Serological tests for COVID-19 – a primer

One of the fundamental pillars in the prevention and control of COVID-19 is timely, scalable and accurate diagnostic testing. Diagnostic testing plays a critical role in defining the epidemiology of the disease, informing case and contact management, and ultimately in reducing viral transmission (1). To date, laboratory testing has comprised detection of SARS-CoV-2 virus using reverse-transcriptase PCR (RT-PCR) assays, predominantly from patients meeting specific epidemiological criteria.  However, the unprecedented scale of RT-PCR diagnostic testing has placed extraordinary demands on healthcare and laboratory systems, with both local and global challenges relating to regulatory frameworks, supply chains of reagents, and the human and financial resource required to support population-level testing.

Early laboratory responses included early characterisation and release of the viral whole genome sequence by Chinese investigators in early January 2020, which enabled rapid development of RT-PCR workflows for the detection of SARS-CoV-2 (2). Since then, a range of commercially available diagnostic tests have been developed, including RT-PCR assays and serological tests. The broad array of tests now available vary both in analytical performance and in their particular utility in the overall public health response to COVID-19.

What does serological testing detect?

Serological tests rely on detection of specific anti-viral antibodies (IgM, IgA, IgG or total antibody) in patient serum, plasma or whole blood (3). Determining the optimal antigenic epitopes to maximise sensitivity, but minimise cross-reactivity, particularly against other human coronaviruses, has meant that the development of high-quality serological testing has been slower than molecular-based diagnostics (4, 5).  Initial candidate epitopes have largely focused on the immunogenic viral structural proteins nucleocapsid (N), and spike protein (S), particularly the S1 subunit and the receptor binding domain (RBD) (5).   To date, a range of serological tests for COVID-19 have been developed, each with particular test characteristics (Table 1 - available in PDF). Broadly, these serological tests can be divided into tests that (i) can be performed at the point-of care; (ii) can be performed in routine diagnostic laboratories, and (iii) can only be performed in specialised reference laboratories (Table 1 - available in PDF).

Initial studies have reported that most patients with COVID-19 seroconvert by day 10-14 (~80%), with almost 100% seroconversion by day 20 (6, 7). However, comparisons across published studies are challenging due to (i) different antigens used in assays; (ii) differences in the complexity of patient populations, and (iii) variations in the RT-PCR assays used as the ‘gold standard’ for determining sensitivity of serological assays. Further, it is not clear whether the type and amount of antibody correlate with severity of disease, or more importantly, with immune protection from re-infection. As noted by the World Health Organisation (WHO), the Public Health Laboratory Network of Australia (PHLN) and the Royal College of Pathologists Australasia (RCPA), a negative result using a serological test does not rule out SARS-CoV-2 infection, particularly in those with strong epidemiological risk factors, and both PHLN and RCPA note that there is no role for serological point of care tests (PoCT) in the acute diagnosis of COVID-19 (8, 9).

At present, the most widely available (and most publicised) serological tests are PoCT, which involve detection of anti-SARS-CoV-2 antibodies through binding to immobilised antigen, generally bound to colloidal gold on a test strip (Figure 1 - available in PDF).  The relatively cheap and simple nature of lateral flow assays means that production is suited to scaling-up for increased testing capacity. However, there are limited published data on the performance characteristics of serological PoCT, and high-quality data are urgently needed to guide laboratories, public health agencies and governments in the appropriate and responsible deployment of PoCT, and serological assays more broadly (4). Currently WHO recommends the use of PoCT immunodiagnostic assays in research settings only, and not for clinical decision making until further evidence is available (10). Ideally, validation of serological assays, including PoCT, should be performed against a serum panel that includes samples from: (i) patients at acute and convalescent stages of infection (to assess sensitivity), and (ii) patients with other human coronavirus infections (to assess specificity).

What serological assays are available in Australia and how is this regulated?

At the time of writing (6^th May, 2020), more than 20 serological PoCT have been approved by the Therapeutic Goods Administration (TGA) for inclusion on the Australian Register of Therapeutic Goods (ARTG), with multiple distributors (available at https://www.tga. gov.au/covid-19-diagnostic-tests-included-artg-legal-supply-australia).  An emergency TGA exemption on 22nd March 2020 allows for COVID-19 diagnostic tests to be supplied to accredited pathology laboratories in Australia (11).  In addition, unapproved diagnostic tests can also be supplied under this exemption, but again, only to an accredited laboratory. Work is ongoing globally to monitor the clinical performance and safety of new diagnostic tests, particularly in the context of emerging reports of limited sensitivity and specificity to serological PoCT (12).

The National Pathology Accreditation Advisory Council (NPAAC) has existing guidelines on the use of PoCT in Australia (13). These guidelines cover issues such as clinical supervision for performing PoCT; ensuring test quality; staff training and competency for performing PoCT, and appropriate reporting of test results.  More recently, this advice has been extended to serological PoCT for COVID-19, with an emphasis on a robust quality framework to support the implementation and deployment of such tests (14).  Of note, in Australia, the supply of self-testing (e.g. testing at home) for many infectious diseases, including COVID-19, is prohibited under another TGA regulation, the Therapeutic Goods (Excluded Purposes) Specification 2010.

Where might serological assays be used?

Given the time lag from symptom onset to detectable antibody, serological PoCT have no role in the detection of acute COVID-19 infection.  However, there are some settings where serological assays, including PoCT, may have potential utility, including defining antibody prevalence in key populations such as frontline workers, and determining the extent of COVID-19 infection within the community.  For other applications, such as identifying individuals for further evaluation of therapeutic immunoglobulin donation and vaccine development and evaluation, assays that assess neutralising antibody response are likely to be required, although as mentioned above, there are still limited available data to support the concept of protective immunity following infection with SARS-CoV-2. 

Regardless of the type of serological assay used, in order to appropriately deploy serological testing, it is critical to understand the limitations of test performance in the epidemiological context in which tests are used.  This is particularly important in a setting such as Australia, which, based on the number of reported cases of COVID-19 (6,849 cases as of 5th May, 2020), has an estimated COVID-19 prevalence of 0.03%. As such, even with serological tests that are highly sensitive and specific, the majority of positive tests are likely to represent false positive results.  When considering the use of serology to inform policies relating to relaxing of physical distancing interventions, specificity of the assay becomes critical. If the majority of those considered immune actually represent false positive results, then the threshold to maintain immunity (if this correlates with antibody detection) within the community will not be achieved.

Conclusions

There has been considerable media and government interest in the promise of relatively low-cost, scalable, and easy to use serological assays, particularly in the context of global shortages for reagents for RT-PCR testing.  The unprecedented demands on laboratories to rapidly upscale testing for COVID-19 has necessarily led to ‘fast-tracking’ of normally stringent regulatory requirements for test approval, both globally and in Australia.  Peer-reviewed and high-quality validation data are urgently required to guide laboratories, public health agencies and governments in appropriate serological test selection and deployment.   Without such data, many countries run the risk of roll-out of sub-optimal tests, which ultimately may cause more harm than good in the COVID-19 response.

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