As coronavirus disease 2019 (COVID-19) raged around the globe in late March, hundreds of San Miguel County, Colorado, residents turned out for a blood test. Standing 6 feet apart outside a Telluride school gym, they waited for the blood draw that would tell them whether they had mounted an immune response to the disease-causing virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—a sign that they’d been infected.

In the first such community-wide campaign in the US, the San Miguel County Department of Health offered the voluntary screening to most of the area’s 8000 residents over 2 weeks. Just 8 of the 986 individuals tested on March 26 and 27 were positive for SARS-CoV-2 antibodies. Another 23 were borderline, suggesting that they’d recently been exposed to the virus and were just starting to make antibodies against it. But those were early days. The screenings, paid for by test manufacturer United Biomedical Inc and the county, eventually would be repeated to see how much things had changed.

Unlike polymerase chain reaction (PCR) tests—also referred to as molecular or nucleic acid–based tests—antibody tests aren’t intended to identify active SARS-CoV-2 infections. Instead of detecting viral genetic material in throat or nasal swabs, antibody tests reveal markers of immune response—the IgM and IgG antibodies that for most people show up in blood more than a week after they start to feel sick, when symptoms may already be waning.

Serologic antibody tests not only can confirm suspected cases after the fact, they can also reveal who was infected and didn’t know it. Up to a quarter of people with SARS-CoV-2 infection may unwittingly spread the virus because they have mild or no symptoms.

Implications for the health care workforce could be substantial, microbiologist Florian Krammer, PhD, of Mount Sinai’s Icahn School of Medicine, said in an interview. “If we find serologically that you are immune, it’s very unlikely that you can get reinfected, which means you can’t pass the virus on to your colleagues or to other patients. And I think it also gives a peace of mind if you have to work with COVID-19 patients to know that you’re probably immune to the infection,” he explained.

Antibody tests are ramping up quickly, with a growing list of commercial kits and test protocols from academic researchers including Krammer’s team and a Dutch team coming online in recent days and weeks. Scientists said the tests will be critical in the weeks and months ahead, when they may be used for disease surveillance, therapeutics, return-to-work screenings, and more. But the tests must be deployed appropriately, they added, and with an acknowledgment of unanswered questions.

Turning Antibodies Into Therapies

In their first therapeutic application, serology tests are being used to screen donor blood for antibodies to SARS-CoV-2. Plasma containing the antibodies from recovered patients is then transfused to gravely ill patients in an experimental treatment known as convalescent plasma. Early results from a small number of Chinese patients, published in JAMA in late March, were promising.

The US Food and Drug Administration (FDA) is coordinating a national effort to develop blood-based, antibody-rich COVID-19 therapies. They include convalescent plasma and the hyperimmune globulin derived from it, which ideally will provide passive immunity to people who have been exposed to the virus.

In an interview, Carlos Cordon-Cardo, MD, PhD, who chairs the Mount Sinai Health System pathology department in New York City, said physicians there have begun to transfuse convalescent plasma to critically ill patients as part of an FDA expanded access program. Krammer’s research team developed the test that’s being used to screen donor blood.

They recently described their new assay in a preprint article (which has not been peer-reviewed). The test detected antibodies in plasma from blood drawn as early as 3 days after patients first developed symptoms. Crucially, it did not react with human coronaviruses already circulating in the population, demonstrating that it’s specific to SARS-CoV-2. “What we've found with our test is that basically everybody’s naive,” Krammer said. “There’s no preexisting immunity. And that makes it very easy to distinguish between people who have been infected and who haven’t been infected.”

Antibody testing could also help to address a potential unintended consequence of receiving convalescent plasma or hyperimmune globulin. It’s possible that some COVID-19 survivors who undergo these treatments won’t develop their own immunity, putting them at risk for reinfection, Lee Wetzler, MD, a professor of medicine and microbiology at the Boston University School of Medicine, said in an interview. Serologic testing could be used to check their antibody status after they’ve recovered; those with low or no immunity would be prime candidates for a vaccine when one becomes available.

Mount Sinai and United Biomedical’s tests are both enzyme-linked immunosorbent assays (ELISAs), a common laboratory platform that can measure antibody titers. Being able to quantify antibodies will be important for identifying convalescent plasma donors with abundant titers and studying how the immune system responds to the virus. “The titers that we measure in ELISA seem to correlate with neutralizing antibodies,” Krammer said. “So basically the idea is the higher these titers, the better you are protected.”

Krammer has shared his test’s reagents and tool kits with about 150 different US clinical labs. These types of quantitative tests will help scientists to understand if there’s a certain antibody type or threshold a person needs to be protected, according to Wetzler, who is also an infectious disease physician at the Boston Medical Center.

