Development and application of a serological assay has helped to establish connections between COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods.

Three clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320.

In Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used to establish past infection.

Elucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (COVID-19) infections are crucial for effective disease containment. We describe an epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links between three clusters of COVID-19.

We present findings of investigations from Jan 29 to Feb 24, 2020, that linked two people with COVID-19 from Wuhan, China, to three clusters of COVID-19 cases in Singapore. Serological testing had a crucial role in establishing a link between clusters, showing its use in identifying convalescent COVID-19 cases and supporting epidemiological investigations.

In Singapore, a globally connected city-state in southeast Asia, health officials have attempted to contain the spread of COVID-19 through intensive epidemiological investigations coupled with isolation of cases and quarantine of close contacts. However, establishing the source of infection to ascertain the possible extent of spread can be difficult, because scant epidemiological data might be available. Even when possible nodes of transmission are retrospectively identified through epidemiological investigations, nucleic acid-based tests would not be diagnostically useful if these infected individuals have recovered and no longer shed the virus. Hence, serological tests are needed to identify convalescent cases and aid investigations and containment efforts.

As of April 15, 2020, more than 1·9 million cases of coronavirus disease 2019 (COVID-19), and more than 120 000 deaths from the disease, have been recorded worldwide.Many initial cases reported outside of China were imported or were linked to travellers from China.However, as community transmission has become widespread, the source of cases of COVID-19 in several countries has not been established.

The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

For laboratory confirmation of COVID-19, we did RT-PCR testing for SARS-CoV-2, using previously published methods.Two serological platforms were developed for confirmation of specific antibody responses to SARS-CoV-2 in people with suspected infection or individuals with PCR-confirmed disease. A virus neutralisation test (VNT) was established at the Duke-National University of Singapore Medical School ABSL3 facility using a SARS-CoV-2 virus isolate (BetaCoV/Singapore/2/2020; GISAID accession number EPI_ISL_407987) cultured from a patient in Singapore; VNT was done using protocols previously published for severe acute respiratory syndrome coronavirus (SARS-CoV).For ELISA assays, we used recombinant nucleocapsid protein from SARS-CoV and SARS-CoV-2 expressed in mammalian cell culture using the pcDNA3.1 vector (ThermoFisher Scientific, Carlsbad, CA, USA), according to previously published methods,and a recombinant receptor binding domain (RBD) of the SARS-CoV-2 spike protein custom-produced by a commercial provider (GenScript, Piscataway, NJ, USA). ELISA wells were coated with 100 ng of the respective protein per well and serum samples were used at dilutions from 1/50 to 1/400, followed by horseradish peroxidase-conjugated goat anti-human IgG (Santa Cruz, Dallas, TX, USA) used at a dilution of 1/2000.

All epidemiological investigations and outbreak containment measures were implemented under the Infectious Diseases Act,which allows use of data for analysis to control outbreaks.

Once individuals with COVID-19 were identified, their activities from 14 days before symptom onset until they were isolated were mapped and their close contacts traced. Contact tracing before symptom onset was done to identify the source of exposure that led to the case being infected, allowing for further active case-finding around the source. Contact tracing after symptom onset until isolation was done to identify exposed individuals for quarantine to break the transmission chain. Both these approaches were part of the containment strategy. A close contact was defined as anyone who had prolonged contact within 2 m of the case. All close contacts with active or recent symptoms were tested, whereas those who were asymptomatic and exposed while the case was symptomatic were quarantined. Activity maps were reviewed and cross-checked to establish potential exposures and identify possible epidemiological links between cases and clusters.

COVID-19 laboratory testing is focused on use of quantitative RT-PCR for diagnosis, and serological testing can be overlooked. We have highlighted the importance of serological testing for epidemiological investigation of COVID-19 cases, and we urge further development of serological testing capabilities.

In our epidemiological investigation, we used RT-PCR and serological testing to diagnose cases of COVID-19 and establish links between clusters. RT-PCR testing alone is limited by its ability to detect convalescent cases of COVID-19, because RT-PCR can only detect severe acute respiratory syndrome coronavirus 2 during the period of viral shedding, which is the acute phase of infection. Serological testing can be useful in detecting previous infection in people with suspected infection who have recovered, assisting in epidemiological investigation and containment efforts.

We searched PubMed on March 3, 2020, for reports on serological testing in individuals with coronavirus disease 2019 (COVID-19). We used the keywords (“COVID-19”, OR “2019-nCoV”, OR “SARS-CoV-2”) AND (“serology” OR “serologic testing”). Our search did not identify any reports of the epidemiological application of serological tests in COVID-19. In one report, researchers described serological characteristics of COVID-19, and in other publications, researchers have commented on the potential importance of COVID-19 serological tests. In another study, epidemiological investigations were reported of the epidemic in Singapore, but serological methods had not been used.

