key: cord-0683406-cdotxizq authors: Arkell, Paul; Gusmao, Celia; Sheridan, Sarah L; Tanesi, Maria Y; Gomes, Nelia; Oakley, Tessa; Wapling, Johanna; Alves, Lucsendar; Kopf, Stacey; Sarmento, Nevio; Barreto, Ismael Da Costa; Amaral, Salvador; Draper, Anthony DK; Coelho, Danina; Guterres, Helio; Salles, Antonio; Machado, Filipe; Fancourt, Nicholas SS; Yan, Jennifer; Marr, Ian; Macartney, Kristine; Francis, Joshua R title: Serological surveillance of healthcare workers to evaluate natural infection- and vaccine-derived immunity to SARS-CoV-2 during an outbreak in Dili, Timor-Leste date: 2022-03-28 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.03.043 sha: 3dd5ed9217bf0c552fc1c55e8e47c09e13b8876c doc_id: 683406 cord_uid: cdotxizq Background : Serosurveillance can be used to investigate the extent and distribution of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a population. Characterisation of humoral immune responses gives insight into whether immunity is infection- or vaccine-derived. Methods : A longitudinal study of healthcare workers (HCWs) in Dili, Timor-Leste was conducted during vaccine roll-out (ChAdOx1) and a concurrent SARS-CoV-2 outbreak. Results : Three-hundred-and-twenty-four HCWs were included at baseline (April-May 2021). Thirty-two (9.9%) were anti-Nucleocapsid protein IgG (anti-N), indicating significant subclinical infection among HCWs early in the local outbreak. One-hundred-and-fifty-seven (48.5%) participants were followed-up (July-September 2021), by which time there had been high uptake of vaccination (91.7%) and 86.0% were anti-S seropositive. Acquisition of anti-N antibodies was observed in partially vaccinated HCWs (30/76, 39.5%), indicating some post-dose 1 infections. Discussion : Serosurveillance of HCWs may provide early-warning of SARS-CoV-2 outbreaks and should be considered in non-endemic settings, particularly where there is limited availability/uptake of testing for acute infection. Characterisation of humoral immune responses may be used to assess vaccine impact as well as coverage. Such studies should be considered in national and international efforts to investigate and mitigate against future emerging pathogens. Results: Three-hundred-and-twenty-four HCWs were included at baseline (April-May 2021). Thirty-two (9.9%) were anti-Nucleocapsid protein IgG (anti-N), indicating significant subclinical infection among HCWs early in the local outbreak. One-hundred-and-fifty-seven (48.5%) participants were followed-up (July-September 2021), by which time there had been high uptake of vaccination (91.7%) and 86.0% were anti-S seropositive. Acquisition of anti-N antibodies was observed in partially vaccinated HCWs (30/76, 39.5%), indicating some post-dose 1 infections. Discussion: Serosurveillance of HCWs may provide early-warning of SARS-CoV-2 outbreaks and should be considered in non-endemic settings, particularly where there is limited availability/uptake of testing for acute infection. Characterisation of humoral immune responses may be used to assess vaccine impact as well as coverage. Such studies should be considered in national and international efforts to investigate and mitigate against future emerging pathogens. Serosurveillance can be used to determine the proportion of a population who have evidence of humoral immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease pandemic. (Bobrovitz et al., 2021; Chen et al., 2021; Kayı et al., 2021; Rostami et al., 2021; Vaselli et al., 2021) Detection of specific antibodies against the viral nucleocapsid protein (anti-N antibodies) indicates previous infection or vaccination with a whole virus vaccine, whereas antibodies against the spike protein may indicate previous infection and/or vaccination with any vaccine. Characterisation using a spectrum of humoral immune responses therefore gives insight into whether immunity is derived from natural infection (where anti-N and anti-S antibodies are usually detectable) or derived from spike protein-based vaccines (where anti-S antibodies are detectable, but anti-N antibodies are not). (Duarte et al., 2022; Wei et al., 2021; Whitaker et al., 2021) Healthcare workers (HCWs), through occupational exposure, are generally at higher risk of infection from SARS-CoV-2, though infection prevention and control measures can mitigated against this. Studies of HCWs in settings with established SARS-CoV-2 transmission have found high seropositivity when compared to general population estimates and have identified significant asymptomatic and/or undiagnosed infection. (Galanis et al., 2021; Grant et al., 2021; Meinus et al., 2022; Rudberg et al., 2020) However, only a minority of studies have been conducted in low-and middle-income countries (and none previously in Timor-Leste). (Rostami et al., 2021; SeroTracker, n.d.) Timor-Leste has a population of 1.3m and occupies the eastern side of an island located between Australia and Indonesia. Throughout 2020 widespread SARS-CoV-2 community transmission