Seroprevalence of Unidentified SARS-CoV-2 Infection in Hong Kong During 3 Pandemic Waves

IMPORTANCE Seroprevalence studies inform the extent of infection and assist evaluation of mitigation strategies for the COVID-19 pandemic. OBJECTIVE To estimate the prevalence of unidentified SARS-CoV-2 infection in the general population of Hong Kong. DESIGN, SETTING, AND PARTICIPANTS A prospective cross-sectional study was conducted in Hong Kong after each major wave of the COVID-19 pandemic (April 21 to July 7, 2020; September 29 to November 23, 2020; and January 15 to April 18, 2021). Adults (age (cid:2) 18 years) who had not been diagnosed with COVID-19 were recruited during each period, and their sociodemographic information, symptoms, travel, contact, quarantine, and COVID-19 testing history were collected. MAIN OUTCOMES AND MEASURES The main outcome was prevalence of SARS-CoV-2 infection. SARS-CoV-2 IgG antibodies were detected by an enzyme-linked immunosorbent assay based on spike (S1/S2) protein, followed by confirmation with a commercial electrochemiluminescence immunoassay based on the receptor binding domain of spike protein. RESULTS The study enrolled 4198 participants (2539 [60%] female; median age, 50 years [IQR, 25 years]), including 903 (22%), 1046 (25%), and 2249 (53%) during April 21 to July 7, 2020; during September 29 to November 23, 2020; and during January 15 to April 18, 2021, respectively. The numbers of participants aged 18 to 39 years, 40 to 59 years, and 60 years or older


Introduction
The COVID-19 pandemic has induced a substantial global health burden. 1,2The diagnosis of COVID-19 is confirmed via detection of SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction among individuals with an exposure history or indicative clinical features. 3However, since the infection can be asymptomatic or involve minimal symptoms and laboratory tests may not be available, a substantial proportion of SARS-CoV-2 infection could be missed. 4Underestimation of the true extent of infection at the population level could bias the evaluation of public health policies. 57][8] At present, most of the available seroprevalence studies were from hotspots in Europe, America, and mainland China, whereas data from cities with lower attack rates are limited. 5Hong Kong is an urbanized metropolitan city ranking the first for population density in the world. 9Its close proximity to mainland China, the first epicenter of COVID-19, together with its extensive international traffic make it susceptible to importation of COVID-19 cases and subsequent local transmission. 10We examined the prevalence of SARS-CoV-2 IgG after 3 major waves of COVD-19 in Hong Kong, the world city of Asia. 11

Study Design and Setting
This prospective cross-sectional study was conducted to assess the seroprevalence of SARS-CoV-2 in Hong Kong.A recruitment session was scheduled after each major wave of COVID-19 in Hong Kong.
In total, 3 recruitment sessions were conducted from April 21 to July 7, 2020; from September 29 to November 23, 2020; and from January 15 to April 18, 2021, after each major wave.Adults 18 years or older were invited through various public channels, including posters, e-mails, and social media.
Individuals with COVID-19 confirmed in Hong Kong or elsewhere were excluded.All individuals were tested once only.Those who had participated in an earlier round of recruitment were excluded to avoid repeated testing.Eligible registrants were stratified according to age, sex, and region of residence and then arranged consecutively for blood sample obtained according to their date of registration.The recruitment started once the COVID-19 wave was under control and was stopped when there was a substantial upsurge of local cases.Registrants were asked to fill in an online survey to record their sociodemographic and medical information; history of travel, contact, quarantine, and COVID-19 testing; and presence of clinical symptoms anytime throughout the pandemic.Written informed consent was obtained from each participant.The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. 12ng Kong practiced stringent containment measures, including compulsory isolation of confirmed cases and quarantine of all close contacts, but without complete city lockdown.The internationally recognized stringency index developed by the University of Oxford was used as an objective indicator of the intensity of government containment measures implemented during the study period. 13This composite measure is based on 9 response indicators including school closures, workplace closures, and travel bans, rescaled to a value from 0 to 100 (100 indicates strictest).The

SARS-CoV-2 IgG Detection
An in-house enzyme-linked immunosorbent assay based on recombinant SARS-CoV-2 spike (S) protein consisting of the S1 and S2 subunits (Sino Biological Inc) was used to screen for SARS-CoV-2 IgG antibodies in plasma samples.The assay has been shown to provide a sensitivity of 100% and specificity of 96.9% based on testing of 120 postinfection samples collected between 21 and 125 days after infection and 196 samples collected from healthy individuals before the COVID-19 pandemic.
All reactive samples were confirmed by an electrochemiluminescence immunoassay based on the receptor binding domain of S protein, which has been shown to provide a sensitivity of 98.8% and a specificity of 99.9% (Elecsys Anti-SARS-CoV-2 S; Roche Diagnostics GmbH).