In Singapore, several surveillance methods are used to identify people with COVID-19. On Jan 2, 2020, a suspect case-definition of COVID-19 was circulated to all doctors in Singapore;doctors are legally required to notify the Ministry of Health of cases of COVID-19.From Jan 31, 2020, Singapore began testing all patients with pneumonia in hospital for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); this testing was later expanded to include people with pneumonia in primary care. The diagnosis of COVID-19 is confirmed either by a respiratory sample testing positive for SARS-CoV-2 using a laboratory-based RT-PCRor by a serum sample testing positive for SARS-CoV-2 on serological analysis.

Results

Figure 1 Transmission map of COVID-19 Show full caption Map shows how COVID-19 was linked to two travellers from Wuhan, China, and two church clusters and a family gathering in Singapore. COVID-19=coronavirus disease 2019. As of April 6, 2020, Singapore had recorded 1375 cases of COVID-19, of which 554 were imported and 821 locally transmitted. Three clusters were identified that involved two churches (Church A and Church B) and one family gathering. These clusters comprised 28 locally transmitted cases. The clusters were linked to two travellers (W1 and W2) from Wuhan, China, who attended a church service at Church A on Jan 19, 2020 ( figure 1 ). The clusters at Church A and Church B were linked by one individual from Church A (A2), who probably transmitted the infection to the primary case of the Church B cluster (F1) at a family gathering on Jan 25, 2020. All cases were confirmed by RT-PCR testing, except for A2, who was diagnosed by serological testing.

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Infectious Diseases Act 2003. The clusters at Church A and Church B were detected in early February and mid-February, respectively. Although W1 and W2 were diagnosed with COVID-19 at the end of January, their possible link to Church A was only discovered after the Church A cluster was identified, through investigation and repeat interviews.By that time, A2 had recovered from COVID-19 and was not immediately linked to either cluster. Family members who had been infected at the family gathering on Jan 25, 2020, were first linked to F1 and were initially regarded as part of the cluster at Church B. However, subsequent investigations into case histories indicated that the family cluster was a distinct cluster and that A2 was most probably the missing link between the two church clusters; this idea was substantiated when A2's serological results were confirmed to be positive.

Figure 2 Incubation period, duration of symptoms, and length of admission, from Jan 14 to Feb 26, 2020 Show full caption Data for 30 people with coronavirus disease 2019 are shown. Individuals are labelled with cluster letter and number, age (years) and sex (M=male; F=female). Median age of affected individuals was 50·5 (IQR 25–79) years and half the cohort (15 of 30) were female. Five locally transmitted cases of COVID-19 (A1–A5) were linked to Church A. These people attended a church service on Jan 19, 2020, the same day W1 and W2 visited the church. Although all five people had developed symptoms by Feb 2, 2020 ( figure 2 ), only A1, A4, and A5 were diagnosed (between Feb 6 and Feb 8, 2020), because they had been hospitalised for pneumonia and tested for SARS-CoV-2 as part of enhanced surveillance measures to test all patients admitted to hospital with pneumonia. A2 and A3 were not diagnosed when symptomatic in late January because their symptoms were mild and they did not meet the suspect case-definition at that time. A2 and A3 were tested only after mapping of activities and movements of other cases suggested that A2 could be the missing link between the clusters at Church A and Church B. An RT-PCR test of a nasopharyngeal specimen taken from A3 on Feb 18, 2020, was positive, although this individual had clinically recovered from the illness, which persisted from Jan 28 to Feb 10, 2020. Serological analysis of a serum sample obtained on the same day as the nasopharyngeal specimen was also positive. Although A2 had two negative RT-PCR tests, the serological result was positive, indicating past infection.

A2 and A3 attended a Chinese New Year family gathering on Jan 25, 2020, at the home of F1. Nine cases (A2, A3, and F1–F7) were linked to this family gathering. A2, whose symptoms started on Jan 23, 2020, was unwell at this event and most probably was the primary source of transmission. A3 developed symptoms later, on Jan 28, 2020, and was, therefore, unlikely to be the source of infection at this gathering.

17 locally transmitted cases were linked to Church B (B1–B16 and F1). Thorough review of activity maps ascertained that F1, who had developed symptoms on Jan 29, 2020, and continued to work at Church B while ill, was the primary case of the Church B cluster.

In mid-February, epidemiological and clinical evidence strongly suggested that A2 was the missing link between the clusters at Church A and Church B through attendance at the family gathering on Jan 25, 2020, while symptomatic. However, by the time this link was ascertained, more than 3 weeks had passed from symptom onset on Jan 23, 2020, and symptoms had resolved completely a week previously, on Feb 8–10, 2020. A2 had two negative RT-PCR test results from samples taken from the nasopharynx, with testing done 1 day apart on Feb 18 and Feb 19, 2020. The sample for serological testing was taken on Feb 18, 2020, and a positive result was confirmed on Feb 22, 2020.