By L.V. Rao PhD, S. Rivello BS, and K. Kuppusamy PhD. Quest Diagnostics, Marlborough, MA

Insights

The COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, has threatened global public health and is associated in particular with respiratory-related morbidity and mortality. This beta coronavirus SARS-CoV-2 is believed to have originated in Wuhan City in China and then spread worldwide. As of May 23, 2020, more than 5.3 million cases have been reported with 350,000 deaths.1

Coronaviruses were first identified in the mid-1960s and named for the crown-like spikes on their surface. They are a large family of single-stranded RNA viruses that can be isolated from different animal species. They are a frequent cause of respiratory infections. There are six major species known to cause human infections besides SARS-CoV-2. These include highly pathogenic SARS-CoV and MERS-CoV along with less virulent but common species HKI1, NL63, OC43, and 229E. These less virulent species are globally distributed. Predominant species may vary by region, year, and season. Infections are mildly asymptomatic and associated with mild to moderate illness in children and adults. They are the second most common cause of the “common cold.” MERS-CoV pathogen illness was first reported in Saudi Arabia in 2012, and only 2 cases were reported in the United States by May 2014. The reported cases of SARS-CoV in 2003 in Asia did not widely spread in the US. Unlike all human coronaviruses that cause mild respiratory symptoms, SARS-CoV, MERS-CoV, and SARS-CoV-2 are associated with severe respiratory diseases.2

Common signs and symptoms of SARS-CoV-2 infection include headache, muscle pain, fever/chills, sore throat, abdominal pain, cough/shortness of breath, loss of smell/taste, diarrhea, etc. Infected people do not always exhibit symptoms and can be asymptomatic throughout the course with viral shedding. Most infected people recover spontaneously. Some progress to viral pneumonia and multiorgan failure. The virus is transmitted through contact with respiratory secretions from an infected person. The median incubation period of COVID-19 is 5.1 days and it is expected that nearly all infected persons will exhibit symptoms within 12 to 14 days of infection.3

 Serology (antibody) assays:
An antibody, also called immunoglobulin, is an adaptive protein produced by the immune system in response to the presence of a foreign substance, called an antigen. Antibodies recognize and latch onto antigens to destroy them and subsequently remove them from the body. The antibodies that are typically produced are grouped into five major classes (ie, isotypes), and each isotype is designated by a letter attached to an abbreviation of the word immunoglobulin: IgG, IgM, IgA, IgD, and IgE.

Serological assays are designed to detect specific antibodies to demonstrate that people have mounted an immune response after being infected with the virus. They can play a critical role in identifying individuals who have mounted an immune response and possibly overcome infection in the past. Current evidence indicates that these antibodies start to generate approximately 6 to 10 days after infection with SARS-CoV-2. There are three isotypes of antibodies:

  • IgM antibodies appear to peak approximately 12 days after SARS-CoV-2 infection. They persist in detectable quantities in different individuals for different lengths of time
  • IgG antibodies have been observed to peak approximately 17 days after SARS-CoV-2 infection and persist for at least 49 days. Further, IgG has been observed in patients two weeks after symptom onset. IgG has been found in patients for up to 2 years, post-recovery from severe acute respiratory distress syndrome CoV (SARS).
  • IgA antibody response appears to begin early and peaks at week 3. It is stronger and more persistent than the IgM response4

What are these antibody tests used for?

  • IgG presence in the blood is an indicator of infection with SARS-CoV-2 and past infection >14 days. In some cases, they may not appear for four weeks post symptom onset if at all. In immunocompromised patients, the response may be delayed or can be below the detection threshold for positivity of the respective test
  • COVID-19 detection in NAAT-negative patients, especially individuals who present 9 to 14 days post symptom onset5
  • Epidemiological studies of disease (tracking the spread of virus)
  • Identification of convalescent plasma donors
  • Potentially verification of vaccine response once antibody correlates of protection are identified

These antibody tests are NOT to be used for:

  • The diagnosis of symptomatic patients or to exclude recent infection
  • Relationship between IgG positivity and subsequent immunity to reactivation or reinfection has NOT yet been established