However, a substantial number of the new commercial COVID-19 antibody tests aren’t ELISA-based. They’re lateral flow assays, which provide a simple positive or negative result, with no quantitative information. These kits are cheap and easy to use and, depending on how they’re employed, may be helpful for disease surveillance, Elitza Theel, PhD, director of the Mayo Clinic Infectious Diseases Serology Laboratory in Rochester, Minnesota, said in an interview.

Palo Alto–based Nirmidas Biotech is one of many companies offering a rapid, point-of-care lateral flow assay. According to Meijie Tang, PhD, the firm’s CEO and president, a state Centers for Disease Control and Prevention (CDC) laboratory and other partners are evaluating the test’s performance. “We plan to collaborate with hospitals, clinics, health care and medical institutions to validate the test and make [it] widely available,” she said in an email.

Diazyme Laboratories in Poway, California, has developed chemiluminescence immunoassays, which are closer in concept to ELISAs than lateral flow assays. The tests generate a light signal proportional to SARS-CoV-2 IgM antibodies. In an email, cofounder and Managing Director Chong Yuan, PhD, said the company would ship about 2 million tests to clinical labs over the over the next month.

The Right Test at the Right Time

On April 1, the FDA granted Emergency Use Authorization (EUA) to a rapid SARS-CoV-2 IgG and IgM lateral flow assay from Cellex Inc in Research Triangle Park, North Carolina. Mount Sinai’s test received EUA 2 weeks later.

The agency by early April had also allowed more than 70 companies to sell COVID-19 antibody tests without this authorization, albeit with some stipulations. Among other requirements, manufacturers operating without EUA must state that they’ve clinically validated their tests using specimens from patients with PCR-confirmed infections. The test reports must note that the FDA has not reviewed the assays and that they should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection or to inform patients of infection status.

Yet, according to Theel, several companies are marketing lateral flow assays as rapid point-of-care tests to identify active COVID-19, something the FDA announced it will take action against. “We do not really know how well these assays work at this point,” Theel said in a follow-up email.

Although commercial manufacturers claim their tests have high sensitivity and specificity, they haven’t published their data yet. This lack of transparency is worrisome, Theel said: “The question is, when following symptom onset were these samples collected to show that sensitivity and specificity?”

Her laboratory has found that most people with SARS-CoV-2 don’t start producing antibodies—or seroconvert—until at least 11 to 12 days after symptom onset. “So, if we were using these rapid lateral flow assays at the point of care to test recently symptomatic patients,…they are more likely than not going to be negative,” she said.

Greater FDA oversight for antibody tests could be coming, according to news reports. In addition, the World Health Organization (WHO) is working with partners and its own global laboratory network to evaluate available assays for diagnostic and research purposes, a spokesperson said in an email. One partner in the effort is the Foundation for Innovating Diagnostics (FIND), a Geneva, Switzerland–headquartered nonprofit that’s evaluating both PCR and serology tests. As of mid-April, the group had selected 27 antibody tests, mostly from China, for its first round of evaluation. The tests’ performance results will be posted on the FIND website as they become available.

According to news reports, newly available rapid, point-of-care PCR tests, like a recently announced 5-minute assay from Abbott Laboratories, won’t substantially increase diagnostic testing capacity in the short-term. Faced with a PCR test shortfall amid incredible demand, health systems may consider subbing in serology tests. But experts strongly underscored that antibody testing generally should not be used to diagnose active cases.

Krammer said that resorting to antibody testing to diagnose active infections is a “complete misuse.” Not only are antibody tests likely to report false-negatives early on, they’ll also miss infections among people who are immunocompromised and don’t produce antibodies.

“Molecular testing is still going to be the go-to preferred method for diagnosis of COVID-19 in symptomatic patients,” Theel said. In her view, the only appropriate use of antibody testing for active infection may be for people who have had symptoms for over a week but are PCR negative. But the precise timing of that still hasn’t been defined.

“I think that it is very important that we understand the limitations of serologic testing, recognizing that it takes time to mount a detectable immune response, and to use them for the right reasons,” Theel said. “A false-negative serologic result in an acutely symptomatic patient with replicating and shedding virus has serious public health consequences.”

WHO has published guidance on adjusting public health and social measures for the next phase of the COVID-19 response.1 Some governments have suggested that the detection of antibodies to the SARS-CoV-2, the virus that causes COVID-19, could serve as the basis for an “immunity passport” or “risk-free certificate” that would enable individuals to travel or to return to work assuming that they are protected against re-infection. There is currently no evidence that people who have recovered from COVID-19 and have antibodies are protected from a second infection.

