Diagnostic Performance of an Antigen Test for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection (COVID-19)
Phase: NA
Conditions: Covid-19
Sponsor: Carilion Clinic

PROTOCOL 
 
NCT04610489 
Diagnostic Performance of an Antigen Test for SARS-CoV-2 Infection (COVID-19) 
October 30, 2020 
 

 
10/30/20 
PROTOCOL 
Diagnostic performance of an antigen test for SARS-CoV-2 infection 
John Epling, MD, MSEd 
Carilion Clinic Department of Family and Community Medicine 
 
Introduction 
Testing for SARS-Co-V-2-related disease is an important and complex topic during the current pandemic.  There are a variety of 
testing solutions available, and the fact that most are released for clinical use under the FDA’s Emergency Use Authorization means 
that the study of their accuracy needs to meet criteria that are less strict than usual.  Of particular concern is testing of symptomatic 
patients, where accuracy is important and time-sensitive, and the need for more rapid-turnaround solutions is growing daily as 
commercial and public health labs face processing delays for the most-favored test, the real-time polymerase chain reaction-based 
assays (rtPCR).  Antigen-based testing, increasingly available from commercial labs, promises a more rapid result – often within an 
hour of the test – but at a cost of lower sensitivity compared to PCR testing.  The Sofia antigen test (Quidel, Inc.) is one of two antigen 
tests approved by the FDA with an Emergency Use Authorization.  The FDA’s EUA letter for the antigen test states that “Negative 
results should be treated as presumptive and confirmed with a molecular assay, if necessary, for patient management.” 
In addition, the ability to do these tests via mid-turbinate nasal swab (rather than by nasopharyngeal swabbing) would enable their use 
in a wider variety of settings with more limited personal protective equipment (i.e., limited availability of N95 respirators). There are 
only a few studies of the diagnostic performance of antigen testing available in the published and pre-print literature.  The sensitivity 
of antigen testing in these studies varies considerably, and most of the studies were done using NP swabs (see Table and References). 
The purpose of this study is to compare the diagnostic performance of SARS-CoV2 antigen test obtained by mid-turbinate nasal swab 
with the reference standard rtPCR testing from nasopharyngeal swabs in a population of adult patients with symptoms consistent with 
COVID-19 disease. 
 
Methods 
Study design: Cross sectional diagnostic testing study 
 
Subjects: Adults (>=18 years) with symptoms of COVID-19 infection (as determined by their treating physicians) for fewer than 5 
days who are sent to a regional testing center for rtPCR testing by nasopharyngeal swab.  Healthcare workers from a large regional 
health system are included in this population. 
 
Setting: Outpatient 

 
Testing Methods: The test of interest is a mid-turbinate nasal swab (both nares) using the Sofia rapid antigen test (Quidel, Inc.). The 
reference standard used will be a Quest rtPCR tests using nasopharyngeal swabbing technique of both nares.  The antigen test will be 
collected first to reduce the chance that the detection will be inappropriately improved due to rhinorrhea produced by nasopharyngeal 
swabbing.  If additional testing – such as influenza, RSV or streptococcal testing is ordered by the PCP, this will be performed after 
the antigen testing also. 
 
Data Collection and Management: The antigen testing samples will be processed on-site at the regional testing centers, and the results 
and identifiers will be entered into a RedCap database for the study.  The rtPCR test results will be processed at Quest Laboratories.  
Results of the rtPCR tests will be sent to the patient’s ordering physician.  The results for the enrolled patients will be extracted from 
the Epic EHR and matched to the antigen test results in the RedCap database.  
 
Sample Size Determination: Because of operational concerns for healthcare workers, it is determined that the lowest acceptable 
sensitivity of the antigen test (relative to the rtPCR) is 90%.  The average test positive rate in the testing centers for “Persons Under 
Investigation” for the month prior to the study was approximately 10%.  A sample size of 800 subjects achieves 80% power to detect a 
difference of 0.099 between two diagnostic tests whose sensitivities are 0.999 and 0.900. This procedure uses a two-sided McNemar 
test with a significance level of 0.05000. The proportion of discordant pairs is 0.101. 
 
Analysis:  The results of the testing will be analyzed using SAS (Cary, NC).  The following statistics will be calculated for the antigen 
test (relative to the reference standard rtPCR test): prevalence, sensitivity, specificity, positive and negative predictive values, 
likelihood ratios for positive and negative tests, and overall test accuracy. 
 
Results 
 
We anticipate presenting descriptive statistics and the diagnostic test characteristics as described in the Methods-Analysis section. 
 
Discussion 
 
We anticipate better understanding of the diagnostic value of SARS-CoV-2 antigen testing in the clinical management of the COVID-
19 epidemic. 
 
