COVID ANIGEN TESTING

COVID-19: Is Antibody Testing Key to Uplifting Quarantine?

Authors:News Author: Debbie Koenig; CME Authors: Charles P. Vega, MD and Esther Nyarko, PharmDFaculty and Disclosures

CME / ABIM MOC / CE Released: 6/5/2020

Valid for credit through: 6/5/2021

Note: This is the twenty-eighth of a series of clinical briefs on the coronavirus outbreak. The information on this subject is continually evolving. The content within this activity serves as a historical reference to the information that was available at the time of this publication. We continue to add to the collection of activities on this subject as new information becomes available.

Clinical Context

With the COVID-19 pandemic in global overdrive, researchers and lab manufacturers are in a frantic search to find solutions to curb the impact on health systems, the economy, and the community. One solution, researchers think, is the development of additional testing methodologies to detect the virus. Experts have indicated this could be a key turning factor in controlling the epidemic and restoring some form of pre-COVID-19 normality in uplifting lockdown restrictions. Knowing how many have been infected or exposed and identifying persons who are immune is essential.

According to a statement from the FDA published on April 7, 2020,^[1] serological tests can play an important role in the clinical and public health response to COVID-19. They can be useful in establishing previous exposure or infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identifying persons who possibly might be protected from a subsequent infection. This can help more persons recovering from infection to return to work or school. Serum antibodies may also play a role in the diagnosis of acute COVID-19, and serological tests may ascertain candidates for convalescent plasma donation in helping others fight infection.

With more than 90% of the country under stay-at-home orders,^[2] officials have said one thing might help us get back to normal: a rapid blood test to see who has developed immunity to COVID-19.

In March, the FDA allowed manufacturers to promote serological tests if they met certain criteria for accuracy and reliability, but they were not required to have full FDA approval.^[1] Currently, more than 70 tests are available under this policy. Thirteen of these tests have been authorized under an Emergency Use Authorization (EUA).^[3]

The efficacy of these serological tests and concerns about current polymerase chain reaction (PCR) testing has been called into question. Can we improve on current testing? How do serological tests perform in clinical practice? The current study by Pan and colleagues addressed some of these questions.

Study Synopsis and Perspective

In an April 11 Bloomberg report,^[4] many doctors expressed concern about patients testing negative with obvious symptoms. Many are told they do not have the disease but later test positive. Researchers have yet to determine the cause of these false negatives, but the report identified possible causes to be lack of appropriate vetting by federal health regulators for hurriedly developed tests, a shortage of test supplies and materials, disease factors such as long incubation times, and inadequate sampling. Whatever the cause, the rising concern has prompted action by a number of lab manufacturers looking for alternative testing methods.

Currently, 2 types of testing are available to detect COVID-19 infection: PCR, which is used in the majority of cases, and antibody (serological) testing.^[5] Neither perfect, they each have pros and cons.

How does serological testing differ from PCR?

Polymerase chain reaction tests are molecular tests and have been the primary tool for diagnosis by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) since the onset of the COVID-19 outbreak. Diagnosing by PCR involves the use of swabs inserted into the back of the nasal passage via the nostril to obtain samples. PCR testing detects RNA in SARS-CoV-2 and is reliable only if the patient is actively infected.

In contrast, serological tests use blood samples to identify antibodies. They analyze the serum component of whole blood and gauge the body’s immune response to foreign pathogens (ie, antigens). Samples for blood testing are obtained through a finger prick or in some cases a blood draw. Samples are tested to detect presence of immunoglobulin (Ig)M antibodies and IgG, which is indicative of recent exposure to SARS-CoV-2 (early-stage [IgM] and late-stage [IgG] infection). There are different kinds of serological tests: neutralization tests, immunofluorescent assay, enzyme-linked immunosorbent assays (ELISAs), and Western blot tests.^[6] Serological testing methods offer the benefits of short time to diagnosis and early identification of active immune response against a virus. It provides a retrospective review of host response to infection and resulting inferred immunity. It can identify persons who have recovered from COVID-19.

How effective are serological tests compared with PCR?