Currently, there are three major different types of SARS-CoV-2 serological assays. Rapid diagnostic tests (RDTs), enzyme-linked immunosorbent assays (ELISA), and chemiluminescence-based tests have been designed to detect antibodies to specific proteins of the SARS-CoV-2 virus. The majority of the available tests mainly target antibodies to one or two main proteins of the SARS-CoV-2—the nucleocapsid (N) protein and the spike (S) protein. The N protein of coronaviruses is a structural component of the helical nucleocapsid. It has an essential function in viral pathogenesis, replication, and RNA packaging. Antibodies to the N protein are frequently detected in COVID-19 patients, suggesting that the N protein may be one of the immunodominant antigens in the early diagnosis of COVID-19. Several assays focus on the S1 subunit or the S1/S2 portion of the S protein, which is relatively specific to the coronavirus strain.6 The S1 subunit hosts the angiotensin-converting enzyme (ACE)-2 receptor which is thought to be the mechanism by which the SARS-CoV-2 virus enters into human cells.7

Quest Diagnostics currently tests IgG antibodies for the SARS-CoV-2 virus with kits developed by three major global diagnostic companies (see Table 1). Their performance is validated by using both exposed and non-exposed SARS-CoV-2 infected patients. Their test performance has been reviewed by the US FDA and designated for Emergency Use Authorization (EUA).8 Quest Diagnostics also verified their analytical performance per manufacturer claims as per CLIA regulations and College of American Pathologists (CAP) guidelines.

Table 1: Quest Diagnostics FDA EUA-designated serology IgG test performance

*PPV= Positive Predictive Value; **NPV= Negative predictive value (95% Confidence Interval)

Developer/Manufacturer Test Technology Target Protein Sensitivity Specificity PPV* at 5% prevalence NPV** at 5% prevalence
Abbott Architect  

High Throughput CLIA

Nucleocapsid 100%
(95.8%; 100%)
99.6%
(99.0%; 99.9%)
92.9%
(83.4%; 98.1%)
100%
(99.8%; 100%)
Ortho-Clinical Diagnostics VITROS  

High Throughput CLIA

Spike 90%
(92.7%; 100%)
100%
(99.0%; 100%)
100%
(83.0.%; 100%)
99.5%
(99.6%; 100%)
Euroimmun ELISA Spike 90%
(74.4%; 96.5%)
100%
(95.4%; 100%)
100%
(46.0%; 100%)
99.5%
(98.6%; 99.8%)

Expected result: Negative

Interpretation of the test result:

  • Positive: IgG antibody presence may indicate exposure and immune response to COVID-19 SARS-CoV-2 coronavirus
  • Negative: IgG antibody absence does not rule out SARS-CoV-2 infection, particularly in those who have been in recent contact with the virus. Follow-up testing with a molecular diagnostic test may be considered to diagnose or rule out current infection if a consideration. Some individuals, especially immunocompromised subjects, may not develop a detectable positive IgG antibody response after exposure to the CoV-2
    virus, confirmed by a molecular diagnostic test

Limitations of the serology tests:

  • The serological test result should always be interpreted together with clinical symptoms and other results

–     False-negative may occur if performed early in the disease course, especially in mild disease. Some individuals, especially immunocompromised subjects, may not develop a detectable positive IgG antibody response after exposure to the CoV-2 virus, confirmed by a molecular diagnostic test

–     False-positive may occur due to potential cross-reaction with common cold coronaviruses HKI1, NL63, OC43, and 229E. According to national respiratory and enteric viruses surveillance systems (NREVSS) stratified by the US census region, the positivity of these viruses is 2.2%, 1.0%, 0.8%, and 0.6% respectively for OC43, NL-63, 229E, and HKU1.9 Many subsequent studies have shown that there is no cross-reactivity with other coronaviruses

–     Results from antibody testing should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection, or to inform infection status

How long do SARS-CoV-2 antibodies last?
Duration of antibody response to SARS-CoV-2 virus is not known. Based on the studies performed with individuals who recovered from SARS-CoV infection, viral-specific antibodies were maintained for an average of 2 years. A significant reduction of immunoglobulin G–positive percentage and titers occurred in the third year.10

Do the antibodies produced have any neutralizing capabilities to prevent reinfection?
Reinfection with SARS-CoV-2 in individuals who have detectable IgG antibodies is not known. But recent studies on monkeys11,12 and humans13 demonstrated that it is likely that these antibodies can provide protection for some time.

