Serology surveillance of anti-SARS-CoV-2 antibodies among asymptomatic healthcare workers in Malaysian healthcare facilities designated for COVID-19 care

about the of infection control procedures, understanding the true prevalence of COVID-19 infection among HCW is vital. Seroprevalence studies are useful to provide information on the proportion of people with past symptomatic or asymptomatic infection. We conducted a serology surveillance of anti-SARs-CoV-2 antibodies among asymptomatic HCW from designated COVID-19 healthcare facilities.


Introduction
On 25 th January 2020, Malaysia had its rst con rmed case of coronavirus disease 2019 (COVID-19), [1] a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [2] Initially, con rmed cases were mainly imported and daily reported cases remained relatively low. However, local transmission began to emerge, following a mass religious event that was held in Kuala Lumpur between late February and early March 2020. This event was attended by an estimated 16,000 people from all over Asia, of whom 14,500 were Malaysians. [3] Since then, massive spikes in local cases and exportation of cases to other countries had been observed. As of 16 th June 2020, over 8 burden. [4] The spectrum of COVID-19 severity ranges from mild to critical. According to the Chinese Center for Disease Control and Prevention, about 80% of con rmed COVID-19 patients suffered from mild disease. [5] Asymptomatic COVID-19 infections have been well documented, but its proportion within total COVID-19 cases remains unclear. [6] Nevertheless, current evidence suggests that asymptomatic and presymptomatic COVID-19 individuals can transmit the infection to others. [7] As of 6 th May 2020, the World Health Organization (WHO) reported that a total of 22,073 HCW from 52 countries had contracted COVID-19 infection. [8] In Malaysia, the rst case of COVID-19 infection among HCW was reported in early March 2020, [9] followed by further clusters. [10][11][12] As they manage suspected and con rmed COVID-19 patients, HCW are presumed to have higher risk for COVID-19 infection and, if infected, can possibly transmit the virus to vulnerable patients and other co-workers. With the uncertainties of the proportion and transmission risk of asymptomatic cases, and ongoing HCW concern about the adequacy of infection control procedures, understanding the true prevalence of COVID-19 infection among HCW is vital. Seroprevalence studies are useful to provide information on the proportion of people with past symptomatic or asymptomatic infection. We conducted a serology surveillance of anti-SARs-CoV-2 antibodies among asymptomatic HCW from designated COVID-19 healthcare facilities.

Study Setting and Participants
A cross-sectional study of HCW from Kuala Lumpur Hospital, Sungai Buloh Hospital and National Public Health Laboratory (NPHL) was conducted from April 13 th to May 12 th , 2020. These represent two of the three public hospitals which were designated to manage con rmed COVID-19 patients in Klang Valley, Malaysia; with NPHL being a referral laboratory for diagnostic reverse transcription polymerase chain reaction (RT-PCR) testing for suspected COVID-19 cases. Quota sampling was applied to ensure the recruited study samples were representative of the HCW involved in provision of care for patients directly (e.g. doctors, nurses and assistant medical o cers), and indirectly (e.g. laboratory technologists, pharmacists, drivers, clerks and so on). Participation in the study was voluntary. All participants had worked in the respective healthcare facility for at least 30 days prior study enrollment, and were asymptomatic at the point of study recruitment. HCW who were previously con rmed with COVID-19 infection or listed as "patient under investigation" for COVID-19 were excluded from this study. A "patient under investigation" has presented with acute respiratory infection (sudden onset of respiratory infection with at least one of shortness of breath, cough or sore throat) with or without fever; and had history of travel to or residence in a foreign country within 14 days prior to the onset of illness, close contact with a con rmed case of COVID-19 within 14 days before illness onset, or who had attended any event associated with a known COVID-19 outbreak. HCW with active symptoms upon study recruitment were excluded as they would have been seen by Occupational Safety and Health team for further management.

