Following the outbreak of SARS-CoV-2 at the end of 2019, the FDA’s policy path has been an evolving balance of risk and benefit assessments, all made in real-time during the unfolding pandemic. When it came to testing, the FDA allowed higher levels of flexibility in performance and processes when it came to serological testing compared with that of the molecular diagnostic testing. This was largely because the serological detection of antibodies is not intended to diagnose active SARS-CoV-2 infection but rather help answer critical questions over the prevalence of the infection within different populations, its rate of spread, and immunity to the disease following infection.
On March 16th, 2020, the FDA released a policy aimed at limiting serological testing to CLIA-certified laboratories with staff trained in high-complexity clinical testing, but only if those serological tests were appropriately validated to demonstrate their clinical performance.
Since then, the FDA has also provided guidelines on when serological testing should and should not be used to mitigate some of the confusion and miscommunication over this type of test versus the molecular diagnostic test.
How COVID-19 Serology Testing Should Be Used
Primarily serological testing should be used to identify individuals who have previously been infected with SARS-CoV-2. This knowledge can be used to guide epidemiology and seroprevalence studies, as well as facilitate contact tracing. In addition:
- Serology tests may be used to identify donors of convalescent plasma.
- Serological testing is a key component in evaluating the immune response to candidate vaccines.
- There is also the potential for serology tests to aid in the diagnosis of COVID-19 in RT-PCR negative patients who present later during the disease course.
How COVID-19 Serology Testing Should NOT Be Used
- Serology testing should not be used to diagnose acute or recent cases of COVID-19.
- With our current knowledge, serology tests cannot be used to definitively determine if a patient has developed protective immunity to the virus.
- As such, SARS-CoV-2 serology testing should not be used to guide personal protective equipment (PPE) use or adherence to social distancing practices.
Overview of Current SARS-Cov-2 Serological Testing Platforms
There are several serological testing platforms ranging from a simple point of care to highly complex testing in BSL3 level facilities. Ideally, a serological test needs to be simple and easy to implement, clinically sensitive, highly specific for the SARS-CoV-2 virus, as well as affordable and scalable for wide-spread use.
Currently, there are no solutions that fulfil all of these requirements.
Serological testing platforms
1. Rapid Diagnostic Tests (RDT)
Time to result 10-30 mins
Rapid Diagnostic Tests (RDT) are typically lateral flow assay, much like pregnancy testing cassettes that show the user colored lines to indicate a positive or negative result. They can either be used at Point-of-Care or potentially even in the safety and privacy of home. Relatively inexpensive ($3-70 per test), these devices use small volumes of blood for testing – however, the global market has been flooded with inexpensive devices that provide poor clinical value, with low specificity to SARS-CoV-2 and low sensitivity to antibody titers.
As such, countries including the UK and Spain raised urgent calls to the life science sector to provide solutions to this unmet medical need. Despite performance issues, the ease of use and low cost will provide a means of scaling up widespread testing, which ultimately is a good thing.
2. Neutralization Assay
Time to Result 3-5 days
On the other end of the complexity spectrum is the neutralization assay. Neutralization assays determine if an individual has produced antibodies that are able to block the activity of the virus and neutralize it from spreading. These so-called neutralizing antibodies (nAb) are most often found in individuals who have recovered from SARS-CoV-2, however levels of nAb vary widely from patient to patient.
Individuals displaying neutralizing antibodies are great candidates for convalescent serum donation programs. The neutralization assay involves mixing cultured human cells and cultured SARS-CoV-2 virus together in the lab with and without the presence of the individual’s serum- if the serum blocks the ability of the virus to infect these cultured human cells, then the serum contained nAbs.
Since the live virus is required, this type of testing is carried out in a BSL3 laboratory. As such this is not a cost-effective and scalable test and is principally only used within the field of clinical vaccine development. However, it is the only testing method that can detect functional nAbs. Genscript Bio is the only company that currently offers a pseudo-virus-based neutralization screen that can be performed in BSL2 laboratories.
3. Enzyme-Linked Immunosorbent Assays (ELISAs)
Time to Result 2-5 hours.