The pathogenicity of SARS-CoV-2 seems to be similar to SARS-CoV in some ways. Studies showed that neutralizing antibodies from a convalescent SARS patient could block the SARS-CoV-2 from entering into target cells in vitro. This implies that there is a potential cross-protective epitope between the two viruses. Thus, the possible immune protection against reinfection with SARS-CoV-2 may share some standard features with convalescent SARS-CoV.14

A small study of rhesus macaques found they do not develop a coronavirus infection the second time they are exposed to a SARS-CoV-2 re-challenge.11 SARS-CoV-2 infection in rhesus macaques induced humoral and cellular immune responses and provided protective efficacy against a SARS-CoV-2 re-challenge.12 A recent research study from Emory University indicated that nearly all people (44) hospitalized with COVID-19 develop virus-neutralizing antibodies within six days of testing positive.13

What is the prevalence of COVID-19 IgG antibody in the Commonwealth of Massachusetts?
Serology testing for SARS-CoV-2 is at increased demand to better quantify the number of cases of COVID-19, including those that may be asymptomatic or have recovered. Per Anthony S. Fauci, MD, NIAID director, these tests provide data regarding the prevalence of a disease in a population by identifying individuals who have developed antibodies to the virus. SARS-CoV-2 IgG antibody prevalence studies provide a more detailed picture of the true magnitude of the COVID-19 pandemic in the US. Antibody prevalence studies can provide better estimates of the extent of COVID-19  in different communities beyond those who are identified by symptomatology alone as many of the infections are very mild, and
can document infections in those who did not access testing while they were sick.15

Commonwealth of Massachusetts:
The SARS-CoV-2 antibody test data for Massachusetts based on Quest Diagnostics test results are broken out by selected counties and displayed in Table 1. Overall, as of May 2020, there was a 7.8% test positivity rate based on 37,096 tests performed. Table 2 displays these data by sex and age categories. Specimens from both males and females and various age groups showed similar positivity rates.

Table 1.  Quest Diagnostics SARS-CoV-2 IgG Antibody Test Results by Massachusetts Counties

 County # of Tests Performed # of Positive Results % Positive Tests
MIDDLESEX 8,757 664 7.6%
NORFOLK 6,133 531 8.7%
PLYMOUTH 5,457 345 6.3%
SUFFOLK 4,072 450 11.1%
WORCESTER 3,239 220 6.8%
BRISTOL 3,038 180 5.9%
ESSEX 2,887 240 8.3%
BARNSTABLE 1,644 110 6.7%
HAMPDEN 1,124 109 9.7%
HAMPSHIRE 379 24 6.3%
BERKSHIRE 162 18 11.1%
FRANKLIN 137 11 8.0%
DUKES 51 <10
NANTUCKET <20 <10
GRAND TOTAL 37,096 2,912 7.8%

 

Table 2.  Quest Diagnostics SARS-CoV-2 IgG Antibody Test Results in Massachusetts Displayed by Sex and Age Groups

State of MA* # of Female # of Male Female % Positive Tests Male % Positive Tests
Age <20 726 647 8% 9%
Age 21-44 7848 6132 8% 9%
Age 45-65 9053 7114 7% 8%
Age >65 3012 2546 8% 7%
GRAND TOTAL 20639 16439 8% 8%
* There are 18 results from specimens where sex was not provided and had a positivity rate of 3%.

State of Connecticut:
The SARS-CoV-2 antibody test data for Connecticut based on Quest Diagnostics test results are broken out by selected counties and displayed in Table 3. Overall, as of May 2020, there was a 10% test positivity rate based on 34,605 tests performed. Table 4 displays these data by sex and age categories. Specimens from both males and females and various age groups showed similar positivity rates.

Table 3.  Quest Diagnostics SARS-CoV-2 IgG Antibody Test Results by Connecticut Counties

County # of Tests Performed # of Positive Results % Positive Tests
FAIRFIELD 12,948 1569 12.1%
HARTFORD 8,086 712 8.8%
NEW HAVEN 7,047 770 10.9%
NEW LONDON 1,908 93 4.9%
LITCHFIELD 1,864 180 9.7%
MIDDLESEX 1,325 82 6.2%
TOLLAND 1,110 72 6.5%
WINDHAM 317 11 3.5%

GRAND TOTAL

34,605 3,491 10.1%

Table 4.  Quest Diagnostics’ SARS-CoV-2 IgG Antibody Test Results in Connecticut Displayed by Sex and Age Groups

State of CT * # of Female tests # of Male tests Female % Positive Tests Male % Positive tests
Age  <20 774 696 11% 12%
Age 21-44 6617 4426 11% 11%
Age 45-65 9003 6674 10% 10%
Age >65 3342 2920 8% 9%
GRAND TOTAL 19736 14716 10% 10%
* There are 153 results from samples where sex was not provided. Those samples had a positivity rate of 14%