Data Collection Procedure
Each HCW who consented to participate in this study was given a self-administered questionnaire to capture sociodemographic characteristics, adherence with recommended infection prevention and control (IPC) measures, history of exposure to SARS-CoV-2 and clinical signs and symptoms in the past month prior to study entry. The questionnaire was modi ed from the protocol "Assessment of potential risk factors for 2019-novel coronavirus (2019-nCoV) infection among HCW in a healthcare setting", published by WHO. [13] For each participant, 5 mL of peripheral venous blood was collected for anti-SARS-CoV-2 antibody serology testing.

De nitions and Personal Protection Equipment (PPE) Guidelines
The history of close contact and prolonged face-to-face exposure with COVID-19 patients were captured in the questionnaire. Close contact was de ned as contact between HCW and patient within one meter distance, with or without PPE. Prolonged face-to-face exposure was de ned as face-to-face exposure within one meter distance which lasted at least 15 minutes.
According to local guidelines, use of PPE should be guided by risk assessment concerning anticipated contact during routine patient care. For inpatient facilities, HCW are required to wear full PPE which includes an N95 mask, an isolation gown, gloves, eye protection and a head cover when providing care to PUI or con rmed COVID-19 patients who are intubated, or those who are not intubated but unable to wear a surgical mask. For patients who can wear a surgical mask, HCW may opt for a surgical mask instead of an N95 mask. Full PPE should be worn when collecting oropharyngeal or nasopharyngeal swabs. [14] Anti-SARS-CoV-2 Antibodies Serological Test Total circulating neutralizing antibodies against SARS-CoV-2 were tested by using the cPass SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) kit, according to the manufacturer's instructions (GenScript Biotech, USA). [15] This test is based on antibody-mediated blockage of virus-host interaction between the receptor binding domain of the viral spike glycoprotein and the angiotensin converting enzyme-2 receptor protein. Its ability to detect total antibodies in COVID-19 patient sera with different level of anti-SARS-CoV-2 IgM/IgG was demonstrated in previous study. [16] It has a sensitivity of 95%-100% and speci city of 100%, and is capable of distinguishing antibody responses from other known human coronaviruses. [16] To con rm assay performance in our laboratory, the test was rst performed on 20 serum samples from recovered patients with PCR-con rmed SARS-CoV-2 infection, and 20 archived serum samples from July 2019, well before the pandemic emerged.

Sample Size and Statistical Analysis
In view of the uncertainty about seroprevalence of antibodies against SARS-CoV-2 among the HCW, we used 50% as the prevalence to provide the most conservative sample size. Hence, with a 95% con dence interval, precision of 5% and 5% non-responses, we estimated we would need at least 383 HCW for this study.
The analysis was carried out using Statistical Package for the Social Sciences (SPSS) version 20.0 (IBM, USA). Categorical variables were expressed in frequency and percentage. As for continuous variables, mean and standard deviation were used. Seroprevalence of anti-SARS-CoV-2 antibodies was calculated as a proportion, and con dence intervals were calculated using Wilson score interval.
Exposure History to SARS-CoV-2 Overall, 115 of HCW claimed to have contact with known COVID-19 cases outside of their workplace in the month prior to study enrollment. Among them, 81 (70.4%) reported the contact as more than 14 days prior to study entry, while ve (4.3%) did not specify how recent the contact occurred.
The majority (68.9%) of participants had potential exposure to SARS-CoV-2 at their workplace, within the month prior to study entry ( Table 2). The source of exposure at the workplace included contact with COVID-19 patients and their bodily uids, as well as contaminated objects and surfaces. A higher proportion of doctors and nurses were found to have various types of exposure compared to other professional categories, ranging from 45-65% and 51-76%, respectively. Laboratory technologists who were not involved in provision of care directly, had contact with patients' body uids most of the time.

