key: cord-0925584-mp4l2upo authors: Cross, Gail B.; Naftalin, Claire M.; Ngiam, Jinghao N.; Bagdasarian, Natasha; Poh, Chek M.; Goh, Yun S.; Chia, Wan N.; Amrun, Siti N.; Tham, Sai M.; Teng, Hazel; Alagha, Rawan; Kumar, Shoban K.; Tan, Shaun S. Y.; Wang, Lin F.; Tambyah, Paul A.; Renia, Laurent; Fisher, Dale; Ng, Lisa F. P. title: Discrepant serological findings in SARS‐CoV‐2 PCR‐negative hospitalized patients with fever and acute respiratory symptoms during the pandemic date: 2022-03-12 journal: J Med Virol DOI: 10.1002/jmv.27656 sha: 41f31e8622016ea882157385967d80479d175226 doc_id: 925584 cord_uid: mp4l2upo Coronavirus Disease 2019 (COVID‐19) serology has an evolving role in the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. However, its use in hospitalized patients with acute respiratory symptoms remains unclear. Hospitalized patients with acute respiratory illness admitted to an isolation ward were recruited. All patients had negative nasopharyngeal swab polymerase chain reaction (PCR) for SARS‐CoV‐2. Serological studies using four separate assays (cPass: surrogate neutralizing enzyme‐linked immunosorbent assay [ELISA]; Elecsys: N‐antigen based chemiluminescent assay; SFB: S protein flow‐based; epitope peptide‐based ELISA) were performed on stored plasma collected from patients during the initial hospital stay, and a convalescent visit 4–12 weeks later. Of the 51 patients studied (aged 54, interquartile range 21–84; 62.7% male), no patients tested positive on the Elecsys or cPass assays. Out of 51 patients, 5 had antibodies detected on B‐cell Epitope Assay and 3/51 had antibodies detected on SFB assay. These 8 patients with positive serological test to COVID‐19 were more likely to have a high‐risk occupation (p = 0.039), bacterial infection (p = 0.028), and neutrophilia (p = 0.013) during their initial hospital admission. Discrepant COVID‐19 serological findings were observed among those with recent hospital admissions and bacterial infections. The positive serological findings within our cohort raise important questions about the interpretation of sero‐epidemiology during the current pandemic. NUHS Clinician Scientist Program (NCSP) award to G. B. C. COVID-19, serology, Singapore In early 2020, Singapore was not spared from the global Coronavirus Disease 2019 (COVID- 19) pandemic. 1 There was the transmission of COVID-19 in the community and large outbreaks in migrant workers residing in dormitories. 2 Patients at risk of COVID-19 who were suspects by broad clinical criteria, were isolated. Once a polymerase chain reaction (PCR) positive case was identified, there was rapid contact tracing and quarantining of all exposed contacts. [3] [4] [5] In the early months of the pandemic, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was limited to reverse (PCR)based tests, while validated and widely accepted serological tests not being available until mid-2020. 6, 7 Based on the experience from the SARS pandemic in 2003, hospitals were found to be particularly vulnerable. Indeed, while some centers managed nosocomial transmission of COVID-19 well, 8 nosocomial transmission of SARS-CoV-2 did contribute to the morbidity of this disease worldwide. [9] [10] [11] [12] Singapore's Ministry of Health (MOH) provided guidance on the criteria for any person to be considered a suspect case with COVID-19. Recognizing that the suspect case definition changed frequently as the epidemiological risk of the disease evolved over time, many public hospitals in Singapore extended isolation precautions to anyone who presented with a febrile illness and had symptoms consistent with an acute respiratory infection. 13, 14 While such an abundance of caution was resource intense, 14, 15 it helped identify cases early on and mitigated the risk of nosocomial transmission. 16 Given the known high false-negative rate of PCR-based tests, particularly early in the disease, 17 we sought to determine what proportion of hospitalized patients with fever and/or respiratory symptoms were PCR negative yet serology positive for SARS-CoV-2 infection, using four separate serological assays, including two licensed assays and two experimental ones. Here, we describe discrepant serological findings among individuals hospitalized with fever and/or acute respiratory symptoms, during the early phase of the pandemic in Singapore and discuss the implications of these discrepancies on the diagnosis of COVID-19. Patients aged 21 and above who were admitted to National University Hospital, Singapore between March 16, 2020, and June 19, 2020, were recruited. Patients had either fever, cough, coryza, sore throat, or shortness of breath and were isolated for evaluation of COVID-19. Due to national restrictions on the processing and analysis of biological samples from confirmed and suspected COVID-19 cases, patients who fulfilled the MOH's case definition of a suspect case for COVID-19 were excluded from the study. All patients had at least one