Two types of COVID-19 tests

There are two types of tests being conducted in addressing the COVID-19 disease pandemic. The more commonly administered test is a qPCR assay designed to detect genomic material (RNA in this case) from the SARS-CoV-2 virus that causes the disease. The sample for this test is mucus and cells collected with a nasal swab inserted far back up a subject’s nose. A positive result indicates an active infection. Current recommendations from public health authorities are to quarantine the infected person for 14 days, after which period they are presumed to be incapable of spreading the virus to others. The other type of test that is important during the pandemic is a serology test to detect anti‑SARS‑CoV-2 antibodies in a subject’s blood. If present, these antibodies would have formed during an active infection sometime in the past from which the subject has recovered. The sample in this case is blood either from a finger prick or drawn from an arm vein. A positive on this test means that the person does not currently have the virus and is presumably no longer contagious, but had the disease and has now recovered. Because of the large percentage of asymptomatic cases of COVID-19, a positive test result can come as a bit of a shock, as the subject may not have been aware that they had been infected and therefore would not even know when they had been experiencing an active infection.

MarinBio offers a high degree of expertise in designing, constructing, and validating molecular biology assays such as qPCR or other advanced techniques for accurate and quantitative detection of viral nucleic acids in samples. In the case of the RNA genome of SARS‑CoV‑2, primers can be designed against genes coding the virus’ RNA polymerase, envelope, or neocapsid proteins, either individually or in some combination, depending on the requirements of the assay.

Problems from combining qPCR (measuring virus) and serology (measuring antibody) test results

Obviously, these two tests provide quite different kinds of information, but both are important. This blog post concerns only the antibody test. Although some state and even federal regulatory agencies have apparently sometimes combined results from these two types of tests to provide data on the spread of the virus, this is not correct. The number of positive qPCR test results to date at any given time is the sum of the number of current, active infections plus the number of infections from the past from which persons have since recovered or, in some cases, died. Subtracting the number of active cases and the number of deaths from the total number of positive qPCR results gives a number representing people in the population who were diagnosed with the disease and have recovered. Adding positive antibody test results, i.e., recovered persons, to positive qPCR results gives an inaccurately inflated total, because there will be some overlap in the two sets of positive results. At the same time, the high rate of asymptomatic cases observed among persons with positive qPCR test results hints that many more cases have occurred than the total number of positive qPCR test results to date would indicate.

The importance of testing for SARS-CoV-2 antibodies

Therefore, the main importance of SARS-CoV-2 antibody testing during the current pandemic is that it can provide not only information to individual test subjects but can also help epidemiologists get a better view of how much the disease has spread in given populations. For individuals, a positive result in particular may mean that they don’t have to worry about contracting the disease in at least the near term, as the antibodies should allow them to fight off reinfection. For how long is still unclear, but most authorities assume there will be some period of immunity conferred by contracting the disease. Let’s hope so, otherwise it is unlikely that a vaccine can do a better job of providing immunity than actually getting COVID-19, and in such a scenario human society would be met with the unwelcome task of achieving herd immunity while struggling to minimize excess serious illnesses and deaths.

False negatives and false positives in COVID-19 antibody testing

The situation with antibody testing has been anything but clear cut to this point. Perhaps the biggest issue has been with the quality of the tests themselves, of whether their sensitivity and specificity are sufficient to prevent false negatives and false positives. A person receiving a false negative result continues to worry that they might contract the disease and need to exercise care not to get infected even though they’ve already had it. A  false positive is worse, as that person will assume that they have already had the disease and likely cannot be infected. If they are subsequently infected due to relaxing personal preventative practices against contracting COVID-19, this can have consequences not only for their own health but in spreading the disease to others. There are numerous SARS-CoV-2 antibody tests available through various sources, yet many of these seem to have unfortunate (and variable) levels of both false negatives and false positives, with frequencies that are still not well understood. But why is this so?

Relaxing FDA regulations: Emergency Use Authorization (EUA)

Diagnostic tests are considered medical devices by regulatory authorities and must normally pass extremely rigorous evaluation by the FDA before they are approved for use on human subjects. Even receiving 510(k) premarket clearance demonstrating that a Class II “device” such as an ELISA test is “substantially equivalent” to existing products and is therefore safe to market is no slam dunk. As a result, antibody tests employing tried and true technology like ELISA that have been subjected to vetting by the FDA are typically quite accurate. However, after initially holding the line on their customary approval protocols, the FDA decided in March 2020 to relax normal regulatory restrictions in the face of overwhelming demand for both qPCR and antibody tests during the ongoing crisis.

