Latest Thoughts on SARS-CoV-2 Antibody Tests
Two types of tests are being distributed for diagnosis of COVID19 infection; molecular (PCR) and serology. Molecular testing is used to detect SARS-CoV-2 in respiratory specimens of patients suspected of being infected. Serologic tests detect antibodies in blood that are formed following infection with SARS-CoV-2. Serology provides evidence that a patient has been exposed to the virus but is not helpful in diagnosis of COVID19.
Antibody tests should not be used for diagnosis of acute COVID19 infections. Individuals with symptomatic COVID19 generally do not have detectable antibodies to SARS-CoV-2 within the first 10 days after the onset of symptoms. The majority of hospitalized SARS-CoV-2 infected individuals with confirmed viral RNA have detectable IgG antibodies at 14 days after the onset of symptoms. IgM antibody becomes detectable only 1 to 2 days earlier than IgG. Tests for COVID19 IgM and IgG will miss patients in the early stages of disease when they are still infectious. Even more worrisome is that some patients continue to shed virus after seroconversion. A negative serologic test might give patients a false sense of assurance that they are not infected and cause them to ignore instructions to shelter in place and maintain social distancing, endangering even more people. Another important limitation of serologic tests is that elderly and immunocompromised patients may not develop detectable levels of antibodies after infection.
Antibody assays validated using specimens from hospitalized patients may not perform as well when testing individuals who were asymptomatic or mildly symptomatic. Patients with milder disease and lower viral loads may produce lower antibody titers.
Antibody tests may play a role in detecting unrecognized past infection and immunity however that role needs to be rigorously evaluated. Currently, no one knows how long antibodies to SARS-CoV-2 will persist. Antibody to seasonal coronaviruses that cause common colds starts declining a couple of weeks after infection and some people become susceptible to reinfection within one year. More encouragingly, antibody to SARS-CoV peaks at approximately four months after infection and offers protection for two to three years.
The presence of antibody to SARS-CoV-2 does not ensure immunity. Currently available tests target antibodies to 1 of 2 viral surface proteins; nucleoprotein (N protein) or spike protein (S protein). Several assays use the S1 subunit of the spike protein, which incorporates the binding domain for angiotensin converting enzyme type 2 (ACE2) receptor, because it is highly immunogenic. Antibodies that bind to the spike protein are likely to be neutralizing antibodies because they would prevent virus from invading human cells. In contrast, non-neutralizing antibodies also bind to SARS-CoV-2 but do not prevent it from entering cells. Currently, it is not known which antibody responses, if any, are protective or sustained. Approximately one third of patients with COVID19 who developed antibodies during hospitalization lack antibodies that neutralize virus in plaque growth assays, which is the standard laboratory test for antibody effectiveness. The best way to investigate immunity is to follow people with and without antibody to determine if they become reinfected.
The Infectious Disease Society of America (IDSA) published their guideline on serologic testing for COVID19 on April 20, 2020. IDSA states that antibody testing has not been clinically verified and should not be used as the sole test for diagnostic decisions. Antibody test results should not be used to make staffing decisions or decisions regarding the need for personal protective equipment until more evidence about protective immunity is available.
Potential utility of SARS-CoV-2 serology includes:
- Detection of PCR-negative cases, especially for patients who present late with a very low viral load below the detection limit of RT-PCR assays, or when lower respiratory tract sampling is not possible;
- Identification of convalescent plasma donors;
- Epidemiologic studies of disease prevalence in the community;
- Verification of vaccine response once antibody correlate(s) of protection identified.
To accelerate the availability of serology tests, FDA has permitted companies to develop and distribute serology tests to hospitals and clinics if they have validated their assays with specimens from patients with PCR confirmed infections and notified FDA of their intent. Notification is not the same as obtaining Emergency Use Authorization (EUA), which requires the manufacturer to submit performance data to the FDA for review. Results must be accompanied by a statement that, “This test has not been reviewed by the FDA.”
Since March 2020, more than 90 test developers have notified FDA that they have serological tests available. Most of these are lateral flow assays and a few are enzyme linked immunoassays. Many of these companies claim their tests have high sensitivity and specificity but have not published any data. FDA has warned that some companies have falsely claimed their tests were FDA approved. Many concerns are being expressed worldwide about the reliability of tests that are being rapidly developed and marketed without rigorous oversight. For example, Spain recalled thousands of rapid antibody tests after realizing they were inaccurate.
No universal standard for reporting antibody results is available and test detection limits are variable. Some assays provide semi-quantitative results and others are qualitative rapid point of care tests. Several assays test for both IgG and IgM (total antibody). These tests have unclear value because of the poor specificity of IgM. For some assays both sensitivity and specificity is poor, or undefined. Clinical laboratories need to have time to perform validation studies of these assays.
Potential drawbacks if serological assays are not well-validated include:
- False negative risks if performed early in disease course, especially in mild disease;
- False positive risks, particularly with tests for Immunoglobulin M (IgM) and potential crossreactivity with common cold coronaviruses (e.g. HKU1, NL63, OC43, 229E).
Many of these EUA tests have stated specificity between 96 and 98%. If the prevalence of COVID-19 in the general population is 4.5%, the positive predictive value of this test is only 50%. In other words, the false positive rate is 50%. If disease prevalence is 10%, then a positive antibody test has a 70% chance of being correct. This test has a greater chance of being accurate when testing a high-risk group such as exposed healthcare workers or family members.
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.
FDA recently clarified that, when it grants an Emergency Use Authorization (EUA) for a point-of-care test, that test is deemed to be CLIA-waived. For the duration of the national emergency declaration for COVID-19, such tests can be performed in any patient care setting that operates under a CLIA Certificate of Waiver or Certificate of Compliance/Certificate of Accreditation.
In addition, FDA clarified that tests for SARS-CoV-2 that are offered prior to or without an EUA have not been reviewed by FDA, are not FDA authorized, and have not received a CLIA categorization. Thus, those tests are considered high complexity by default until they receive an EUA or other FDA approval that indicates they may be performed as moderate complexity or waived tests.
References
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