Adherence to Infection Prevention and Control Measures
Generally, adherence of the study participants to infection prevention and control (IPC) measures was found to be satisfactory. High compliance with PPE usage (>97%) were reported by HCW who had prolonged face-to-face exposure with COVID-19 infected patients and contact with infected patients' body uids, contaminated objects and surfaces (Table 3). Among them, the level of compliance towards hand hygiene was satisfactory (Table 4).

Seroprevalence of Total Antibodies Against SARS-CoV-2
The sVNT was found to detect antibodies in all 20 samples from convalescent COVID-19 cases, and did not detect antibodies in any of the 20 pre-pandemic samples. None of the study participants had anti-SARS-CoV-2 antibodies detected (95% con dence interval (CI): 0, 0.0095).

Discussion
In Malaysia, the massive SARS-CoV-2 transmission started since the end of February and peaked in mid-March to mid-April 2020 (Fig. 1). [17] During this period, both medical resources and healthcare workers around the country were redirected to combat the disease. Healthcare staff from various hospitals of different departments were deployed to manage suspected and con rmed COVID-19 patients in designated healthcare facilities. This study was conducted during the post-peak period. Our nding of zero seroprevalence among asymptomatic HCW suggests that there is a low risk of asymptomatic COVID-19 infection in our healthcare setting. Another Malaysian study of 310 HCW at a non-COVID designated healthcare facility in Sarawak showed a seroprevalence rate of 4.5%, using a rapid antibody test kit, and its seroprevalence among the asymptomatic HCW was 2.7%. However, the authors reported that two study subjects with faint IgM positive results were actually false positives. [18] The sVNT used in our study is likely to have a higher speci c than a rapid antibody test kit, thus reducing the risk of false positives. [19] Besides, we hypothesized HCW from designated COVID-19 hospitals would have a better perception of the potential risk of infection involved due to higher occupational exposure, which would lead to better adherence to IPC measures and reduce the transmission risk within the facility itself.
A similar study in China reported zero serological response among 420 HCW who were deployed to Wuhan during this pandemic. [20] The study coupled with our nding and suggest that appropriate PPE usage protects HCW from contracting the infection while caring for patients. However, studies conducted in other countries observed higher seroprevalence rates among HCWs. A study of 316 employees in a German tertiary hospital showed that 1.6% were seropositive for antibody against SARS-CoV-2. [21] In Kyoto, seroprevalence of anti-SARS-CoV-2 antibody among HCW were reported to be as high as 5.4%. [22] An even higher seropositivity rate was observed in an otolaryngology unit in Italy, where 5/58 (8.6%) HCW tested seropositive for IgG. [23] In a tertiary hospital in Barcelona, 45 (9.3%) out of 578 HCW were seropositive for antibody against SARS-CoV-2. [24] We postulate the discrepancy between our study and others could be multifactorial. First of all, Malaysia had a relatively low COVID-19 incidence compared to these countries during the study period. As of 17 th June 2020, the incidence of COVID-19 in these countries were about 8 to 23 times higher than Malaysia, except Japan which had a lower incidence. [25] HCW seroprevalence may correlate with wider community circulation. [26,27] Secondly, the variation of participants' eligibility criteria between the studies may also contribute to the discrepancy. Our study focused only on asymptomatic HCW, but studies from Italy and Spain included both symptomatic HCW and those who were infected with COVID-19 previously. [23,24] The testing method was not standardized across all studies. Any differences between the performances of each testing method can also affect the seroprevalence result.
In terms of the history of SARS-CoV-2 exposure, more than a quarter of participants claimed to have contact with known COVID-19 person outside of workplace. The participants may assume any contact with infected co-workers within hospital compound (i.e. pantry, praying area, etc.), but outside of ward as a form of contact beyond workplace. On the other hand, we observed a connection between professional categories and history of SARS-CoV-2 exposure at the workplace. This study found that doctors and nurses have more exposure to SARS-CoV-2 through all types of contact, from patients to respiratory droplets, objects and surfaces. This was in line with the nature of their work, which involves direct provision of care to patients. Laboratory technologists whose main job scope is to process patient specimens were found to have contact with patients' body uids most of the time.