The measurement of antibodies specific to COVID-19

The development of immunity to a pathogen through natural infection is a multi-step process that typically takes place over 1-2 weeks. The body responds to a viral infection immediately with a non-specific innate response in which macrophages, neutrophils, and dendritic cells slow the progress of virus and may even prevent it from causing symptoms. This non-specific response is followed by an adaptive response where the body makes antibodies that specifically bind to the virus. These antibodies are proteins called immunoglobulins. The body also makes T-cells that recognize and eliminate other cells infected with the virus. This is called cellular immunity. This combined adaptive response may clear the virus from the body, and if the response is strong enough, may prevent progression to severe illness or re-infection by the same virus. This process is often measured by the presence of antibodies in blood.

WHO continues to review the evidence on antibody responses to SARS-CoV-2 infection.2-17 Most of these studies show that people who have recovered from infection have antibodies to the virus. However, some of these people have very low levels of neutralizing antibodies in their blood,4 suggesting that cellular immunity may also be critical for recovery. As of 24 April 2020, no study has evaluated whether the presence of antibodies to SARS-CoV-2 confers immunity to subsequent infection by this virus in humans.

Laboratory tests that detect antibodies to SARS-CoV-2 in people, including rapid immunodiagnostic tests, need further validation to determine their accuracy and reliability. Inaccurate immunodiagnostic tests may falsely categorize people in two ways. The first is that they may falsely label people who have been infected as negative, and the second is that people who have not been infected are falsely labelled as positive. Both errors have serious consequences and will affect control efforts. These tests also need to accurately distinguish between past infections from SARS-CoV-2 and those caused by the known set of six human coronaviruses. Four of these viruses cause the common cold and circulate widely. The remaining two are the viruses that cause Middle East Respiratory Syndrome and Severe Acute Respiratory Syndrome. People infected by any one of these viruses may produce antibodies that cross-react with antibodies produced in response to infection with SARS-CoV-2.

Many countries are now testing for SARS-CoV-2 antibodies at the population level or in specific groups, such as health workers, close contacts of known cases, or within households.21 WHO supports these studies, as they are critical for understanding the extent of – and risk factors associated with – infection.  These studies will provide data on the percentage of people with detectable COVID-19 antibodies, but most are not designed to determine whether those people are immune to secondary infections.

Other considerations

At this point in the pandemic, there is not enough evidence about the effectiveness of antibody-mediated immunity to guarantee the accuracy of an “immunity passport” or “risk-free certificate.” People who assume that they are immune to a second infection because they have received a positive test result may ignore public health advice. The use of such certificates may therefore increase the risks of continued transmission. As new evidence becomes available, WHO will update this scientific brief.

References

In response to the growing COVID-19 pandemic and shortages of laboratory-based molecular testing capacity and reagents, multiple diagnostic test manufacturers have developed and begun selling rapid and easy-to-use devices to facilitate testing outside of laboratory settings. These simple test kits are based either on detection of proteins from the COVID-19 virus in respiratory samples (e.g. sputum, throat swab) or detection, in blood or serum, of human antibodies generated in response to infection.

WHO applauds the efforts of test developers to innovate and respond to the needs of the population.

However, before these tests can be recommended, they must be validated in the appropriate populations and settings. Inadequate tests may miss patients with active infection or falsely categorize patients as having the disease when they do not, further hampering disease control efforts. At present, based on current evidence, WHO recommends the use of these new point-of-care immunodiagnostic tests only in research settings. They should not be used in any other setting, including for clinical decision-making, until evidence supporting use for specific indications is available.

WHO continues to evaluate available immunodiagnostics tests for COVID-19 and will update this scientific brief when necessary.

Rapid diagnostic tests based on antigen detection

One type of rapid diagnostic test (RDT) detects the presence of viral proteins (antigens) expressed by the COVID-19 virus in a sample from the respiratory tract of a person. If the target antigen is present in sufficient concentrations in the sample, it will bind to specific antibodies fixed to a paper strip enclosed in a plastic casing and generate a visually detectable signal, typically within 30 minutes. The antigen(s) detected are expressed only when the virus is actively replicating; therefore, such tests are best used to identify acute or early infection.

