• Summary
  • Specimen Collection
  • How is SARS-CoV-2 RNA Testing Performed?
  • Diagnostic Testing
  • Screening Testing
  • How Early Will a Test Be Positive?
  • Performance of RT-PCR Viral Tests
• Antibody Testing
  • General CDC Antibody Guidance
  • What Are the Different Types of Antibody Tests?
  • When Can Antibody Testing be Helpful?
  • Vaccination and Test Interpretation
• Learn More – Primary Sources

NOTE: Information and guidelines may change rapidly. Check in with listed references in ‘Learn More – Primary Sources’ to best keep up to date.

SUMMARY:

The CDC has provided guidance on both viral testing for SARS-CoV-2 as well as the role of antibody testing. Testing for the presence of the virus during the pandemic remains the current diagnostic standard. While antibody testing can play a role for public health teams to understand the spread of the disease, currently its use as a diagnostic test for individuals remains limited. A COVID-19 vaccine will not affect the results of SARS-CoV-2 viral tests.

Viral Testing

Specimen Collection

• Obtain an upper respiratory specimen for initial diagnostic testing
  • A nasopharyngeal (NP) specimen collected by a healthcare professional  or
  • An oropharyngeal (OP) specimen collected by a healthcare professional  or
  • A nasal mid-turbinate swab collected by a healthcare professional or by a supervised onsite self-collection (using a flocked tapered swab)  or
  • An anterior nares (nasal swab) specimen collected by a healthcare professional or by onsite or home self-collection (using a flocked or spun polyester swab)  or
  • Nasopharyngeal wash/aspirate or nasal wash/aspirate (NW) specimen collected by a healthcare professional
• Lower respiratory tract specimens
  • Collect and test sputum in patients who develop a productive cough | Induction of sputum is not recommended
  • Under certain clinical circumstances (e.g., those receiving invasive mechanical ventilation), a lower respiratory tract aspirate or bronchoalveolar lavage sample should be collected and tested as a lower respiratory tract specimen

How is SARS-CoV-2 RNA Testing Performed?

RT-PCR

• Usually performed using real-time reverse transcription polymerase chain reaction (RT-PCR)
  • Qualitative detection of RNA
• Multiple tests on the market that can target various genes
  • Envelope (env) | Nucleocapsid (N) | Spike (S) | RNA-dependent RNA polymerase (RdRp) | ORF1
• A positive test can only determine presence of SARS-CoV-2 RNA and not whether the virus is intact and capable of infecting others

Antigen

• Antigen tests can quickly detect fragments of proteins found on or within the virus by testing samples collected from the nasal cavity using swabs
• The benefit of antigen testing is speed, with results potentially available within minutes
• However, antigen tests, while very specific for the virus, are not as sensitive as molecular PCR tests
  • Positive antigen results: Highly accurate but higher chance of false negatives | Negative antigen results may still need PCR confirmation prior to treatment decisions or to prevent inadvertent spread of SARS-CoV-2

Note: Prior receipt of a COVID-19 vaccine should not affect the results of SARS-CoV-2 viral tests (NAAT or antigen)

Breath Sample Analysis

• FDA has issued an emergency use authorization (EUA) for a diagnostic test that detects chemical compounds in breath samples associated with a SARS-CoV-2 infection
• Test is performed by a qualified, trained operator under the supervision of a health care provider licensed or authorized by state law to prescribe tests
• Results available in <3 minutes

Diagnostic Testing

Signs or Symptoms of COVID-19

• Positive test
  • NAAT: Indicates infection regardless of vaccine status
  • Positive antigen test result may need confirmatory testing if the person has a low likelihood of SARS-CoV-2 infection (e.g., no known exposure to a person with COVID-19 within the last 14 days or is fully vaccinated or has had a SARS-CoV-2 infection in the last 3 months)
  • Isolate if positive test: Discontinue isolation 5 days after symptom onset and at least 24 hours after the resolution of any fever (without the use of fever-reducing medications) | Continue to wear mask around others for 5 additional days
    • Some individuals may require extended isolation and precautions (e.g., severely immunocompromised)
    • Testing is not recommended to determine when infection has resolved
    • Loss of taste and smell may persist for weeks or months after recovery and need not delay the end of isolation​
• Negative test
  • If symptoms are consistent with COVID-19, may be a false negative | Isolation and further discussion with healthcare professional recommended

Testing to determine resolution of infection

• May be appropriate for severe illness or immunocompromise
• “For all others, a test-based strategy is no longer recommended except to discontinue isolation or precautions earlier than would occur under the symptom-based strategy”

Screening Testing

No Symptoms and No Close Contact with Someone Known to Have a COVID-19 Infection

• Asymptomatic or presymptomatic infection contribute to community SARS-CoV-2 transmission
  • May help with re-opening of businesses, communities, and schools
• Point-of-care tests (e.g., antigen tests) can be particularly helpful due to short turn-around times
• Quarantine not required while results are pending
• Examples of screening programs
  • Testing employees in a workplace setting
  • Testing students, faculty, and staff in a school or university setting
  • Testing a person before or after travel

How Early Will a Test Be Positive and How Long Until Negative?