The FDA process by which such a relaxation occurs is called an Emergency Use Authorization (EUA). Tests for which an EUA application has been submitted can be marketed during its review, and if such authorization is granted, the normal Clinical Laboratory Improvement Amendments (CLIA) standards for the test are waived. The original announcement on easing regulations for release of COVID-19 tests for sale in the US only required companies to notify the FDA that they intended to sell their tests in the US, to self-certify the tests worked, and to assert that they would apply for an EUA. As a result, more than 200 antibody tests from manufacturers and distributors (over half from China) became available in a matter of weeks. However, as complaints have surfaced regarding the quality of some of these antibody tests, the FDA issued modified rules on May 4 mandating that vendors apply for an EUA for their test within 10 days of notifying the FDA of their intent to sell it. The FDA also set minimum standards of an overall sensitivity of 90% and overall specificity of 95% for these tests. Then in late May the FDA followed up on these restrictions by removing more than 30 tests from their list of commercially available COVID‑19 serology kits, saying these “should not be distributed” for sale. On the other hand, as of June 1, 15 antibody tests have received approval for their EUA applications.

MarinBio scientists have decades of experience in the development of ELISAs and other immunological assays. MarinBio can design and deliver antibody tests with low rates of false negatives and false positives that not only meet but exceed current FDA COVID-19 standards for overall specificity (≥95%) and sensitivity (≥90%). Contact MarinBio today if you are planning a project involving ELISA testing, and not just for SARS-CoV-2 antibodies but for any antigen of interest. Multiplex ELISAs for measuring levels of multiple antigens in a single assay are also a specialty of MarinBio.

The need for randomized, representative testing for antibodies against SARS-CoV-2

Large-scale, randomized antibody testing administered systematically to various populations in the US could provide solid estimates of the actual spread of the virus. Epidemiologists are expert at designing such large-scale clinical tests. Better estimates of past prevalence would also afford a much better idea of the actual lethality rate of the disease across the various age cohorts and for the different comorbidities that hold increased risk for developing serious symptoms or even dying. Such information is crucial for public health policy makers to how to best protect at-risk groups and to know what type of directives to recommend regarding public behavior during the pandemic. The National Institutes of Health (NIH) announced April 10 that enrollment would begin on a 10,000 person “sero-survey” involving use of at-home serology tests performed on volunteers with results reported to federal public health authorities on a rolling basis. While extremely useful and far better than nothing, relying on volunteers as in a typical biopharma clinical trial does not provide a high level of randomization for measuring progress of a pandemic in the US population.

Large-scale antibody testing studies conducted to date

Attempts have been made by various groups to conduct randomized, representative testing for SARS-CoV-2 antibodies in individual urban areas of the US with somewhat divergent results. The University of Miami and Miami-Dade County completed a round of antibody testing on April 24 that did random sampling weighted across the county. Of 1,800 individuals tested, 6% were positive for SARS-CoV-2 antibodies. At the time of the survey there had been 10,000 positive qPCR tests reported in a county of 2.7 million people; 6% translates to 165,000 residents, or 16.5 times higher than qPCR results would indicate. Similar data was released from researchers at the University of Southern California and the Los Angeles County Department of Public Health. Based on results from testing 863 people for SARS-CoV-2 antibodies, researchers estimated that between 2.5% and 4.2% of L.A. County’s over 10 million people had been previously infected. The low end of the range translates to 260,000 people, 20-fold higher than the 13,000 qPCR positive results that had been reported by the date of the survey.

Researchers at Stanford University tested 3,330 residents of Santa Clara County for antibodies to SARS‑CoV-2 in early April. Results showed a raw antibody prevalence of 1.5% that was then adjusted for participants’ zip codes, gender, and ethnicity. Following criticism that their initial weighting was too aggressive, the Stanford researchers revised their initial estimates downward to a prevalence of 2.8%. In a county of 1.9 million that had only reported 1000 infections at the time of the antibody testing, this translates to an underreporting of infections by a factor of 54. Meanwhile, SARS-CoV-2 antibody testing done on 1,300 people in New York City in late April by state health officials there showed 21% testing positive. With a population of 8.4 million this would translate to 1.7 million cases having occurred in the city, which would be approximately seven times higher than the 250,000 cases that had been reported there at the time of the antibody study.

There are currently no widely agreed-upon explanations for the divergences observed in the results of the studies, and various criticisms have been made of their design and of the particular antibody tests used. However, it seems certain that there have been many times more COVID-19 infections than qPCR test results would indicate, doubtless due to the high numbers of asymptomatic cases.

Clearly there is a continuing need for the development of highly accurate, specific, and sensitive antibody ELISA assays for determining whether a subject has or has not experienced a COVID-19 infection. MarinBio is staffed by scientists who are well-versed in all aspects of designing and conducting immunological assays, and who are familiar with the many technical details that must be considered in generating high levels of specificity and sensitivity. If you are developing or even considering developing such a test, MarinBio stands ready to assist in your projects. With a strong track record of successful collaborations with biopharma companies, MarinBio is an ideal partner. Contact MarinBio at 415-883-8000 www.marinbio.com.