Performance of an Antigen-Based Test for Asymptomatic and Symptomatic SARS-CoV-2 Testing at Two University Campuses

Wisconsin, September-October 2020

Ian W. Pray, PhD; Laura Ford, PhD; Devlin Cole, MD; Christine Lee, PhD; John Paul Bigouette, PhD; Glen R. Abedi, MPH; Dena Bushman, MSN, MPH; Miranda J. Delahoy, PhD; Dustin Currie, PhD; Blake Cherney, MS; Marie Kirby, PhD; Geroncio Fajardo, MD; Motria Caudill, PhD; Kimberly Langolf, MS; Juliana Kahrs, MS; Patrick Kelly, MD; Collin Pitts, MD; Ailam Lim, PhD; Nicole Aulik, PhD; Azaibi Tamin, PhD; Jennifer L. Harcourt, PhD; Krista Queen, PhD; Jing Zhang, PhD; Brett Whitaker, PhD; Hannah Browne; Magdalena Medrzycki, PhD; Patricia Shewmaker, PhD; Jennifer Folster, PhD; Bettina Bankamp, PhD; Michael D. Bowen, PhD; Natalie J. Thornburg, PhD; Kimberly Goffard, MBA; Brandi Limbago, PhD; Allen Bateman, PhD; Jacqueline E. Tate, PhD; Douglas Gieryn; Hannah L. Kirking, MD; Ryan Westergaard, MD, PhD; Marie Killerby, VetMB

Disclosures

Morbidity and Mortality Weekly Report. 2020;69(5152):1642-1647. 

In This Article

Abstract and Introduction

Introduction

Antigen-based tests for SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), are inexpensive and can return results within 15 minutes.[1] Antigen tests have received Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in asymptomatic and symptomatic persons within the first 5–12 days after symptom onset.[2] These tests have been used at U.S. colleges and universities and other congregate settings (e.g., nursing homes and correctional and detention facilities), where serial testing of asymptomatic persons might facilitate early case identification.[3–5] However, test performance data from symptomatic and asymptomatic persons are limited. This investigation evaluated performance of the Sofia SARS Antigen Fluorescent Immunoassay (FIA) (Quidel Corporation) compared with real-time reverse transcription–polymerase chain reaction (RT-PCR) for SARS-CoV-2 detection among asymptomatic and symptomatic persons at two universities in Wisconsin. During September 28–October 9, a total of 1,098 paired nasal swabs were tested using the Sofia SARS Antigen FIA and real-time RT-PCR. Virus culture was attempted on all antigen-positive or real-time RT-PCR–positive specimens. Among 871 (79%) paired swabs from asymptomatic participants, the antigen test sensitivity was 41.2%, specificity was 98.4%, and in this population the estimated positive predictive value (PPV) was 33.3%, and negative predictive value (NPV) was 98.8%. Antigen test performance was improved among 227 (21%) paired swabs from participants who reported one or more symptoms at specimen collection (sensitivity = 80.0%; specificity = 98.9%; PPV = 94.1%; NPV = 95.9%). Virus was isolated from 34 (46.6%) of 73 antigen-positive or real-time RT-PCR–positive nasal swab specimens, including two of 18 that were antigen-negative and real-time RT-PCR–positive (false-negatives). The advantages of antigen tests such as low cost and rapid turnaround might allow for rapid identification of infectious persons. However, these advantages need to be balanced against lower sensitivity and lower PPV, especially among asymptomatic persons. Confirmatory testing with an FDA-authorized nucleic acid amplification test (NAAT), such as RT-PCR, should be considered after negative antigen test results in symptomatic persons, and after positive antigen test results in asymptomatic persons.[1]

Paired nasal swabs were collected from students, faculty, staff members, and other affiliates at two Wisconsin university campuses during university-based testing programs. At university A, all persons tested (screening or diagnostic) at the university testing center during October 1–9 were eligible to participate. At university B, only students who were quarantined during September 28–October 6 after exposure to persons with COVID-19 could participate.

All participants completed a questionnaire and provided information on demographic characteristics, current and past (14 days) symptoms,§ and recent exposure to persons with COVID-19. For each participant, two mid-turbinate nasal swabs were collected by health care personnel at university A and were self-collected under supervision at university B. Both nostrils were sampled with each of the two swabs. Swabs for antigen testing were analyzed according to the manufacturer's instructions.** Swabs for real-time RT-PCR were stored in viral transport media at 39°F (4°C) and analyzed within 24–72 hours of collection. At university A, real-time RT-PCR was performed using the CDC 2019-nCoV real-time RT-PCR diagnostic panel,[6] with cycle threshold (Ct) values reported for the N1 and N2 viral nucleocapsid protein gene regions. At university B, real-time RT-PCR was performed using the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific). Viral culture†† [7] was attempted on residual RT-PCR specimens if the RT-PCR or antigen test result was positive.

Statistical analyses were performed using Stata (version 16.1; StataCorp). Sensitivity, specificity, PPV, and NPV were calculated for antigen testing compared with real-time RT-PCR results. Ninety-five percent confidence intervals (CIs) were calculated using the exact binomial method; t-tests were used for Ct value comparisons§§; p-values <0.05 were considered statistically significant. This investigation was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.¶¶ Ethical review boards at both universities determined the activity to be nonresearch public health surveillance.[2]

Among a total of 1,105 nasal swab pairs submitted, seven (0.6%)end highlight were excluded for having inconclusive antigen or real-time RT-PCR results. Test comparisons were performed on 1,098 paired nasal swabs (2,196 total swabs), including 1,051 pairs (95.7%) from university A and 47 pairs (4.3%) from university B (Table 1). Among the 1,098 pairs evaluated, 994 (90.5%) were provided by students aged 17–53 years (median = 19 years), 82 (7.5%) by university faculty or staff members aged 22–63 years (median = 38 years), and 22 (2.0%) by other university affiliates aged 15–64 years (median = 29 years). Fifty-seven persons participated more than once on different testing days. Overall, 453 (41.3%) participants were male, and 917 (83.5%) were non-Hispanic White. At specimen collection, 227 (20.7%) participants reported experiencing one or more COVID-19 symptoms, and 871 (79.3%) reported no symptoms.