Enzyme-Linked Immunosorbent Assays (ELISAs) tests are carried out in a lab rather than Point-of-Care and can provide a quantitative or semi-quantitative readout of an individual’s antibody level or ‘titer’ against the SARS-CoV-2 virus. The test uses a plate coated with a specific viral protein to capture the viral-specific antibodies, these are then measured using a colorimetric detection method. This method is typically more reliable than lateral flow methods. However, the sensitivity and specificity are reliant on the quality of the viral protein on the plate.
The first generation of ELISA testing kits that came out of China were generally poorly designed and manufactured leading to low sensitivity and specificity to SARS-CoV-2. Since then, the quality of ELISA testing has been improved by the engineering and manufacturing of high-quality SARS-CoV-2 protein antigens with high specificity to this virus, and both structural and physical characteristics more similar to that of the native viral proteins.
4. Chemiluminescent Immunoassays
Time to Result 1-2 hours.
The Chemiluminescent Immunoassays platform is similar to ELISA but uses beads coated with viral antigen to capture virus-targeting antibodies in an individual’s serum. These antibodies are detected using a chemical reaction producing light. The radiance (or amount of light produced) is proportional to the amount of viral antigen-specific antibodies in the individual’s serum.
Table 1. Examples of Serological Tests Approved for use under EUA in the US and CE marked for use in the EU. As of May 15th in the US only a handful of tests had been approved by the FDA for diagnostic use under the EUA, since then the number of serological testing kits carrying the EU CE mark of quality has doubled to more than 200 according to the EU Commission.
For a more comprehensive overview of Global SARS-CoV-2 tests, here are two great reference sources.
Understanding the Limitations of Serological Testing
Although serology tests will play a significant role in the fight against SARS-CoV-2, in order to maximize their clinical value, it is important to understand the performance characteristics and limitations of the various serological tests currently available. Part of this is understanding the profile of the immunoglobulin immune response, or so-called seroconversion, which is why many of these tests look for the presence of both IgM (early antibody response) and IgG (second wave- antibody response) (see Fig. 1).
IgM & IgG Screening for Anti-SARS-CoV-2 Antibodies
A combination of IgM and IgG testing might, at first sight, provide more clinical clarity, however, by design, IgM typically lacks the specificity of IgG and so tests including and IgM profile may provide misleading results. In fact, the FDA recently stated that anti-IgM screening provides no real benefits, and as of today, no IgM test has received EUA approval from the FDA. Whether identifying IgG, IgM, or IgA responses to SARS-CoV-2, the timing of the test is everything, so repeated testing is recommended.
Quality of viral antigen material
Central to the performance of all serological tests is the quality of the viral antigen material. Generally, this is recombinantly generated in the lab, but in order to be folded and modified to a more-native state, high-quality serological tests should only use viral antigens generated in human cell lines, and typically only produce the portions of those proteins that are highly specific to this virus. It takes significant expertise, time, and resources to select and design SARS-CoV-2 specific expression vectors, and then express and purify these antigens from human cell lines. The first wave of serological testing kits from Chinese manufacturers almost certainly did not employ these quality characteristics for the generation of their screening antigens. As more companies are engaged in this initiative, the quality of the viral antigens, and therefore the quality of the serological testing that they support, has markedly improved.
Fig 1. The profile of a typical immune response to SARS-CoV-2. The incubation period of the virus has been described as 2-14 days, after which the first response of the immune system is the production of IgM immunoglobulins targeting the virus. This, coupled with cell-mediated immune responses, results in a reduction of a detectable virus. After 1-2 weeks, the immune system begins to generate more specific IgG immunoglobulins to target the virus. This IgG response may endure for weeks to months.
Key Serological Test Performance Indicators
There are two key performance criteria for SARS-CoV-2 serological testing:
Sensitivity refers to the test’s ability to correctly identify a positive sample (sample with anti-SARS-CoV-2 antibodies), whereas Specificity describes the ability of the test to correctly identify a negative result (sample without anti-SARS-CoV-2 antibodies). These work inversely against one another, and frequently in clinical testing, the threshold for a positive result is lowered in order to detect more positive specimens (higher false-positive signals). Conversely, if a test is designed to return as few false-negatives as possible, it might also miss some positive samples.
