key: cord-1033936-ok33bqqg
authors: Stadlbauer, D.; Tan, J.; Jiang, K.; Hernandez, M.; Fabre, S.; Amanat, F.; Teo, C.; Asthagiri Arunkumar, G.; McMahon, M.; Jhang, J.; Nowak, M.; Simon, V.; Sordillo, E.; van Bakel, H.; Krammer, F.
title: Seroconversion of a city: Longitudinal monitoring of SARS-CoV-2 seroprevalence in New York City
date: 2020-06-29
journal: nan
DOI: 10.1101/2020.06.28.20142190
sha: b5d9d83fd761e84dc3efe8d97645b950e486eecf
doc_id: 1033936
cord_uid: ok33bqqg

By conducting a retrospective, cross-sectional analysis of SARS-CoV-2 seroprevalence in a sentinel group (enriched for SARS-CoV-2 infections) and a screening group (representative of the general population) using >5,000 plasma samples from patients at Mount Sinai Hospital in New York City (NYC), we identified seropositive samples as early as in the week ending February 23, 2020. A stark increase in seropositivity in the sentinel group started the week ending March 22 and in the screening group in the week ending March 29. By the week ending April 19, the seroprevalence in the screening group reached 19.3%, which is well below the estimated 67% needed to achieve community immunity to SARS-CoV-2. These data potentially suggest an earlier than previously documented introduction of SARS-CoV-2 into the NYC metropolitan area.

3

The first case of Coronavirus Disease 2019 (COVID-19) was identified in NYC at Mount Sinai 45

Hospital on February 29, 2020 (1). A sharp rise in severe acute respiratory syndrome coronavirus 46 2 (SARS-CoV-2) infections started to occur shortly afterwards during the week ending on March 47 8, followed by a significant increase of COVID-19 deaths during the week ending on March 15 48 (Figure 1) . New York State implemented a stay-at-home order called the 'New York on Pause 49

Program' effective at 8pm on March 22, 2020, and, as a consequence, daily case numbers in 50 both New York State and NYC started to plateau and then decreased in April 2020. Although 51 nucleic acid amplification testing (NAAT) is now widely available in New York State, there was 52 little testing capacity at the beginning of the local epidemic in early March, and many mild to 53 moderate cases likely went undetected. In addition, asymptomatic cases might have been missed 54 since, in the absence of symptoms, NAAT would not have been recommended. 55

Although it is currently unknown if previous infection with SARS-CoV-2 can protect from 56 reinfection, there are data from SARS-CoV-2 infection of non-human primates as well as from 57 studies with other human coronaviruses suggesting that infection may confer immunity (2, 3). It 58 is therefore important to determine the true infection rates in a population in order to assess how 59 close this population is to potential 'community immunity' (4). Knowing the true infection rate also 60 allows calculation of the infection fatality rate (IFR), which is very likely much lower than the case 61 fatality rate (CFR). To estimate true infection rates, serosurveys can be used that measure the 62 presence of antibodies that have been mounted to past virus infections, rather than the presence 63 of virus. Several serological assays for measuring antibodies to SARS-CoV-2 have been 64 developed (5). Many focus either on the virus nucleoprotein, the spike protein on the virus surface, 65 or the receptor binding domain (RBD), which is an important part of the spike protein that interacts 66 with angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2 (6). We have 67 recently established a two-step enzyme-linked immunosorbent assay (ELISA) in which 68 4 serum/plasma samples are prescreened at a set dilution for reactivity to RBD. Positives in this 69 first step are confirmed and the antibody titer assessed in a second step against a stabilized 70 version of the full-length spike protein (7, 8). The use of two sequential assays reduces the false 71 positive rate and favors high specificity. The assay used here has a workflow that closely 72 resembles an assay established in the Mount Sinai Health System (MSHS) CLIA-certified Clinical 73

Pathology Laboratory, which received an FDA emergency use authorization (EUA) for this ELISA 74 in April 2020. However, the assay used in this study was performed in a research laboratory 75

setting. An initial test of the assay performance with a panel of negative and positive samples 76 suggested that the research-grade assay has a sensitivity of 95% and a specificity of 100% (S. 77 Table 1 ). This results in a positive predictive value of 1 (PPV, 95% confidence interval (CI): 0.908-78 1.000) and a negative predictive value of 0.97 (NPV, 95% CI: 0.909-0.995). In the week of 79