was avoided, largely due to border controls and quarantine measures. By 1 st January 2021, 44 COVID-19 cases had been detected using Nucleic Acid Amplification Tests (NAAT), mostly among returned international travellers. However, no onward community transmission was detected despite widespread use of NAAT in the capital city, Dili. In This study took place at three sites: The single large referral hospital in Dili (Hospital Nacional Guido Valadares, HNGV), the national health laboratory in Dili (Laboratório Nacional de Saúde, LNS), and the regional ambulance service in Dili (Servico Nacional Ambulancia e Emergencia Medica, SNAEM, with the study taking place in the headquarters building). All individuals working in patient-facing and/or clinical sample-processing roles at any of these facilities were eligible. Individuals attended one of three research clinics set up at each site, for collection of serum sample and to record epidemiological information: Demographic data (age, gender, nationality), self-reported vaccine status (doses, dates of administration), occupational data (occupation, perceived risk of COVID-19 at work, known PPE-breach) and clinical data (history of recent illness, symptoms (if applicable), known COVID-19 diagnosis) were collected during a 'baseline visit' (occurring between 7 th April and 31 st May 2021) using a structured interview-questionnaire. After three months participants attended a 'follow-up visit' (occurring between 9 th July and 8 th September 2021) where collection of data and serum were repeated. Figure 1 shows the timing of study visits in relation to confirmed cases of COVID-19 in Dili Municipality and the timing of HCW vaccination.
Samples were stored and analysed at LNS. Anti-S IgG antibodies were detected using the qualitative Ortho Clinic Diagnostics chemiluminescent assay on the Vitros ECiQ platform. Those which were anti-S positive were also tested using the qualitative Epitope Diagnostics Individuals' vaccination status was stratified by the number of doses received, and whether their most recent dose had been within 21 days prior. Anti-S and anti-N seropositivity were determined for each time-point and compared across demographic, vaccine-related, occupational, and clinical variables in univariate analyses. To assess for bias caused by loss-to-follow-up, baseline characteristics of those who attended follow-up were compared with those who did not, in univariate analyses. Fisher's exact and Mann Whitney U tests were used to test associations between categorical and ordinal data, respectively. Results were considered significant if p<0.05. All participants gave written informed consent and were informed of their serology results. One-hundred-and-one (31.2%) were nurses, 64 (10.5%) cleaners, 51 (15.7%) doctors, 37 (11.4%) laboratory scientists, 18 (5.6%) midwives, 13 (4.0%) ambulance staff and 40 (12.3%) miscellaneous healthcare staff; 191 (59.0%) were female; the median age was 29 years (interquartile range (IQR) 34-43 years), and 317 (97.8%) described themselves as having Timorese nationality. Of those who were anti-S seropositive, 72 were also anti-N seropositive (indicating previous infection), representing 45.9% of those who had a follow-up visit. On univariate analysis, increasing age (p=0.013) and previous laboratory-confirmed SARS-CoV-2 infection (p=0.008) were significantly associated with anti-N seropositivity. More complete vaccination status (p=0.039) and occupation as a laboratory scientist (p=0.033) were inversely associated with anti-N seropositivity. Table 1 shows seropositivity in different groups at both timepoints. performed on the 157 individuals in whom follow-up data were available. Out of 129 (82.2%) who were anti-S seronegative at baseline, 9 (7.0%) had unchanged serology at follow-up, 64 (49.6%) had become anti-S seropositive but were anti-N seronegative, and 56 Table 2 shows associations between baseline vacation status and serostatus with markers of SARS-CoV-2 infection during the follow-up period. This longitudinal serosurveillance study characterised humoral immunity to SARS-CoV-2 among HCWs during the period of vaccine roll-out and a concurrent outbreak in Dili Municipality, Timor-Leste. At baseline (between in April to May 2021), a significant proportion of HCWs (9.9%) had evidence of previous infection. This was early in the local outbreak when cases of laboratory-confirmed SARS-CoV-2 infection in Dili Municipality were low (see figure 1 ). Most of these individuals did not report having a recent illness, nor a previous laboratory-confirmed diagnosis of COVID-19. This highlights the propensity for SARS-CoV-2 to cause sub-clinical disease and the importance of serosurveillance in detecting transmission, particularly where there is low access/uptake of testing for acute infection. Other studies frequently described high numbers of cases detected through serosurveillance, compared with those diagnosed acutely and reported through systems of