In a recent study, with results published in the April 10 issue of the Journal of Infection,^[5] Pan and colleagues pointed out the concerns with false negatives with the PCR testing method. Researchers showed chest computed tomography scanning from 5 suspected patients in China who demonstrated ground-glass opacity and/or mixed consolidation; however, this was inconsistent with initial real-time reverse-transcriptase polymerase chain reaction (RT-PCR) testing that came back as negative. These 5 cases eventually tested positive after repeat examinations. In an effort to address the concern of the increasing rates of false negative using PCR diagnostics, Pan et al conducted a study comparing serological Ig (IgM and IgG) testing using colloidal gold-based immunochromatographic (ICG) technology with RT-PCR from throat swab samples among 105 patients with COVID-19 admitted to one hospital in China.

Among patients with positive RT-PCR testing, the rate of positive IgM testing during periods from 1 to 7 days after illness onset, 8 to 14 days after illness onset, and more than 15 days after illness onset were 11.1%, 78.6%, and 74.2%, respectively. The respective positive rates for IgG throughout illness were 3.7%, 57.1%, and 96.8%. The sensitivity of the ICG assays peaked around day 15 of illness. Combining IgM and IgG would improve the sensitivity of the ICG assay. Among cases with positive radiographic findings but negative RT-PCR tests, 23.1% of samples were positive for IgM and 38.5% were positive for IgG. As in confirmed cases of COVID-19, IgM and IgG levels peaked at more than 15 days after the onset of illness among patients with negative RT-PCR results.

Authors concluded there was good correlation between IgM and IgG detection in both whole blood and plasma. Authors indicated serological testing sensitivity to be comparable with that of PCR but was more beneficial because of the additional time saved without use of specialized equipment. Other benefits cited include the serological test’s detection of antibodies, which can be a sign of recovery, and ease of collection compared with PCR, which uses oral swab sampling that can induce retching and coughing, increasing exposure risk for healthcare workers.

Limitations to this study include lack of assay-specific analysis; difficulty of getting a truly negative control because the study was conducted in the heart of the Wuhan outbreak area with many possibly already infected; the fact that ELISA assay was not used, and, as such, ICG results were only qualitative.

Are serological tests currently in use?

Serological tests are already in use in other countries. In the United States, it is currently unknown how many people have been infected with SARS-CoV-2. Experts believe adding serological tests to current testing strategies would increase availability and sensitivity of testing. Seeing the possibility of this, the CDC has designed and is researching a new serological test with the intention to use these study results to estimate how many people have been infected nationally, as well as persons who have not had it and may still be at risk for infection.^[7] According to the CDC, having an additional test will enable confirmation of infection and will help determine factors that confer protection against the virus. On April 11, the FDA issued its first authorization for a serological test.^[8]

How accurate are both diagnostic methods?

Standard molecular tests provide results ranging from a couple of hours to a few days, depending on the manufacturer. Some are in development that are said to produce results in 30 to 45 minutes. Serological tests are said to be faster than molecular tests and can provide results in as little as 15 minutes.^[9]

A positive COVID-19 result with a molecular test does not rule out infections from other bacteria or viruses.^[10] In addition, a negative test does not completely rule out the possibility of the virus in the body at undetectable levels. With the saliva molecular tests, the FDA recommends a second testing method to confirm initial negative results. Serological tests may not detect active infection, as it takes the body some time to develop an antibody response to SARS-CoV-2. As a result, it may produce false negatives even though there is an active infection. It can provide useful information on patients who have had the infection and recovered. This can be valuable, as currently, data are lacking on re-infection of SARS-CoV-2 and length of immunity.

Have serological tests been used in previous coronavirus outbreaks?

In the case of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), although serological tests were used, it took a long time to develop them and required a lot of resources. Historical data show that survivors of the SARS outbreak in 2003 had antibodies in their bloodstream years after they recovered.^[11] It is unclear whether COVID-19 will produce a similar occurrence in immune response, as recent data have shown re-infection with COVID-19 after recovery, indicating these particular patients did not develop immunity against the virus. Some governments have suggested the idea of an "immunity passport" or "risk-free certificate" for persons who had recovered from COVID-19 on the basis they cannot be re-infected.^[12] This pass would enable them to travel without restriction; however, currently, no evidence supports this. The WHO has issued a statement against this idea, warning that such practice can harm the public by increasing risks for continued spread and exposure because of relaxation of standard precautions against COVID-19. The WHO’s concern arises from available study data that show people who have recovered from the infection, have very low levels of neutralizing antibodies to the virus, suggesting low cellular immunity that can affect complete recovery.

Will antibody tests help us leave quarantine?

Antibodies develop when one has been exposed to a particular infection, and experts believe many COVID-19 cases are so mild people may not know they had it. That means there is a chance that a significant number of people have developed antibodies already.