nasopharyngeal PCR-negative swab at the entry to the study. Swabs were tested for SARS-CoV 2 by real-time PCR (RT-PCR) on the Roche cobas ® platform at the hospital clinical laboratory. The detection of the ORF1ab gene target with or without the E-gene target was interpreted as a positive result. A single patient who did not undergo PCR testing was excluded from the analysis. Clinical information, including symptomatology, medical/drug history, results of investigations performed, demographic history, and history of risk factors for COVID-19 exposure, was collected at the first study visit (acute phase). Patients returned for a second study visit between 3 and 12 weeks later (convalescent phase). Ten milliliters of blood was collected in BD Vacutainer serum separating tubes (SST) (Becton Dickinson) during both acute and convalescent phases. After clotting, serum was separated using centrifugation for 10 min at 1000 rcf, harvested, aliquoted into 500 µl, and stored at −80°C. Frozen aliquots were transferred with cold-chain maintained to testing laboratories in batches for the four assays listed below. For the S protein flow-based (SFB) and Epitope assays, plasma was isolated from blood taken from 10 healthy volunteers who reported no intercurrent illness at the time of blood collection, This assay is approved for SARS-CoV-2 diagnosis by Singapore's Health Sciences Authority (HSA). The commercial Elecsys Anti SARS-CoV-2 assay uses a recombinant protein representing the nucleocapsid (N) antigen for the determination of antibodies against SARS-CoV-2. This test principle is that of a sandwich electro-chemiluminescent immunoassay. 18 antibodies. This assay is approved by both Singapore's Health Science's Authority and the US Food and Drug Administration (FDA). Before use, sera were inactivated with 10% Triton X-100 (Thermo Fisher Scientific) as above. The sera were screened for antibodies specific for the S protein as previously described. 19 S proteinexpressing cells were seeded at 1. with the means of both taken to be true. Optical density (OD) values of samples were normalized to a positive control to account for plate-toplate variations, and background signals were subtracted. A cut-off value above mean + 3 SD of healthy control samples was taken to be positive. The determination of the threshold of a positive result has been previously described. 21 Briefly, ROC curves for peptides to differentiate between SARS-CoV-2 infections and others were performed using the best thresholds determined as the maximum of the Youden's J statistic. Areas under the curve (AUC) were calculated for each peptide. For peptide combination analyses, logistic regression models were used to model the combinatory effects of 2, 3, and 4 peptides' OD readings toward the prediction of SARS-CoV-2 infection from others as a binary outcome. The logistic regression model fitted values were then used for ROC analysis to identify the optimal thresholds as well as AUCs. This was conducted using R version 3.6.2. Patient characteristics were examined, with continuous variables presented as the median and interquartile range (IQR), while categorical variables were presented as frequencies and percentages. Results of each serology test were tabulated against the initial and convalescent visit, by patient category (e.g., healthy controls, recovered SARS, acute COVID-19 illness, hospitalized cohort) and days postillness onset (pio). Characteristics of patients who tested positive for SARS-CoV-2 using any of the serological assays were described in greater detail, including their occupation and clinical presentation. Those with positive assay findings were then compared against those who tested negative using None of the patients was found to have total IgG or IgG3 against the S protein. However, three participants were found to have raised IgG1 at both the acute and convalescent visits against the full-length spike protein (Figure 1 ). The magnitude of binding for these three participants was above the predetermined positivity threshold but was lower than the median percentage binding seen in COVID-19 cases with mild disease previously. 19 Five out of 51 (9.8%) patients had SARS-CoV-2-specific antibodies recognizing epitopes on the Spike (S) protein; S14P5, S20P2, and S21P2, and on the Nucleocapsid (N) protein; N4P5, as shown in Figure 1 . One participant had antibodies that recognized two epitopes (S20P2, N4P5), while the remaining four participants had antibodies to single epitopes against SARS-CoV-2 ( The clinical and serological characteristics of these eight patients with various antibodies against SARS-CoV-2 is shown in Table 2 Table 3) . A statistically significant difference was found between the proportions of patients who had positive serology with neutrophilia during the acute admission (5/8), compared with neutrophilia seen in the negative serology patients (7/43, p = 0.013) ( Table 3 ). Since the advent of this study, serological assays have proven to be of great utility given the high proportions of COVID-19 patients who are asymptomatic, or who have subclinical infections. 