February 9, 2020, we started to collect random, de-identified, cross-sectional plasma samples 80 and stored for standard of care medical purposes by the MSHS Clinical Pathology laboratories. 81

These samples were divided into two distinct patient groups. The first group included samples 82 from patients seen in Mount Sinai's emergency department (ED) and from patients that were 83 admitted to the hospital from the ED during the period beginning with the week ending on February 84 9 till the week ending on April 19. This group, termed the 'sentinel group', served as an indicator 85 for SARS-CoV-2 infection since we assumed that individuals with moderate to severe COVID-19 86 would come to the ED and would be admitted to the hospital at increasing rates as the epidemic 87 progressed. The second group of samples, termed the 'screening group', were obtained from 88 patients at OB/GYN visits and deliveries, oncology-related visits, as well as hospitalizations due 89 to elective or planned surgeries, transplant surgeries, pre-operative medical assessments and 90 related outpatient visits, cardiology office visits, and other regular office/treatment visits. We 91 reasoned that these samples might resemble more closely the general population because the 92 purposes for these scheduled visits were unrelated to COVID-19. The sentinel group comprised 93 43.6% females while the screening group included 65.8% females ( Table 1) . The majority of 94 5 individuals in the sentinel group were 61 years of age or older while the screening group had a 95 more balanced age distribution resembling the general population (Table 1) . Except for the weeks 96 ending February 9 and February 16, for which only 16 samples were obtained across the two 97 weeks (3 in the sentinel group, 13 in the screening group), the sentinel group size ranged between 98 195 and 274 samples per week and the screening group included 230-493 samples per week (S. 99 Table 2 ). A total of 5,485 samples obtained from patients between the weeks ending February 9 100

and April 19 were tested: 3,412 samples in the screening group and 2,073 in the sentinel group. 101

In the sentinel group, no positives were detected in the week ending February 9 and 16 and low 102 seroprevalence was found between the weeks ending February 23 to March 15 (ranges between 103 1.4 and 3.2%, Figure 1A ). While we believe these positives are true positives, the prevalence is 104 low and within the confidence intervals of the PPV. A sharp increase to 6.1% was detected in the 105

week ending March 22. This increase continued in the weeks ending March 29 (17.4%), April 5 106 (46.7%) and April 12 (56.4%); however, seropositivity in the sentinel group seemingly plateaued 107 in the week ending April 19 at 58.1%. In the context of COVID-19 case and death rates reported 108 for NYC, the seroprevalence values we report reflect hospital admissions due to COVID-19, 109 although the uptick in positive serology results lagged approximately one to two weeks behind 110 the increased molecular detection of SARS-CoV-2 infections. This is expected since there is 111 usually a delay between infection and seroconversion. In summary, the numbers in the sentinel 112 group are a reflection of hospital admissions due to Similar to the sentinel group, the seroprevalence found in the screening group was very low during 114 the weeks ending February 9 through March 29 (0% to 2%, Figure 1B ). Of note, some samples 115 during that time had moderately high reactivity (endpoint titers of 1:150-1:400) ( Figure 1D ). An 116 increase in seroprevalence from 1.6% to 2.2% was detected in the week ending March 29, 117 followed by increases to 10.1% and 11.7% in the following weeks, up to 19.3% seroprevalence 118 in the week ending April 19. These numbers are significantly lower than the percentages 119 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . 6 calculated in the same weeks for the sentinel group, which makes sense since the visits from 120 which the screening samples are derived are not related to COVID-19 infections and therefore 121 are not enriched in SARS-CoV-2 infected patients. In addition, the delay between the sharp 122 increase in SARS-CoV-2 detection by NAAT in NYC and the increase in seroprevalence in our 123 screening group is longer than the delay between the increase in confirmed cases and the 124 increase in seroprevalence in the sentinel group. This can be attributed to different antibody 125 kinetics in mild cases, which likely constitute the majority of infections in the screening group. We 126 have recently shown that for mild cases, induction of measurable and significant antibody levels 127 often takes several weeks (9). The antibody titers detected in both groups were initially lower and 128 gradually increased to titers as high as 1:51,200 ( Figure 1C and D). Although positive samples 129 with lower antibody titers could constitute false positives, we believe that this again is a function 130 of antibody kinetics, exemplified by the gradual increase in titers that can be clearly observed in 131 the screening group ( Figure 1D ). Seropositive samples collected in the weeks ending February 132 23 and March 1 may potentially indicate that SARS-CoV-2 already had been introduced into the 133 population of NYC earlier than initially detected. Overall, the titers in the sentinel group were 134 significantly higher than in the screening group ( Figure 1F) , which is likely a function of disease 135 severity in individuals in the sentinel group. 136