At the core of these performance characteristics is the selection of the viral antigen materials used in the screen, to ensure no cross-reactivity with other coronaviruses.
Potential for false positives with serological testing
There are four common coronaviruses (cCoV) responsible for the common cold:
Studies have shown that 60-75% of children under 12 and 90% of adults have circulating antibodies against all or some of these viruses. SARS-CoV-2 shares approximately 30% homology across its genome with these cCoVs viruses. This equates to ~90% amino acid homology across the viral N proteins, and 77% homology across the viral Spike proteins. What this means is that cross-reactivity of antibodies targeting the cCoVs and SARS-CoV-2 protein used in serological testing could result in high false positives.
This characteristic is represented by the percentage specificity, which varies widely among different testing platforms and manufacturers. For a test such as the SARS-CoV-2 serological test, a false positive represents a higher risk to the population, so the specificity of this test for SARS-CoV-2 virus is essential, and effectively more important from a surveillance perspective than the sensitivity of the test.
The Foundation for Innovative New Diagnostics (FIND) is a global non-profit driving innovation on diagnostics who are conducting an independent evaluation of the emerging serological tests coming to the market in order to compare real-performance of these tests with the manufactures validation studies. It is important to acknowledge that all tests, whatever they are testing for, intrinsically have some level of false positive and false-negative results. And in fact, many of the current serological tests for SARS-CoV-2 are performing as well, if not better, than tests for other virus infections such as HIV.
Positive and Negative Predictive Values (PPV & NPV)
Positive and Negative Predictive Values (PPV and NPV) help in the interpretation of these tests but are highly dependent on the prevalence of what the test is detecting. Even with the improvement of these SARS-CoV-2 serological tests, performance characteristics run in the ranges of: sensitivity of 90-99%, specificity of 95-100%, and although these sound impressive, it is important to understand that testing predictive values (positive or negative) are dependent upon sensitivity, specificity and disease prevalence.
How accurate are serological tests for Sars-CoV-2?
So why does this matter? Well, let’s take a test that has a sensitivity of 95% (i.e. will accurately provide a positive result for 95% of infected individuals) and a specificity of 95% (i.e. will accurately provide a negative result for the 95% of the uninfected individuals). In our scenario, we screen a State with a population of 1 million people, and a prevalence of 15% infection rate (150,000 positives and 950,000 negatives) a test with 95% sensitivity and 95% specificity will identify 185,000 individuals as positive (142,500 true positives and 42,500 false positives).
These 42,500, representing 23% of the positive test results, will believe they have some immunological protection against SARS-CoV-2, which they will not. Furthermore, 815,000 individuals will receive negative test results, but 7,500 of these will be false negatives.
Healthy & safety guidance for those who test positive for COVID-19 antibodies
As mentioned earlier, in the case of serological testing, a false positive has potentially much more impact on the health and safety of the population than a false negative, so in this context, it is important to encourage even individuals who test positive to continue safe practices such as frequent handwashing, mask-wearing, and social distancing behaviors.
The FDA has now set their expectations for serological test performance within the EUA requirements, under a prevalence of 5% infection, and providing access to a calculator to help standardize how new serological test performance is accessed.
What we still don’t know about serological testing for SARS-CoV-2
There remain many unknowns that impact the utility of serological testing for SARS-CoV-2, and below I’ve listed several areas where additional research is critically needed to maximize the value of serological testing.
1. Cross-Reactivity of Patient Antibodies
As previously outlined, Covid-19 serological tests are meant to only detect anti-SARS-CoV-2 antibodies in an individual’s serum, but with so many other related coronaviruses, including the most common 4 cCoV, many individuals may have pre-existing circulating antibodies that may cross-react if the test is not appropriately designed and validated. Ironically, there are some reports suggesting that previous infection with one of the cCoVs may invoke some immunological protection against SARS-CoV-2 (Srivastava and Saxena, 2020). However, developing serological tests with both the highest sensitivities and specificities for the SARS-Cov-2 virus is critical to their use.2. Does a positive serological test result correlate with immunity?