Among 227 paired specimens from symptomatic participants, 34 (15.0%) were antigen-positive, and 40 (17.6%) were real-time RT-PCR-positive. The median interval from symptom onset to specimen collection was 3 days (interquartile range = 1–6 days; 7.5% missing). Among symptomatic participants, antigen testing sensitivity was 80.0% (32 of 40), specificity was 98.9% (185 of 187), PPV was 94.1% (32 of 34), and NPV was 95.9% (185 of 193) (Table 2). For specimens collected within 5 days of reported symptom onset (72.4%; 152 of 210), sensitivity was 74.2% (23 of 31), and specificity was 99.2% (120 of 121).

Among 871 paired specimens from asymptomatic participants, 21 (2.4%) were antigen-positive and 17 (2.0%) were real-time RT-PCR-positive. Antigen testing sensitivity was 41.2% (seven of 17), specificity was 98.4% (840 of 854), PPV was 33.3% (seven of 21), and NPV was 98.8% (840 of 850). Test performance was not significantly (p>0.05) different when excluding 53 (6.1%) of 871 participants who were asymptomatic at the time of testing but had reported one or more symptoms in the preceding 14 days.

Sixteen paired swabs were antigen-positive and real-time RT-PCR–negative (i.e., false-positive), including 14 (66.7%) of 21 positive antigen results from asymptomatic participants and two (5.9%) of 34 from symptomatic participants. Eight of the 16 false-positive results were recorded during a 1-hour period at university A. In this instance, a series of consecutive positive results in asymptomatic persons was noted, and investigators offered repeat antigen testing to the affected participants. Six of eight participants were reswabbed within 1 hour, and all six received negative test results on a second antigen test. All eight initial paired swabs from these participants were negative on real-time RT-PCR. Because no user errors could be identified, the false-positive results were included in analysis. Eighteen false-negative antigen test results were obtained, including 10 (58.8%) of 17 real-time RT-PCR–positive tests from asymptomatic participants, and eight (20.0%) of 40 from symptomatic participants. All false-negative results from symptomatic participants were from specimens collected <5 days after onset of symptoms (median = 2 days). Ct values for specimens with false-negative antigen results were significantly higher compared with antigen- and real-time RT-PCR-positive specimens (mean N1 Ct = 32.3 versus 23.7; p<0.01) (Figure).

Figure.

Viral culture results among participants with positive Sofia SARS Antigen Fluorescent Immunoassay or positive SARS-CoV-2 real-time reverse transcription–polymerase chain reaction (RT-PCR) results (n = 69),* by cycle threshold (Ct) value and the interval between specimen collection and reported symptom onset or asymptomatic status — university A, Wisconsin, September–October 2020
*n = 30 antigen- and culture-positive; n = 22 antigen-positive and culture-negative; n = 15 antigen- and culture-negative; n = two antigen-negative and culture-positive.
†Ct values represent cycle thresholds for the N1 target probe during SARS-CoV-2 real-time RT-PCR; Ct values are represented on the y-axis in descending order to indicate that lower Ct values represent higher levels of RNA in the specimen.

Virus was recovered from 34 (46.6%) of 73 positive specimens, including 32 (82.1%) of 39 specimens with concordant positive results and two (11.1%) of 18 with false-negative antigen results; no virus was recovered from 16 specimens with false-positive antigen test results. The two specimens with false-negative antigen results that were culture-positive were from two symptomatic participants who had specimens collected at day 2 and day 4 after symptom onset.***

*These authors contributed equally to this report.
Other affiliates were participants who did not mark "student" or "staff" on the questionnaire (they selected "other" or did not respond); the majority of these persons were family members of staff members.
§Symptom list was based on the interim position statement for COVID-19 case definitions from the Council of State and Territorial Epidemiologists, updated August 7, 2020. Clinical criteria for COVID-19 included fever, cough, shortness of breath, fatigue, sore throat, headache, muscle aches, chills, nasal congestion, difficulty breathing, diarrhea, nausea, vomiting, abdominal pain, rigors, loss of taste, and loss of smell. https://cdn.ymaws.com/www.cste.org/resource/resmgr/ps/positionstatement2020/Interim-20-ID-02_COVID-19.pdf.
Recent exposure was defined as being within 6 feet of a person with a COVID-19 diagnosis for ≥15 minutes in the past 14 days.
**https://www.fda.gov/media/137885/download.
††Specimens were used to perform a limiting-dilution inoculation of Vero CCL-81 cells, and cultures showing evidence of cytopathic effect (CPE) were tested by real-time RT-PCR for the presence of SARS-CoV-2 RNA. Viral recovery was defined as any culture in which the first passage had an N1 Ct at least twofold lower than the corresponding clinical specimen.
§§Ct values from real-time RT-PCR were only compared for specimens collected at university A that were analyzed with the CDC 2019-nCoV real-time RT-PCR diagnostic panel for detection of SARS-CoV-2.
¶¶45 C.F.R. part 46.102(l)(2), 21 C.F.R. part 56; 42 U.S.C. Sect. 241(d); 5 U.S.C. Sect. 552a; 44 U.S.C. Sect. 3501 et seq.
***The participant with a false-negative result 2 days after symptom onset had a repeat specimen 2 days later; the results of testing were positive by antigen test and by real-time RT-PCR.

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