Statistical Analysis
The difference in positive seroprevalence rates observed among recruitment periods was examined by the Fisher exact test with a 2-tailed P value of Յ.05 regarded as significant.The 95% CI of the seroprevalence rate was estimated by the Pearson-Klopper method to account for the low rate of IgG positivity.To estimate the observed seroprevalence rate for the whole Hong Kong population, we applied weighted adjustment for the sex and age distribution of the general population in Hong Kong.
The estimated demographics of the Hong Kong population by the end of 2020 obtained from the Census and Statistics Department were used. 9The daily recorded COVID-19 case numbers were obtained from the Centre for Health Protection of the Government of Hong Kong Special Administrative Region. 14 Overall, 6 participants were confirmed to be positive for anti-SARS-CoV-2 IgG.Two participants with positive results were from the first recruitment, 1 from the second recruitment, and 3 from the third recruitment, which corresponded to positivity rates of 0.22% (2 of 903), 0.10% (1 of 1046), and 0.13% (3 of 2249), respectively, with no significant difference in the positivity rates among the 3 recruitment periods (P = .74by Fisher exact test).The overall observed IgG positivity rate was 6 of 4198 individuals (0.14%; 95% CI, 0.05%-0.31%).All but 1 participant with a positive result had traveled overseas, quarantined, and was tested had negative COVID-19 test results (Table 2).
To estimate the number of unidentified SARS-CoV-2 infections, we applied weighted adjustment according to the sex and age distribution of the general adult population in Hong Kong.

Third recruitment
The daily recorded COVID-19 case numbers (vertical bars) were obtained from the Centre for Health Protection of the Government of Hong Kong Special Administrative Region. 14The 7-day moving mean containment stringency index (horizontal line) was obtained from Our World in Data. 13The stringency index is for comparative purposes across countries but not meant for assessment of the appropriateness or effectiveness of government response to the pandemic.The mean (SD) stringency index in Hong Kong across its pandemic waves was high (62.3[10.2]) compared with the worldwide mean index (18.0;SD not available).

Discussion
This study examined the prevalence of hidden SARS-CoV-2 infections in the general population that were not identified by the official case findings and reporting system.Our findings represent the prevaccination era in Hong Kong, where the vaccination program was rolled out on February 26, 2021, with a slow uptake.As at the end of the current study on April 18, 2021, only 5% of the population had received 2 doses of COVID-19 vaccine. 15We estimated a low adjusted prevalence (0.15%) of unidentified infection compared with the prevalence reported elsewhere. 5Our findings suggest that the stringent policies on pandemic mitigation, in particular the compulsory isolation of patients with confirmed cases and quarantine of all close contacts, were successful even without complete city lockdown. 10Most of the individuals with unidentified cases revealed in this study had received SARS-CoV-2 RNA testing, but all results were negative.Although these subjects might not have been tested at the right time, the possibility of false-negative results should not be neglected.
Of note, the deep throat saliva sample, which was widely used in Hong Kong, has been shown to carry a false-positive rate of up to 31%. 16

Limitations
This study has limitations.The study population could have been biased because more healthconscious individuals may have preferred to participate.We could have underestimated the Role of the Funder/Sponsor: The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Figure .
Figure.Daily Confirmed COVID-19 Case Numbers, Containment Stringency Index, and Recruitment Period

Table 1 .
14roprevalence of Unidentified SARS-CoV-2 Infection in Hong Kong During 3 Pandemic Waves Ն20 years) population of 6 341 600.Based on this rate, we estimated that 9512 (95% CI, 3805-20 293) adults in Hong Kong could be positive for SARS-CoV-2 IgG in addition to the official record of 10 729 confirmed adult cases as of April 18, 2021, the last day of study enrollment.14Toestimate the number of unidentified SARS-CoV-2 infections in children, referenced the proportion of recorded confirmed cases among children 17 years or younger as of April 18, 2021, Characteristics of Study Participants JAMA Network Open.2021;4(11):e2132923. doi:10.1001/jamanetworkopen.2021.32923(Reprinted) November 15, 2021 3/7 Downloaded From: https://jamanetwork.com/ on 09/17/2023 whole adult (age a Hong Kong population data are from the Census and Statistics Department, the Government of the Hong Kong Special Administrative Region. 9b Six registrants who had SARS-CoV-2 infection confirmed in Hong Kong or overseas were excluded.