This is a really tough question to answer right now. We still don’t fully understand the limits of protective immunity with regard to antibody levels and duration of response as it correlates with disease severity. What we do know is that not all antibody responses are equal. Immunity requires the presence of those key neutralizing antibodies, as well as cell-mediated immunity (T cell) responses that are required for term immunological protection. Understanding the immune response to the virus will be critical in the development of vaccines against SARS-CoV-2.3. Antibody titers
Even when it comes to antibody responses, we still don’t understand how antibody titers come into play for immunity, and how long antibody levels persist and remain effective after infection. Only with long term studies that employ accurate serological testing, will these questions be answered.
How is Serological Testing Being Used Globally to Address COVID-19?
There is no question that serological testing can provide key intelligence on the evolution of the pandemic throughout the global population. With such a relatively low number of people so far infected across the world, we are far from herd immunity, and only with the use of high-quality serological testing strategies, coupled with contact tracing, or as the OECD refers to this process as Testing, Tracking and Tracing (TTT), can we lift restrictions and safely return to normality. Different countries are taking different approaches in formulating a roadmap to return to normal life.
Serological testing in Germany
Germany was one of the first to initiate large-scale adoption of serological testing for SARS-CoV-2 through the Helmholtz Center for Infection Research. The initial intention was to use population serological profiles to make informed decisions around the opening of schools and relaxing social distancing practices, as well as to distribute ‘Immunity Passports’ to individuals who tested positive for antibodies.
Ethical implications of immunity passports
There are however several ethical and logistical issues with the use of immunity passports. In addition, it would require serological testing with high negative predictive values and high specificity to SARS-CoV-2, to help ensure that these passports are not given to people who do not have SARS-CoV-2 antibodies. High numbers of false positives with immunity passports would increase the overall population’s epidemic risk.
There are also issues of accessibility to serological testing, and the security and privacy of the data from these tests, particularly in relation to the use of this data across socioeconomic groups. There would also need to be legal clarification around the use of these documents across state and country borders, and clarification of the implications on those without immunity. The UK, France and Italy all called for immunity passports programs to be implemented, an effort that would require many millions of serological tests to be made available. However, no such programs have been formally introduced, and with only 2-4% of most European populations testing positive, there are real limitations on the value of this strategy at this time.
Serological testing in Singapore and South Korea
Singapore and South Korea also employed extensive serological testing programs coupled with Contact Tracing to identify epidemiological links between cases that were not identified through molecular testing. Contact Tracing proved to be very effective in New Zealand, where it was supported by private and publicly-funded efforts using a team of reporters, tracing apps and wearables that received buy-in from the general population. New Zealand was one of the first countries to announce that they were free of COVID-19 cases in early June 2020; however, less than two weeks later, new infections were traced back to two women returning from the UK. Health officials are now tracing 320 close contacts to these individuals in the hope of stemming any further spread in the country.
How Serological Testing Can Support Epidemiological Studies for SARS-CoV-2
As we all hear in the daily news cycle, there are many unanswered questions regarding the epidemiology of SARS-CoV-2, including the percentage of asymptomatic and pauci-symptomatic cases, and understanding why fatality rates vary so much from country and country. A powerful resource for daily updated global case counts, mortality rate comparisons and epidemiological trends can be found on the Johns Hopkins University website
COVID-19 global prevalence as of June 2020
To date, the only continent to escape the pandemic is Antarctica. Within the USA, COVID-19 has been reported in all 50 states and Washington DC (CDC Covid-19 Report, April 7th, 2020 69:339), however, there are large differences in cumulative incidence between states, most likely based on population density, demographics and the accessibility of testing and reporting. In the last two weeks, we have seen a surge in cases across the USA, particularly in areas where the infection was least prevalent, and individuals were less prone to engage in safe practices such as social distancing and wearing face-masks. As a result, the USA is now at a tipping point, where the infection rate may progress to unprecedented levels, severely stretching healthcare resources. We need to acknowledge that this is a marathon, not a sprint, and that every individual in the country needs to take responsibility and act accordingly.