"We don't currently have good numbers for the numbers of people who are infected now, much less people who were infected before who were never tested," Angela Rasmussen, MD, a virologist at Columbia University Mailman School of Public Health in New York, New York, told The New York Times.^[13] "So it's really important from an epidemiological perspective to do these types of serology assays."

Hoping to discover just how widespread the virus has become, last week, the National Institutes of Health (NIH) announced they are looking for 10,000 volunteers with no confirmed history or current symptoms of COVID-19.^[14] Researchers want to see how many have antibodies in their blood.

Experts warn that the presence of antibodies alone does not necessarily indicate you are cleared to reenter the world. First of all, there are several types of antibodies. Only a subset known as "neutralizing" antibodies can block the virus.

"We don't know which antibody response neutralizes the virus," said Joseph Vinetz, MD, a professor of infectious diseases at Yale University in New Haven, Connecticut. "Once you have an antibody test, you have to study people with antibody responses for their risk for getting a new infection."

That will not happen, he said, until enough people who test positive for antibodies return to the workforce -- and then either get re-infected or do not. It is a waiting game.

How many serological COVID-19 tests are currently available?

In collaboration with the Biomedical Research and Development Authority, the FDA, the NIH, the US Department of Defense, and the White House Office of Science and Technology Policy, the CDC is evaluating commercially manufactured serological tests.^[7]

In March, the FDA announced a policy that would allow commercial labs meeting certain conditions to market their tests without waiting for the FDA approval known as an "EUA."^[1]

Testing Challenges and Safety Precautions

The magnitude of persons who need testing will pose a significant challenge. With more than 300 million people in the United States, the 12.6 million diagnostic tests performed to date represent just about 4% of the population.^[15]

Unreliable tests may pose another problem. In the United Kingdom,^[16] Spain, and elsewhere, false test results have dashed hopes for a quick, accurate test, and in the United States, the FDA's decision to relax oversight for antibody tests may not be a good idea.

"It really has created a mess that's going to take a while to clean up," Eric Blank, DrPH, senior director at the Association for Public Health Laboratories, told ABC News.^[17] "In the meantime, you've got a lot of companies marketing a lot of stuff and nobody has any idea of how good it is."

Given all the uncertainty surrounding antibody tests, "what we really need is a pill that reduces the amount of time that somebody is ill or infectious for other people, or that might prevent people from getting infected to begin with," Vinetz said.

The Yale School of Medicine, where he works, and Yale New Haven Health System are working on that. They are establishing a drive-thru COVID-19 clinical research platform for patients with mild or asymptomatic cases, which Vinetz hopes will open in the next few weeks. Persons who test positive will be given either an experimental antiviral drug or a placebo, then asked to return for daily swabs for one week to check the amount of the virus in their throats, with weekly monitoring after that.

Vinetz anticipates having preliminary results within 2 weeks of the platform launching.

The FDA cautioned that it has not evaluated all marketed serological tests.^[18] The recommendation to healthcare providers is to continue to use serological (antibody) tests, as appropriate, but to be aware of their limitations. They should be used as an adjunct to determine exposure to SARS-CoV-2 but not to be used as a sole basis to diagnose COVID-19.

Serological (antibody) tests should not be used as the sole basis to diagnose COVID-19 but instead as information about whether a person may have been exposed. Tests authorized by the FDA can be found on the EUA page.^[8]

Currently, the good news with testing is that the cost for most COVID-19 testing is free.^[19] Point-of-care tests, rapid response, at-home tests, and saliva-based molecular tests are currently in development. In April, the FDA issued clearance for use of molecular saliva testing to diagnose coronavirus.^[20] This emergency clearance was an effort to aid testing efforts in the United States. To increase patient access to testing, on April 21, the FDA authorized an at-home collection option of nasal swab specimens for COVID-19.^[21]

Updates on Who Should Be Tested

On April 26, the CDC added 6 new symptoms that may be diagnosed along with other COVID-19 symptoms.^[22]

Symptoms added to the current list are

• Chills
• Repeated shaking with chills
• Muscle pain
• Headache
• Sore throat
• New loss of taste or smell

On May 3, the CDC updated the prioritization of who should be tested for COVID-19 with available tests.^[23] Priorities for testing are as follows:

High Priority

1. Hospitalized patients with symptoms
2. Healthcare facility workers; workers in congregate living settings
3. First responders with symptoms
4. Residents in long-term care facilities or other congregate-living settings, including prisons and shelters, with symptoms

Priority

• Persons with symptoms of potential COVID-19, including: fever, cough, shortness of breath, chills, muscle pain, new loss of taste or smell, vomiting or diarrhea, and/or sore throat
• Persons without symptoms who are prioritized by health departments or clinicians, for any reason, including but not limited to public health monitoring, sentinel surveillance, or screening of other asymptomatic individuals according to state and local plans

Authors cited no conflict of interest.