How well the tests work depends on several factors, including the time from onset of illness, the concentration of virus in the specimen, the quality of the specimen collected from a person and how it is processed, and the precise formulation of the reagents in the test kits. Based on experience with antigen-based RDTs for other respiratory diseases such as influenza, in which affected patients have comparable concentrations of influenza virus in respiratory samples as seen in COVID-19, the sensitivity of these tests might be expected to vary from 34% to 80%.1

Based on this information, half or more of COVID-19 infected patients might be missed by such tests, depending on the group of patients tested. These assumptions urgently require further study to understand whether they are accurate. Additionally, false-positive results – that is, a test showing that a person is infected when they are not – could occur if the antibodies on the test strip also recognize antigens of viruses other than COVID-19, such as from human coronaviruses that cause the common cold. If any of the antigen detection tests that are under development or commercialized demonstrate adequate performance, they could potentially be used as triage tests to rapidly identify patients who are very likely to have COVID-19, reducing or eliminating the need for expensive molecular confirmatory testing.

With the limited data now available, WHO does not currently recommend the use of antigen-detecting rapid diagnostic tests for patient care, although research into their performance and potential diagnostic utility is highly encouraged.

Rapid diagnostic tests based on host antibody detection

There is another, more common type of rapid diagnostic test marketed for COVID-19; a test that detects the presence of antibodies in the blood of people believed to have been infected with COVID-19.2-5 Antibodies are produced over days to weeks after infection with the virus. The strength of antibody response depends on several factors, including age, nutritional status, severity of disease, and certain medications or infections like HIV that suppress the immune system.6-8 In some people with COVID-19, disease confirmed by molecular testing (e.g. reverse transcription polymerase chain reaction: RT-PCR), weak, late or absent antibody responses have been reported.6,7,9 Studies suggest that the majority of patients develop antibody response only in the second week after onset of symptoms.2,6,7,10-14 This means that a diagnosis of COVID-19 infection based on antibody response will often only be possible in the recovery phase, when many of the opportunities for clinical intervention or interruption of disease transmission have already passed. Antibody detection tests targeting COVID-19 may also cross-react with other pathogens, including other human coronaviruses.7,15,16 and give false-positive results.  Lastly, there has been discussion about whether RDTs detecting antibodies could predict whether an individual was immune to reinfection with the COVID-19 virus. There is no evidence to date to support this.

Tests to detect antibody responses to COVID-19 in the population will be critical to support the development of vaccines, and to add to our understanding of the extent of infection among people who are not identified through active case finding and surveillance efforts, the attack rate in the population, and the infection fatality rate. For clinical diagnosis, however, such tests have limited utility because they cannot quickly diagnose acute infection to inform actions needed to determine the course of treatment. Some clinicians have used these tests for antibody responses to make a presumptive diagnosis of recent COVID-19 disease in cases where molecular testing was negative but where there was a strong epidemiological link to COVID-19 infection and paired blood samples (acute and convalescent) showing rising antibody levels.

Based on current data, WHO does not recommend the use of antibody-detecting rapid diagnostic tests for patient care but encourages the continuation of work to establish their usefulness in disease surveillance and epidemiologic research.

Next steps

• Molecular (e.g. PCR) testing of respiratory tract samples is the recommended method for the identification and laboratory confirmation of COVID-19 cases. COVID-19 molecular diagnostic products are being evaluated for quality and safety through the WHO Prequalification Emergency Use Listing Procedures and through a collaboration with the Foundation for Innovative New Diagnostics (FIND). WHO guidance documents for detection of COVID-19 have been published: WHO Guidance on Laboratory testing for COVID-19 in suspected human cases.  In addition, guidance on how testing might be rationalized when lack of reagents or testing capacity necessitates prioritization of certain populations or individuals for testing is also available.
• To inform WHO policy on the use of immunodiagnostic rapid tests for COVID-19, WHO is working with our global laboratory expert network, and closely reviewing the results of laboratory and clinical studies planned and implemented by reference laboratories, academic groups and non-governmental organizations.
• Target product profiles for desired COVID-19 diagnostics to inform research and development efforts are in development.
• WHO will continue to work with research groups, other agencies, and Member States to develop and interpret data that might indicate specific areas where such tests can be useful for clinical management of cases, epidemiologic understanding, and/or infection control. 

References

Antibody response to COVID-19 infection

Yes, the IgM and IgG SARS-CoV-2 specific antibodies have been detected in patients during the recovery phase of COVID-19 infection.

However, the quantification of such antibodies, immune status of the recovered patients against reinfection with COVID-19, and the duration of the immunity still remain unknown.

Further reading

Preprint Antibody responses to SARS-CoV-2 in COVID-19 patients: the p...

Article Breadth of concomitant immune responses prior to patient rec...

https://www.medrxiv.org/content/10.1101/2020.03.18.20038059v1.full.pdf

This research provides a scientific basis for immunity certificates (cards/passports). Individual immunity is a resource: It must employed, certified, searched for, found, and it may be actively produced. https://doi.org/10.1111/kykl.12227