How might the COVID-19 pandemic unfold in the summer?
Now as we enter into the summer months, it is evident that the role of mitigation protocols such as social distancing is having a significant impact on incident rates. We now know that person-to-person transmission is the primary means of transmission, through respiratory droplets (van Doremalen et. al. 2020), although it is also transmissible through contact with contaminated surfaces through touching to mucous membranes of the eyes, mouth or nose.
Indirect data indicates that individuals are more highly infectious during the early stages of infection, but how viral load relates to symptoms and infectivity have yet to be determined. The SARS-CoV-2 virus has been detected in non-respiratory specimens (including stool, semen, blood, spinal fluid and ocular secretions), long after the virus is no longer detectable from nasal swabs (Wang et. al. 2020), but the transmission risks that this may present are far from understood.
We now have a handle on high-risk groups for SARS-CoV-2 infection. The elderly, and those with underlying chronic health conditions are overwhelmingly most at risk of poor outcomes from COVID-19, but as the prevalence of the infection rises in the USA, more and more cases of younger people dying from complications of COVID-19 are being reported. There may also be some genetic component to susceptibility; recent studies have demonstrated that COVID-19 patients with blood type A face a 50% greater risk of needing ventilator or oxygen support, in contrast with patients with blood type O, who have a 50% reduced risk of severe symptoms (Wu, Z et. al. 2020; Ellinghaus, D., et. al. 2020). Studies are now underway to examine IL-6 and ACE2 variants within populations to determine if other risk genetic factors exist.
How will Serological Testing for Neutralizing Antibodies will Shape the Development of Effective Vaccines
The impact of genetic factors on immune responses to SARS-CoV-2 is not only important to understand from an epidemiological perspective, but also when it comes to vaccine development. We now know that all patients who develop severe respiratory failure from SARS-CoV-2 infection, have immune dysregulation or macrophage-activation syndrome (DAS) (Evangelos, J. et. al. 2020).
We simply don’t understand the underlying susceptibility of an individual having this type of response to SARS-CoV-2 infection, and this raises concerns about potentiation of cytokine release syndrome by a vaccine or even convalescent plasma administration. We should be concerned that a vaccine against the “wrong” viral antigens or infusion of convalescent plasma from COVID-19 survivors, could actually worsen the inflammatory immune response in patients who may be then exposed to SARS-CoV-2.
This concept isn’t without precedent, since immune enhancement has been observed for several flaviviruses, including dengue (Campos et. al., 2018). Where serological tests can help is as a tool for screening specifically for neutralizing antibodies, which target the RBD region of the Spike protein and block the entry pathway of the virus into the cell via the ACE-2 receptor. Focusing our efforts on treatments and vaccines around the generation of these neutralizing antibodies may hold some of the answers to both safer convalescent serum treatments and vaccination approaches (Wu, F. et. al. 2020; Salazar, E et. al. 2020).
It is widely acknowledged within the public health sector that our current serological testing systems are flawed and insufficient. But right now, they are the best that we have, and the decision by the regulatory agencies to prioritize access to testing over the rigor of validation has undoubtedly had a positive impact overall. There are deficient tests out there, and every day the FDA is revising the list of serological tests approved under the EUA and eliminating from review all of those tests whose performance criteria don’t meet specificity and sensitivity requirements.
The plan is that large scale serological testing will provide key information around epidemiological and behavioral variables within populations, as well as support strategies to deploy immune-healthcare workers most effectively. Testing will also enable us to track the scope and impact of asymptomatic infection rates and help establish control policies across different countries and regions to reduce the impact of any second wave of COVID-19 into 2021. Serological testing-based surveillance programs are really the best way to study the spread of SARS-CoV-2, and only by understanding the characteristics of this pandemic’s past, can we work to predict its future impact across the globe.
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|Authored by: Dr. Julie Bick
Dr Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Tory Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.