Frequently Asked Questions About COVID-19 Testing for Providers & Clients

• Ordering, Reports, and Turnaround Time
• What SARS-CoV-2 (COVID-19) tests do you offer?
• I am not a UW provider. Will you accept specimens from my hospital?
• What is the turnaround time for RT-PCR testing?
• I am at an outside location. Does it matter what carrier (e.g. FedEx, UPS) I send my samples by?
• Who is eligible for testing?
• Sampling for RT-PCR testing
• What specimen types do you accept?
• What swab types do you accept?
• I have ordered multiple respiratory tests. How many swabs do I send?
• What is the rate of positivity for sampling with nasopharyngeal vs. oropharyngeal swabs? One swab only vs. two swabs? NP swab vs. OP vs. sputum vs. BAL?
• RT-PCR Assay Performance
• How sensitive is the SARS-CoV-2 RT-PCR test?
• How specific is the COVID RT-PCR test? Does it cross-react with the “coronavirus” test on an extended respiratory panel?
• What is the positive and negative predictive value?
• What does an “inconclusive” result mean?
• Is confirmatory testing performed for inconclusive results?
• What gene targets are used in each assay?
• Serological (antibody) testing
• What are the indications for serological testing for SARS-CoV-2 (COVID-19)
• What COVID-19 serology tests does UW offer?
• What are the target antigens used in the Abbott immunoassays?
• How are the results reported for the anti-nucleocapsid antibody test, and what is the clinical significance?
• How are the results reported for the anti-spike antibody test, and what is the clinical significance?
• What are the performance characteristics of the anti-nucleocapsid antibody test?
• What are the performance characteristics of the anti-spike antibody test?
• What are the limitations of these antibody tests?
• What is the turnaround time?
• My patient has a positive serology result, and is interested in being a potential plasma donor. Where can I refer this patient for more information?
• Clinical Performance and Considerations
• What is the rate of co-infection with COVID-19 and other respiratory viruses?
• Does UW Virology publish information about testing volumes or rates of positivity?

Ordering, Reports, and Turnaround Time

What SARS-CoV-2 (COVID-19) tests do you offer?

We currently offer RT-PCR for detection of SARS-CoV-2 SARS-CoV-2 (COVID-19) Qualitative PCR [NCVQLT], an anti-nucleocapsid IgG antibody test as an indication of prior infection with SARS-CoV-2 SARS CoV 2 Nucleocapsid Antibody, IgG [NCVIGG], and a semi-quantitative anti-spike IgG antibody test as an indication of vaccination status or prior infection with SARS-CoV-2 SARS CoV 2 Spike Antibody, IgG [NCVIGQ].

I am not a UW provider. Will you accept specimens from my hospital?

Yes.

What is the turnaround time for RT-PCR testing?

The turnaround time depends on the testing situation and order received. Our median turnaround times have been approximately 10-15 hours once the sample is received.

I am at an outside location. Does it matter what carrier (e.g. FedEx, UPS) I send my samples by?

It does not matter as long as appropriate specimen handling conditions are met.

Who is eligible for testing?

• Test orders must be medically necessary and accompanied by physician orders.
• UW Medicine phlebotomists only draw blood from patients with a UW Medicine-related requisition and provider.

Sampling for RT-PCR testing

What specimen types do you accept?

Please see the “Ordering & Collection” section of SARS-CoV-2 (COVID-19) Qualitative PCR [NCVQLT] or SARS CoV 2 Nucleocapsid Antibody, IgG [NCVIGG] for up to date information.

What swab types do you accept?

We accept nasal swabs, mid-turbinate swabs, and nasopharyngeal swabs, whether flocked or spun nylon, collected into PBS or VTM/UTM. Our preferred specimen type is a flocked nasopharyngeal swab in 3mL of PBS/VTM/UTM.

I have ordered multiple respiratory tests. How many swabs do I send?

In addition to SARS-CoV-2, UW Laboratory Medicine offers Rapid Flu/RSV testing and an extended respiratory virus panel. If multiple tests are ordered:

• SARS-CoV-2 and Rapid Flu/RSV: One swab. The sample will first go to Microbiology, an aliquot is taken, and then the sample will be sent to Virology. Expect a 30 min delay in SARS-CoV-2 turnaround time for this extra step. Use code FABRMC for inpatient testing at UWMC. Virology would prefer to limit their own Flu/RSV testing.
• SARS-CoV-2 and Respiratory panel: Separate swabs highly preferred; with test volume so high, this is surest way to make sure nothing gets missed and has the fastest TAT. It is technically possible to do both tests from same swab or add on a test to a post-nucleic acid extraction if necessary.
• SARS-CoV-2, Rapid Flu/RSV, and Respiratory Panel: Either two swabs (One for SARS-CoV-2 and Rapid Flu/RSV, one for respiratory panel) or 3 swabs if the patient can tolerate it.

