS-CoV-2 IgG assay was done using scatter plots (Graph Pad Prism 5.02 statistical software; GraphPad, La Jolla, CA, USA).

Methods
Ethical considerations. This study was approved by the Ethics Committee of Hospital Gregorio Marañón (CEIm; studies no. MICRO.HGUGM.2020-023 and MICRO.HGUGM.2020-021). All research was performed in accordance with relevant guidelines/regulations. Informed consent was obtained from patients in Cohort 2; a waiver for informed consent from patients in Cohort 1 and Cohort 3 was obtained given the retrospective designed of the samples collection from those patients.