22 In our study, we demonstrated that eight out of 51 (15.7%) patients admitted with a febrile illness or with acute respiratory illness (ARI), and with a negative swab PCR for SARS-CoV-2, were found to have low titers of specific antibodies against SARS-CoV-2, despite no known previous (Table 2 ). All patients demonstrated the presence of antibodies against the same epitope or the same subclass at both visits, even if it was below the positive threshold (Figure 1) . We explored possible explanations for the low-positive titers of SARS-CoV-2 antibodies found in these eight PCR negative patients. F I G U R E 1 Serological analysis by S protein flow-based (SFB) and B-cell epitope assays. Sera from symptomatic patients (n = 51), isolated for evaluation of COVID-19, were collected at acute and convalescent (between 3 and 12 weeks later) timepoints. Serum samples were screened at 1:100 dilution (A) in an SFB assay for specific total IgG, IgG1, and IgG3 against full-length SARS-CoV-2 S protein expressed on the surface of HEK293T cells, and (B) in a peptide-based enzyme-linked immunosorbent assay (ELISA) against four IgG linear B-cell epitopes of SARS-CoV-2: spike S14P5, S20P2 and S21P2, and nucleocapsid N4P5. Sera or plasma samples from healthy donors (n = 22 for SFB; n = 10 for epitope assay), recovered SARS patients (n = 20 for SFB; n = 10 for epitope assay), and COVID-19 patients (n = 15; median 23 days postillness onset) were included as controls. Data are shown as mean ± SD of two independent experiments, with dotted lines indicating mean + 3 SD of healthy donors. An isotype response was defined as positive by SFB assay when the binding is more than mean + 3 SD of the healthy controls 23 days pio (Table S1 ), which bolsters confidence that these results represent a true infection in 6/8 patients with positive serology at the convalescent visits. At least one patient produced antibodies against two epitopes S20P2 and N4P5-2 (Table 2) , the combination of which was previously shown to have a sensitivity and specificity of 100%. 21 On the SFB assay, three patients had IgG1 binding to SARS-CoV-2 at both the acute and convalescent timepoints, with a specificity of 96.72% for SARS-CoV-2 (Table 3) . We found a statistically significant difference in the proportion of patients who had high-risk occupations for the acquisition of SARS-CoV-2 (p = 0.036, Table 3 ). These patients were a hotel receptionist, taxi driver, teacher, security guard, and another who worked as a container equipment specialist and thus visited construction sites where he had contact with migrant dormitory workers, the latter being a group who experienced a staggeringly high prevalence of SARS-CoV-2 infection at 56.1% 23, 24 (Table 3) . While none in the positive serology group were HCW, who are often perceived as the occupation group at highest risk of exposure, the other occupations represented within the positive serology group approximated that risk. 25 Titers of antibodies in the positive serology group were quantitatively lower than those from mild cases of COVID-19 our group has shown previously. 19, 21 Since IgG levels in asymptomatic COVID-19 infection have been found to be significantly lower than those who develop symptoms, 26 Five out of eight patients in the positive serology group were found to have antibodies against SARS-CoV-2 during the acute hospital visit, at a median of 3 days pio (range 2-5) ( Table 3 ). Since the production of antibodies against SARS-CoV-2 typically takes days to weeks, 27 we considered the possibility that the results seen were due to cross-reactivity to other coronavirus infections. SARS was responsible for more than 200 cases in Singapore in 2003. 28 Prior studies have shown cross-reactive binding between antibodies from SARS patients to SARS-CoV-2, 29,30 and both the B-cell Epitope and SBF assays were tested for cross-reactivity against sera collected from patients with previous SARS infection. 31, 32 While no cross-reactivity was found, the literature on whether recovered SARS patients can sustain the production of SARS-CoVspecific antibodies is inconclusive. 30 Epitope and SFB assays is low for the low-risk community cases enrolled in our study, but the PPV would have been higher if used among high-risk groups such as in a migrant worker dormitory (Table S1 ). This was a single-center small-sized cohort of patients of hospitalized patients with ARI. It was a heterogeneous cohort, and we only examined patients who were hospitalized and could not examine is low. The authors would like to thank the patients who provided clinical information and their blood samples for this study. The authors would also like to thank the medical teams caring for these patients for their help with patient recruitment. 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