What is the rate of positivity for sampling with nasopharyngeal vs. oropharyngeal swabs? One swab only vs. two swabs? NP swab vs. OP vs. sputum vs. BAL?

The clinical sensitivity depends on whether an individual is symptomatic or asymptomatic and the viral load present at that time. Nasopharyngeal swabs have the highest sensitivity. A study of repeat sampling in March/April, suggested that false negatives are rare among symptomatic patients (<3%). Nasopharyngeal swabs are preferred, though we accept nasal swabs and midturbinate swabs; patients with a high clinical index of suspicion and a negative NP or OP swab result may benefit from sampling of the lower respiratory tract or repeat sampling.

RT-PCR Assay Performance

How sensitive is the SARS-CoV-2 RT-PCR test?

The UW laboratory developed test (LDT) using the CDC kit has been shown to be at least as sensitive as any other test that to which it has been compared. Side-by-side comparison with the Washington State Public Health Lab showed 100% concordance in validation. The test is highly sensitive in an analytic sense; if viral RNA is present in the sample, it is very likely to be detected. However, either because of sampling error or the biology of the disease (e.g. virus present in lower but not upper respiratory tract), there have been known cases of patients with negative RT-PCR results who later were RT-PCR positive.

We also offer Roche cobas, Hologic Panther/Panther Fusion, and Abbott alinitym testing. These tests have similar analytical sensitivities and perform well. Of note, the Hologic Panther test does not offer Ct values.

How specific is the COVID RT-PCR test? Does it cross-react with the “coronavirus” test on an extended respiratory panel?

All of the offered assays are highly specific for the SARS-CoV-2 virus, with no cross-reactivity to either other human coronaviruses or to other human respiratory pathogens. The coronavirus assay on our extended respiratory panel does not detect SARS-CoV-2.

What is the positive and negative predictive value?

Because the test is highly specific, positive results have a very high positive predictive value and should be treated as true cases of COVID-19 infection. Negative results must be interpreted within the patient’s context and should not be taken as the sole determining factor in ruling out the disease. Patients with a high index of clinical suspicion may require additional testing.

What does an “inconclusive” result mean?

The UW SARS-CoV-2 Real-time RT-PCR assay targets two distinct gene regions (see SARS-CoV-2 (COVID-19) Qualitative PCR [NCVQLT] for details). When one of the two targets, but not both, is present above the threshold for positivity, the test is reported as “inconclusive”. This is usually seen with low amounts of viral DNA. In practice, “inconclusive” results should be treated as presumptive positive COVID cases with a low viral load present.

Is confirmatory testing performed for inconclusive results?

No.

What gene targets are used in each assay?

The UW SARS-CoV-2 Real-time RT-PCR assay targets two distinct regions within the N gene of SARS-CoV-2. Amplification of both targets results in a presumptive positive (detected) test result, while amplification of one of two targets results in an inconclusive result, and amplification of neither target results a negative (not detected) test result. Samples with inconclusive test results should be considered as positives; due to capacity constraints follow-up testing is currently not available.

The Hologic Emergency Use Authorization (EUA) SARS-CoV-2 assays (Fusion and Aptima) target two conserved regions of the of SARS-CoV-2 (the causative agent for COVID-19) ORF1ab gene. The two regions are not differentiated; amplification of either or both regions is a presumptive positive (detected) test result and amplification of neither target results a negative (not detected) test result.

The Roche cobas Emergency Use Authorization (EUA) SARS-CoV-2 Real-time RT-PCR assay targets the E gene and ORF1ab genes. Amplification of both targets results in a presumptive positive (detected) test result, while amplification of one of two targets results in an inconclusive result, and amplification of neither target results a negative (not detected) test result. Samples with inconclusive test results should be considered as positives; due to capacity constraints follow-up testing is currently not available.

The Abbott Alinity m Emergency Use Authorization (EUA) SARS-CoV-2 Real-time RT-PCR assay targets the RdRp and N genes. Amplification of either or both genes is a positive (detected) test result and amplification of neither target results a negative (not detected) test result.

Serological (antibody) testing

What are the indications for serological testing for SARS-CoV-2 (COVID-19) SARS CoV 2 Nucleocapsid Antibody, IgG [NCVIGG]?

Indications for serological testing include:

• Testing patients who may have had COVID-19 or exposure to SARS-CoV-2 more than 10 days ago.
• As part of a potential “return to work” algorithm.
• Determination of prior vaccination.
• Serological testing is NOT indicated for diagnosis of acute infection.

What COVID-19 serology tests does UW offer?

UW Virology is performing the nucleocapsid antibody test on the Abbott SARS-CoV-2 IgG immunoassay and the spike antibody test on the Abbott AdviseDx SARS-CoV-2 IgG II immunoassay. Both tests are performed on the ARCHITECT instrument. These are chemiluminescent microparticle immunoassays (CMIA) used for the detection of IgG antibodies to either SARS-CoV-2 nucleocapsid or spike protein in human serum and plasma.

What are the target antigens used in the Abbott immunoassays?