In order to determine which subgroup(s) were driving the rise in seroprevalence, we further 137 separated the screening group into four subgroups: (i) "OB/GYN" visits and deliveries (n=1,366 138 samples); (ii) "Oncology" visits and treatment hospitalizations (n=1,319); (iii) "Surgery", including 139 various elective surgeries, transplant surgeries, pre-op medical assessments and related visits 140 (n=544); and, (iv) "Cardiology", including cardiology office visits and other regular office/treatment 141 visits (n=183). This rise was mostly driven by "OB/GYN" visits and deliveries, which showed an 142 early increase in seroprevalence in the week of March 29 (9.6%) followed by continued rise to 143 15.6% and 26.6% in the weeks ending April 12 and 19, respectively (Figure 2A) . Seroprevalence 144 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 29, 2020. . 7 in "Oncology" patients increased during the same time frame, but appears to plateau between 8-145 9% during these three weeks ( Figure 2B) . Similar seroprevalence trends were observed in the 146 "Surgery" and "Cardiology" and other office visits subgroups, although the small number of 147 specimens limited conclusions for these subgroups individually since confidence intervals were 148 very wide (Figure 2C and D) . 149

Although our specimen sampling is biased and is not a true representation of the entire population, 19 is consistent with a report from the New York-Presbyterian Allen Hospital and Columbia 160

University Irving Medical Center that found 15.4% of pregnant women who delivered infants at 161 their facilities between March 22 and April 4, 2020 were infected with SARS-CoV-2 and were 162 mostly asymptomatic (10). This tracks well with seroprevalence in the screening group, which 163 was between 10.1 and 19.3% in the weeks following April 5. A serosurvey conducted by the New 164

York State Department of Health (NYSDOH) determined that between April 19 and 28, the 165 seroprevalence for SARS-CoV-2 in the NYC metropolitan region was 22.7% (11), matching very 166 well with the data for our screening samples from the week ending April 19. Of note, these 167 numbers fall significantly below the threshold for community immunity, which has been estimated 168 to require at least a seropositivity rate of 67% for SARS-CoV-2 (4). Based on the population of 169 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 19 (11,413) , this suggests a preliminary IFR of 0.704%. This is in stark contrast to 172 the IFR of the 2009 H1N1 pandemic which was estimated to be between 0.01% and 0.001% (12). 173

We will continue to conduct this longitudinal serosurvey for at least one year, and expect that the 174 seroprevalence for the screening group will slightly increase and then plateau for the time frame 175 between the end of April and June, due to rapidly declining numbers of cases in NYC. 

Mount Sinai has licensed serological assays to commercial entities and has filed for patent 228 protection for serological assays. weeks ending February 9 (first two weeks combined) to April 19, 2020. 244 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and other regular office visits (see Table S1 for detailed numbers and breakdown per cohort).

About 200 plasma samples per week were selected from an inpatient cohort setting, consisting 259

of plasma from patients that were admitted to the emergency department or to inpatient care 260

('sentinel group'). Plasma samples were chosen in a blinded and unbiased manner. Longitudinal 261 samples from the same patients were included in the analysis if the time points between sampling 262

were at least seven days or more apart since this was seen as independent sampling of the 263 population. For some individuals, a PCR test for viral RNA was performed to diagnose COVID-19 264

infection. The collection and testing of plasma was approved by the Mount Sinai Hospital 265

Institutional Review Board, protocol HS# 20-03253.