Study Highlights

• There has been a rising concern regarding patients testing negative with obvious symptoms of COVID-19.
• PCR tests are molecular tests and have been the primary tool for diagnosis by the WHO and the CDC since the onset of the outbreak. These involve the use of swabs inserted into the back of the nasal passage via the nostril to obtain samples to detect COVID-19. PCR testing detects RNA in the virus and is reliable only if the patient is actively infected.
• Serological tests use blood samples to identify antibodies. They analyze the serum component of whole blood and gauge the body’s immune response to foreign pathogens (ie, antigens). Samples for blood testing are obtained through a finger prick or blood draw.
• Serological tests can play an important role in the clinical and public health response to COVID-19.
• Serum antibodies may also play a role in the diagnosis of acute COVID-19, and serological tests may identify candidates for convalescent plasma donation in helping others fight infection.
• The study compared immunoglobulin testing (IgM and IgG) using colloidal gold-based ICG technology with reverse transcriptase polymerase chain reaction from throat swab samples among 105 patients with COVID-19 infection admitted to one hospital in China.
• ICG can be completed within 15 minutes without the need for extensive specialized equipment; however, it provides only qualitative data it cannot determine the amount of circulating antibody.
• 54.3% of patients were female, and the median age of patients was 58 years.
• 76 patients were diagnosed with a positive RT-PCR on their initial test, and results for another 8 patients were initially negative but were positive on subsequent RT-PCR testing; 37 patients were diagnosed with COVID-19 infection because of typical findings of pneumonia without a positive RT-PCR test.
• Blood samples were conducted between February 6 and February 21.
• Both IgM and IgG became detectable at day 4 after the onset of illness.
• Among patients with a positive RT-PCR, the rate of positive IgM testing during the periods from 1 to 7 days after illness onset, 8 to 14 days after illness onset, and more than 15 days after illness onset were 11.1%, 78.6%, and 74.2%, respectively. The respective positive rates for IgG throughout illness were 3.7%, 57.1%, and 96.8%.
• The sensitivity of the ICG assays peaked around day 15 of illness. Combining IgM and IgG would improve the sensitivity of the ICG assay.
• Among cases with positive radiographic findings but a negative RT-PCR test, 23.1% of samples were positive for IgM and 38.5% were positive for IgG.
• As in confirmed cases of COVID-19, IgM and IgG levels peaked at more than 15 days after the onset of illness among patients with a negative RT-PCR result.
• There was good correlation between IgM and IgG detection in both whole blood and plasma.
• Serological (antibody) tests as the sole basis to diagnose COVID-19 but instead as information about whether a person may have been exposed. Tests authorized by the FDA can be found on the EUA page.
• The following 6 new symptoms have been added to the current list: chills, repeated shaking with chills, muscle pain, headache, sore throat, new loss of taste or smell.
• The CDC recommends the highest prioritized testing for the hospitalized patients with symptoms, healthcare facility workers; workers in congregate living settings; first responders with symptoms; and residents in long-term care facilities or other congregate living settings, including prisons and shelters, with symptoms. The CDC recommends priority testing for persons with symptoms of potential COVID-19 infection and persons without symptoms who are prioritized by health departments or clinicians for any reason.

Clinical Implications

• Currently, 2 types of testing is available to detect COVID-19 infection: PCR, which is used in majority of cases and antibody (serological) testing. Neither perfect, they each have pros and cons. In the current study by Pan and colleagues, ICG testing for IgM and IgG against COVID-19 became more sensitive as symptoms continued past 2 weeks. Both IgM and IgG became detectable at day 4 after the onset of illness. ICG testing was more sensitive when patients had a positive RT-PCR test.
• Serological tests can be useful in establishing previous exposure or infection with COVID-19, deciding which persons recovering from infection can return to work, and identifying potential donors of convalescent plasma.
• Implications for the Healthcare Team: The healthcare team should continue to use tests recommended by the CDC and the WHO. Use of serological tests for COVID-19 should be performed with caution and with much research on the accuracy and potential application of the testing method.