The Abbott SARS-CoV-2 IgG immunoassay detects antibodies to the viral nucleocapsid protein (NP). The Abbott AdviseDx SARS-CoV-2 IgG II immunoassay detects antibodies to the viral spike protein (S).

How are the results reported for the anti-nucleocapsid antibody test, and what is the clinical significance?

• The results for the nucleocapsid are either "reactive" (positive) or "nonreactive" (negative) based on the manufacturer-indicated cutoff.
• A negative result indicates that either a person has not been infected with SARS-CoV-2 or there is not a detectable level of antibody present. Explanations for this may include a very recent exposure such that not enough time has elapsed to generate an immune response, or the immune response has decreased below the detectable level. A negative result does not rule out current or past infection with SARS-CoV-2.
• A positive result indicates previous or current infection. Recent studies examining serial plasma samples in hospitalized patients with SARS-CoV-2 infection suggest that the median time to seroconversion is about 10 days in moderately ill patients, and 14 days in severely ill patients. It is important to note that a positive serology test cannot distinguish between active or past COVID-19. If there is concern for active infection, molecular testing (PCR) with a nasopharyngeal swab is recommended.
• At this time, it is not known the degree to which the presence of antibodies confers protection from reinfection with SARS-CoV-2 or exactly how long the antibody response will last.

How are the results reported for the anti-spike antibody test, and what is the clinical significance?

• The results for the spike antibody assay are "reactive" (positive) or "nonreactive" (negative) based on the manufacturer-indicated cutoff. In addition there is a semi-quantitative value providing a relative concentration of antibody in the sample.
• A negative result indicates that either a person has not been infected with SARS-CoV-2, nor vaccinated, or there is not a detectable level of antibody present. Explanations for this may include a very recent exposure such that not enough time has elapsed to generate an immune response, or the immune response has decreased below the detectable level. A negative result does not rule out current or past infection with SARS-CoV-2.
• A positive result indicates previous or current infection or vaccination. Table 3 in this pre-print shows the values of anti-spike IgG antibodies seen by week of vaccination in an otherwise healthy cohort and may aid in interpretation of this test. The result from this test should not be interpreted as an indication or degree of protection from infection after vaccination.
• It is important to note that a positive anti-spike serology test cannot distinguish between active or past COVID-19. If there is concern for active infection, molecular testing (PCR) with a nasopharyngeal swab is recommended.

What are the performance characteristics of the anti-nucleocapsid antibody test?

• This depends on the time after infection. The Abbott product insert reports a high sensitivity, with 91% sensitivity before 14 days after symptom onset and 100% sensitivity more than 14 days after symptom onset. Our internal data from 125 patients recovered 100% sensitivity at 17 days after symptom onset and 13 days after PCR positivity. The sensitivity of the test in subjects with asymptomatic infection is unclear, and the duration of positive results is unknown at this time.
• This assay does not appear to cross-react with other human coronaviruses, but this type of cross-reactivity cannot be completely ruled out.
• The product insert of the assay reports a specificity of 99.6%.
• Our internal testing in pre-COVID-19 sera has detected 1 false positive in 1,020 specimens for a specificity of 99.90%.

What are the performance characteristics of the anti-spike antibody test?

This depends on the time after infection. The Abbott product insert reports that 98% of PCR-positive patients tested more than 15 days after symptom onset had a positive spike antibody test. Among serum samples collected prior to September 2019 (pre-COVID-19), 99.55% of samples tested negative using the spike antibody test.

What are the limitations of these antibody tests?

• These tests are not intended for acute diagnosis early in the course of disease. As stated above, negative results do not rule out a SARS-CoV-2 infection. For patients in which there is a high clinical suspicion for COVID-19 is high, PCR-based testing is recommended to evaluate infection. Antibody testing should not be used alone to diagnose COVID-19.
• False-positive results rarely may occur as the result of infection with non-COVID-19 human coronaviruses.
• Immunocompromised patients with COVID-19 may not have detectable levels of antibodies, or have a delayed antibody response.

What is the turnaround time?

Within 1 day.

My patient has a positive serology result, and is interested in being a potential plasma donor. Where can I refer this patient for more information?

This assay is not meant for the screening of donated blood. However, if a patient is interested in being a potential convalescent plasma donor, please refer him or her to the following website: https://newsroom.uw.edu/news/plasma-donors-sought-among-those-recovered-covid-19. At the bottom of this website is the contact information patients can use to get more information about participating.

Clinical Performance and Considerations

What is the rate of co-infection with COVID-19 and other respiratory viruses?

Because UW testing guidelines have de-emphasized up front co-testing for multiple respiratory viruses, there is relatively little internal data to support a generalized conclusion at this time other than that co-infection with other viruses can occur. A positive result for another virus does not definitively rule out the presence of SARS-CoV-2.

Does UW Virology publish information about testing volumes or rates of positivity?

Yes, see our public COVID-19 Testing Dashboard