As of early May 2020, 359 HCW from Malaysian MOH had been diagnosed with COVID-19. Of these, 73% had acquired COVID-19 from the community, including from coworkers who were infected in the community, 19% had acquired the virus from patients, and the remaining 8% were still under investigation. [28] Public fear of hospitals and HCW remains high. Many countries observed that patients have avoided seeking medical care for other health problems during this pandemic, thus resulting in late presentation with adverse outcomes. [29][30][31][32] Stigmatization and abuse of HCW has also been reported. [33][34][35][36] A study from Spain in March 2020 observed no difference in the COVID-19 infection rate of HCW by professional categories and risk of exposure in respective work departments. Other studies in Spain [24] , Italy [23] and Netherlands [37] also suggested the risk to a HCW of contracting COVID-19 infection in a healthcare facility is minimal. Although our study was unable to fully rule out additional risks of COVID-19 infection in a healthcare facility, zero seroprevalence of anti-SARS-CoV-2 antibody among asymptomatic HCW should reassure the public that the risk of being infected by a HCW is very low.
Numerous studies have stressed the critical importance of strict adherence to IPC measures to prevent patient-to-HCW infections, [21,23,[38][39][40]  infection rate among the HCW in United States. [41] However, our ndings of high adherence to infection control guidelines and zero seroprevalence of anti-SARS-CoV-2 antibody in HCWs reinforces the importance of strict infection control and PPE usage to reduce the risk of SARS-CoV-2 transmission within healthcare facilities.
SARS-CoV-2-speci c IgM and IgG antibodies start to appear during the rst week of illness, and peak between two to three weeks. [42] After taking into consideration the timeline of seroconversion, serostatus of the HCW in this study most likely re ects COVID-19 transmission in Malaysia between middle of March and April 2020. This period corresponds to the second wave of COVID-19 outbreak in Malaysia following the mass religious event held in Kuala Lumpur [43] , with a doubling time of con rmed COVID-19 cases ranging from two to 110 days. [44] Folgueira et al. found a close link between the driving forces of transmission in the community and HCW infection, which suggested that the rates of SARS-CoV-2 infection among HCW could be an indicator of transmission dynamics in the community. [27] If we adopt a similar principle, this study shows that the proportion of the general public in Malaysia infected as of mid-March to April could be very low, and PPE for HCW have been effective in preventing nosocomial transmission. This also signi es Malaysia's successful public health efforts in combating this pandemic via extensive contact tracing and early admission of con rmed COVID-19 cases for isolation and close monitoring.
This study has several limitations. Firstly, this study was limited to selected healthcare facilities, so its ndings may not be generalizable to HCW in other workplaces, especially non-COVID designated healthcare facilities. Secondly, we acknowledge the existence of selection bias. Participation in the study was voluntary and all participants were sampled by a non-probabilistic sampling method. HCW who refused to participate or were not sampled might be seropositive for antibody against SARS-CoV-2. As the study only recruited asymptomatic HCW, we could have potentially missed out symptomatic individuals with SARS-CoV-2 infection. Nevertheless, the literature shows that a substantial proportion of COVID-19 infected individuals can remain asymptomatic. [45][46][47][48][49] Thirdly, detection of anti-SARS-CoV-2 antibody in a single sample may potentially miss any SARS-CoV-2 infected HCW yet to seroconvert, although the number is likely to be minimal.

Conclusion
This study suggested a low risk of asymptomatic COVID-19 infection in our healthcare setting, even at facilities designated to handle COVID-19 patients and specimens for suspected cases.

Consent for Publication
During the process of consent taking, all participants were informed regarding the need of publishing the results. All participants' personal information were anonymized during the write up, and none of them will be identi ed when the ndings of this study are published.

Availability of Data and Materials
The dataset analyzed during the current study available from the corresponding author on reasonable request.

Competing Interest
None of the authors have any con ict of interest