• In patient with COVID-19 infection who tested positive using a nasopharyngeal swab
  • Earliest detection: Day 1 of symptoms
  • Peak levels highest within week 1 and therefore probability of detection will be highest during that time
  • Viral load declines by week 3 and therefore virus more likely to be undetectable in to week 4
  • Infection severity: More virus may be present in patients with severe disease and therefore it may take longer to obtain a negative test result vs someone with a mild COVID-19 infection

Performance of RT-PCR Viral Tests

• RT-PCR specificities are close to 100% because they target specific RNA sequences of the SARS-CoV-2 virus
• False negative results may be due to
  • Inappropriate timing of collection vs symptom onset
  • Poor sampling technique (need to sample at the back of the nose)
• False positive results may occur due to lab error or contamination
• However, even with good analytic performance, PPV and NPV are related to prevalence and therefore can differ between geographic regions
  • In a setting with high COVID-19 prevalence, a negative test does not necessarily rule out the possibility that an individual is infected with SARS-CoV-2

Antibody Testing

General CDC Antibody Guidance

• According to the CDC

Antibody testing does not replace virologic testing and should not be used to establish the presence or absence of acute SARS-CoV-2 infection

Antibody testing is not currently recommended to assess for immunity to SARS-CoV-2 following COVID-19 vaccination, to assess the need for vaccination in an unvaccinated person, or to determine the need to quarantine after a close contact with someone who has COVID-19

Some antibody tests will not detect the antibodies generated by COVID-19 vaccines

Because these vaccines induce antibodies to specific viral protein targets, post-vaccination antibody test results will be negative in persons without history of previous infection, if the test used does not detect antibodies induced by the vaccine

• In general, antibodies will be detectable 7 to 14 days after illness onset and will be present in most people by 3 weeks
  • Infectiousness likely decreased by that time
  • Evidence suggests some degree of immunity will have developed
• IgM and IgG can appear together, usually within 1 to 3 weeks
  • IgG antibodies appear to persist for at least several months
  • Some individuals may be infected but will not develop antibodies
• Neutralizing antibodies can also be identified and are associated with immunity
• FDA requires companies providing antibody testing to obtain an EUA

What Are the Different Types of Antibody Tests?

• Antigenic Targets
  • Spike glycoprotein (S): Present on viral surface and facilitates virus entry
  • Nucleocapsid phosphoprotein (N): Immunodominant and interacts with RNA
  • Protein targeting is important to reduce cross-reactivity (cause of false positives which may occur with other coronaviruses like the common cold) and improve specificity
• Types of Antibody Testing
  • Binding antibody detection that use purified SARS-CoV-2 (not live virus)
    • Point-of-care (POC) tests
    • Laboratory tests that usually require skilled personnel and specialized equipment
  • Neutralizing antibody detection (none currently FDA authorized)
    • Serum or plasma is incubated with live virus followed by infection and incubation of cells
    • Can take up to 5 days to complete the study

When Can Antibody Testing be Helpful?

Antibody testing may be helpful in the following situations

• Seroconversion: In a patient who did not receive a positive viral test
  • A positive antibody test at least 7 days following acute illness onset but a previous negative antibody test may indicate new onset SARS-CoV-2 infection
• To support a diagnosis in the presence of a complex clinical situation, such as patients who present with COVID-19 complications (e.g., multisystem inflammatory syndrome and other post-acute sequelae of COVID-19)
  • Note: Due to antibody persistence, a single positive antibody test result may reflect previous SARS-CoV-2 infection and not a recent illness
• Clinical, occupational health, and public health purposes, such as serologic surveys

Vaccination and Test Interpretation

• In a person never vaccinated
  • testing positive for antibody against either N, S, or RBD indicates prior natural infection
• In a vaccinated person
  • Testing positive for antibody against the vaccine antigen target, such as the S protein, and negative for other antigen: Suggests vaccine-induced antibody and not SARS-CoV-2 infection
  • Testing positive for any antibody other than the vaccine-induced antibody, such as the N protein: Indicates resolving or resolved SARS-CoV-2 infection that could have occurred before or after vaccination
• The CDC states that

SARS-CoV-2 antibodies, particularly IgG antibodies, might persist for months and possibly years

Therefore, when antibody tests are used to support diagnosis of recent COVID-19, a single positive antibody test result could reflect previous SARS-CoV-2 infection or vaccination rather than the most recent illness

Learn More – Primary Sources:

CDC: Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19)

Interim Guidelines for COVID-19 Antibody Testing in Clinical and Public Health Settings

CDC: Overview of Testing for SARS-CoV-2

CDC: Interim Public Health Recommendations for Fully Vaccinated People

Interpreting SARS-CoV-2 Test Results

The Promise and Peril of Antibody Testing for COVID-19

EUA Authorized Serology Test Performance

CDC Updates and Shortens Recommended Isolation and Quarantine Period for General Population

CDC: Quarantine and Isolation

Coronavirus (COVID-19) Update: FDA Authorizes First COVID-19 Diagnostic Test Using Breath Samples

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