passive surveillance. (Byambasuren et al., 2021; Rostami et al., 2021) Infections following vaccination are frequently asymptomatic or mild, which may further reduce the likelihood that diagnostic testing is performed. (Voysey et al., 2021) Studies in other settings have shown high SARS-CoV-2 seroprevalence among HCWs relative to other groups. (Galanis et al., 2021; Rudberg et al., 2020) This is likely to be driven by occupational as well as community exposure. Among HCWs included in a systematic review and meta-analysis (conducted before widespread availability of vaccines), those with male gender and those doing patient-related work (in addition to those with self-reported previous infection or a positive PCR test) were more likely to be seropositive. (Galanis et al., 2021) These were also identified as risk-factors for anti-N seropositivity in our study. In June-December 2020, a SARS-CoV-2 seroprevalence study was conducted in Java, Indonesia (an island nearby to Timor-Leste) and found that laboratory technicians had higher seroprevalence than other HCWs. (Megasari et al., 2021) This contrasts with the findings of our study and may reflect differences in occupational role, working practice, infection prevention and control procedures and/or availability of PPE between the two settings. Surveillance of HCWs may be useful in providing early-warning of more widespread SARS-CoV-2 outbreaks and should be considered in settings that have not yet reached a state of endemicity. By July-September 2021, almost half of HCWs enrolled in the present study were anti-N seropositive. In settings where spike-based vaccines are exclusively used, anti-N seroconversion is a specific indicator of naturally occurring SARS-CoV-2 infection. (Duarte et al., 2022; Whitaker et al., 2021) This includes detection of breakthrough infections among individuals who are fully vaccinated but not previously infected, although anti-N seroconversion may be less sensitive in this group. (Allen et al., 2021; Whitaker et al., 2021) Therefore, studies which make this determination can provide information about the extent of SARS-CoV-2 transmission which is independent from that of vaccination. Longitudinal surveillance of anti-N antibodies among cohorts of individuals who have received different spike-based vaccines (and/or those who choose to be unvaccinated), correlated with data on clinical outcomes, may be a useful way to estimate real-life vaccine impact in such settings. Furthermore, individuals who are seropositive typically experience a rise in anti-N antibody titre upon re-infection, which could be used to monitor transmission in populations which already have high levels of exposure. (Atti et al., 2022) Over the same period as increasing numbers of infections occurred, there was rollout of vaccination with high uptake among healthcare staff, which resulted in 91.7% with a history of having at least one vaccine dose at follow-up. Acquisition of anti-N antibodies was observed in HCWs who had been partially vaccinated at baseline, indicating some post-dose 1 infections. It was not possible to ascertain how many individuals were infected post-dose 2, because none had received 2 doses at baseline. However, it is most likely that infections occurred just prior to dose 1 or post-dose 1. Ultimately, high anti-S seropositivity was observed (94.3%), reflecting a combination of natural infection-and vaccine-derived immunity in this important cohort. This study provided an opportunity for strengthening surveillance, and serological testing capacity within LNS, as part of wider program of work which will examine seroprevalence to SARS-CoV-2 and other vaccine preventable diseases in both HCW and other population groups across Timor-Leste. Limitations include a small sample size, which precluded multivariate analysis to look for associations with vaccination and/or SARS-CoV-2 infection. There was a significant dropout rate, and followed-up individuals were significantly different to those who were not followed-up in terms of occupation. Association between baseline serostatus and markers of subsequent SARS-CoV-2 infection were non-significant and could therefore have been due to chance of confounding by other factors. Additionally, there was lack of adjustment of serological results using prior-determined estimates of test sensitivity and specificity, meaning a small minority of individuals could have been assigned falsenegative or false-positive results. The role of HCW sentinel-site surveillance including the routine collection and storage of serum should be considered as part of national and international efforts to investigate and mitigate against emerging pathogens, including SARS-CoV-2. 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To date, findings have not been presented or published at academic meetings. *Only samples which were positive for anti-S IgG underwent testing for anti-N IgG. Therefore, all anti-Npositive samples were also anti-S-positive