According to new interim guidelines5 published by the CDC on May 23, 2020, SARS-CoV-2 serologic assays that have Emergency Use Authorization (EUA) are preferred for public health or clinical use since their test performance data have been reviewed by the FDA. Serologic test results should be interpreted in the context of the expected positive and negative predictive values in the population being tested. Serologic testing can be offered as a method to support diagnosis of acute COVID-19 illness for persons who present 9 to 14 days after illness onset. In those patients, serologic testing can be offered in addition to direct detection methods such as molecular tests. This will maximize clinical diagnostic sensitivity as the quantity of nucleic acid is likely decreasing in a respiratory specimen and antibody levels are likely increasing during this time period. Serologic testing should be offered as a method to help establish a diagnosis when patients present with late complications of COVID-19 illness, such as multisystem inflammatory syndrome in children.

References

  1. John Hopkins University School of Medicine. COVID-19 dashboard by the Center for Systems Science and Engineering (CSSE) at John Hopkins University. Last updated June 11, 2020. Accessed June 11, 2020. https://coronavirus.jhu.edu/map.html
  2. Centers for Disease Control and Prevention. SARS basics fact sheet. Last reviewed December 6, 2017. Accessed June 11, 2020. https://www.cdc.gov/sars/about/fs-sars.html
  3. Lauer SA, Grantz KH, Bi Q, Jones FK, Zheng Q, Meredith HR, et al. The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application. Ann Intern Med. 2020;172:577-582. doi:10.7326/M20-0504
  4. Padoan A, Sciacovelli L, Basso D, et al. IgA-Ab response to spike glycoprotein of SARS-CoV-2 in patients with COVID-19: a longitudinal study. Clin Chim Acta. 2020; 507:164‐ doi:10.1016/j.cca.2020.04.026
  5. Centers for Disease Control and Prevention. Interim guidelines for COVID-19 antibody testing in clinical and public health settings. Last reviewed May 23, 2020. Accessed June 11, 2020. https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines.html
  6. Gronvall G, Connell N, Kobokovich A, et al. Developing a national strategy for serology (antibody testing) in the United States. The Johns Hopkins Center for Health Security. Published April 22, 2020. Accessed June 11, 2020. https://www.centerforhealthsecurity.org/our-work/pubs_archive/pubs-pdfs/2020/200422-national-strategy-serology.pdf
  7. Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol. 2019:17:181-192. doi: 10.1038/s41579-018-0118-9
  8. US Food and Drug Administration. EUA authorized serology test performance. Last reviewed June 9, 2020. Accessed June 11, 2020. https://www.fda.gov/medical-devices/emergency-situations-medical-devices/eua-authorized-serology-test-performance
  9. Killerby ME, Biggs HM, Haynes A, et al. Human coronavirus circulation in the United States 2014-2017. J Clin Virol. 2018;101:52‐ doi:10.1016/j.jcv.2018.01.019
  10. Wu LP, Wang NC, Chang YH, et al. Duration of antibody responses after severe acute respiratory syndrome. Emerg Infect Dis. 2007;13(10):1562‐ doi:10.3201/eid1310.070576
  11. Bao L, Deng W, Gao H, et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. 2020. bioRxiv. doi:10.1101/2020.03.13.990226
  12. Chandrashekar A, Liu J, Martinot AJ, et al. SARS-CoV-2 infection protects against re-challenge in rhesus macaques. Science. 2020;eabc4776. doi: 10.1126/science.abc4776
  13. Suthar MS, Zimmerman MG, Kauffman RC, et al. Rapid generation of neutralizing antibody responses in COVID-19 patients. medRxiv. doi: 1101/2020.05.03.20084442
  14. Lin Q, Zhu L, Ni Z, Meng H, You L. Duration of serum neutralizing antibodies for SARS-CoV-2: lessons from SARS-CoV infection. J Microbiol Immunol Infect. 2020;S1684-1182(20)30075-X. doi:10.1016/j.jmii.2020.03.015
  15. National Institutes of Health. NIH begins study to quantify undetected cases of coronavirus infection. Published April 10, 2020. Accessed June 11, 2020. https://www.nih.gov/news-events/news-releases/nih-begins-study-quantify-undetected-cases-coronavirus-infection