Recombinant proteins 268

The recombinant RBD and spike protein of SARS-CoV-2 were generated and expressed as 269 previously described (7, 8) . In brief, the mammalian cell codon-optimized nucleotide sequences ELISAs with RBD-specific monoclonal antibody CR3022 (13, 14) or 2B3E5.

Enzyme-linked immunosorbent assay (ELISA) 285

The serological assays were performed as previously described in detail following a two-step 286 ELISA protocol (7, 8) . In the first step, plasma samples were screened in a high-throughput assay 287

using the recombinant RBD protein.

Ninety-six-well microtiter plates (Thermo Fisher) were coated with 50 µL recombinant RBD protein 289 at a concentration of 2 µg/mL overnight at 4°C. The next day, the plates were washed three times 290 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. .

with PBS (phosphate-buffered saline; Gibco) supplemented with 0.1% Tween-20 (T-PBS; Fisher 291

Scientific) using an automatic plate washer (BioTek). The plates were blocked with 200 µL 292

blocking solution consisting of PBS-T with 3% (w/v) milk powder (American Bio) and incubated 293

for 1 h at room temperature. As a general safety precaution, plasma samples were heat 294 inactivated for 1 h at 56°C. The blocking solution was thrown off the plates and 100 µL of plasma 295 samples diluted 1:50 in PBS-T containing 1% (w/v) milk powder were added to respective wells 296 of the microtiter plates. After 2 h the plates were washed three times with PBS-T and 50 µL anti-297

human IgG (Fab-specific) horseradish peroxidase antibody (HRP, produced in goat; Sigma, 298 #A0293) diluted 1:3,000 in PBS-T containing 1% milk powder was added to all wells and 299 incubated for 1 h at room temperature. The microtiter plates were washed three times with PBS-300

T and 100 µL SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma) was added to all 301 wells. The reaction was stopped after 10 min with 50 µL per well 3M hydrochloric acid (Thermo 302

Fisher) and the plates were read at a wavelength of 490 nm with a plate reader (BioTek). Plasma 303 samples that exceeded an OD 490 cutoff value of 0.15 were categorized as presumptive positives 304

and were tested in a second step in confirmatory ELISAs using full-length, recombinant spike 305

protein.

To perform the confirmatory ELISAs, the plates were coated and blocked as described above 307 except full-length spike protein at a concentration of 2 µg/mL was added to the plates. After 1 h 308 the blocking solution was removed, presumptive positive plasma samples serially diluted in 1% 309 milk prepared in PBS-T were added and the plates incubated for 2 h at room temperature. The 310

remainder of the assay was performed as described above. The data were analyzed in Microsoft 311

Excel and GraphPad Prism 7. The cutoff value was set as an OD490 of 0.15 and true positive 312 samples were defined as samples that exceeded an OD490 value of 0.15 at a 1:80 plasma dilution. 313

The endpoint titer was calculated and defined as the last dilution before the signal dropped below 314 an OD490 of 0.15. For samples that exceeded an OD490 of 0.15 at the last dilution (1:12,800 for 315 samples of weeks ending on March 29 and April 5; 1:6,480 for samples of weeks April 12 and 316

April 19), a four-parameter curve fit (variable slope) was applied and the endpoint titer determined 317 by interpolation.

The sensitivity and specificity of the assay were determined using a panel of serum and/or plasma 319 of 40 patients that had PCR-confirmed SARS-CoV-2 infection (true positives) and 74 negative 320

control samples (56 samples that were taken before the pandemic and 18 samples without 321

confirmed SARS-CoV-2 infection; true negatives). The PPV and NPV were determined taking into 322 account the ratio of true positives and true negatives (seroprevalence of 35%) in the panel.

Importantly, using the 100% specificity determined using the panel and assuming a low (e.g. 1%) 324

true seroprevalence in the test group would not change the PPV.

Statistical analysis 327

The 95% CI of the seroprevalence was calculated assuming binomial data based on methods by 328

Wilson/Brown (15). Significant differences in endpoint titers between the sentinel and screening 329 groups were identified by the Mann-Whitney U test. The 95% CI for assay sensitivity, specificity, 330 positive predictive value and negative predictive value were determined using methods by 331

Wilson/Brown. 332 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20142190 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 29, 2020. . https://doi.org/10.1101/2020.06.28.20142190 doi: medRxiv preprint

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