References 

1.  Lambert-Niclot S, Cuffel A, Le Pape S, Vauloup-Fellous C, Morand-Joubert L, Roque-Afonso A-M, et al. Evaluation of a Rapid 
Diagnostic Assay for Detection of SARS-CoV-2 Antigen in Nasopharyngeal Swabs. J Clin Microbiol [Internet]. 2020 Jul 23 [cited 
2020 Aug 11];58(8). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383555/ 
2.  Nagura-Ikeda M, Imai K, Tabata S, Miyoshi K, Murahara N, Mizuno T, et al. Clinical evaluation of self-collected saliva by RT-qPCR, 
direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19. J Clin Microbiol [Internet]. 2020 Jul 7 [cited 2020 Aug 
11]; Available from: https://jcm.asm.org/content/early/2020/07/03/JCM.01438-20 
3.  Diao B, Wen K, Chen J, Liu Y, Yuan Z, Han C, et al. Diagnosis of Acute Respiratory Syndrome Coronavirus 2 Infection by Detection 
of Nucleocapsid Protein. medRxiv. 2020 Jan 1;2020.03.07.20032524.  
4.  Mak GC, Cheng PK, Lau SS, Wong KK, Lau C, Lam ET, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J 
Clin Virol. 2020 Aug;129:104500.  
5.  Scohy A, Anantharajah A, Bodéus M, Kabamba-Mukadi B, Verroken A, Rodriguez-Villalobos H. Low performance of rapid antigen 
detection test as frontline testing for COVID-19 diagnosis. J Clin Virol. 2020 Aug;129:104455.  
6.  Porte L, Legarraga P, Vollrath V, Aguilera X, Munita JM, Araos R, et al. Evaluation of novel antigen-based rapid detection test for 
the diagnosis of SARS-CoV-2 in respiratory samples. Int J Infect Dis [Internet]. 2020 Jun 1 [cited 2020 Aug 11]; Available from: 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263236/ 
7.  Blairon L, Wilmet A, Beukinga I, Tré-Hardy M. Implementation of rapid SARS-CoV-2 antigenic testing in a laboratory without 
access to molecular methods: Experiences of a general hospital. J Clin Virol. 2020 Aug;129:104472.  
8.  Mertens P, De Vos N, Martiny D, Jassoy C, Mirazimi A, Cuypers L, et al. Development and Potential Usefulness of the COVID-19 
Ag Respi-Strip Diagnostic Assay in a Pandemic Context. Front Med [Internet]. 2020 May 8 [cited 2020 Aug 11];7. Available from: 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227790/ 
9.  Castro R, Luz PM, Wakimoto MD, Veloso VG, Grinsztejn B, Perazzo H. COVID-19: a meta-analysis of diagnostic test accuracy of 
commercial assays registered in Brazil. Braz J Infect Dis. 2020;24(2):180–7.  
 
 
 

Table. Prior Diagnostic Testing Study results – review August 11, 2020 
 
Study 
Pre-
print? 
Ag test 
Comp 
Setting 
Symptoms? 
Results 
Comments 
Lamber-
Niclot(1) 
 
CORIS 
rtPCR 
(several 
brands) 
France, lab-
based 
Unclear 
(likely yes) 
Sens – 50% 
Spec – 100% 
(sens 82.2% with Ct 
< 25) 
 
Nagura-
Ikeda(2) 
 
ESPLINE 
SARS-CoV-
2 
rtPCR 
(several) 
Japan, 
inpatient 
Diagnosed 
COVID-19 by 
swab – 15% 
asymptomatic.  
Range of 
duration of 
symptoms (up 
to 10 days). 
Sens (antigen test) 
11.2% in saliva 
specimen 
 
(rtPCR sensitivity 
from saliva test c/w 
NP swab rtPCR 
ranged from 50-
81.6%) 
self-collected saliva 
specimens tested with 
both PCR and Ag testing 
against NP swab PCR. 
Diao(3) 
Yes 
Direct 
antigen test 
(NP swan) 
rtPCR 
Hospital 
yes 
Sens – 68%, spec – 
100% (Ct <=40) 
sens 98%, spec 
100% (Ct <=30) 
 
Also tested urine antigen 
– not as good. 
Mak(4) 
 
BIOCREDIT 
COVID-19 
(variety of 
sources – 
throat saliva, 
NP aspirate 
+ throat 
swab, 
sputum) 
rtPCR 
Lab-based: 
samples 
from 
COVID 
positive 
patients 
Yes 
Sensitivities: 
Low+high viral load 
– 11.1-45.7% 
High viral load – 
28.6-81.8% 
Low viral load – 0-
21.1% 
NP swab+throat saliva or 
NP aspirate+throat saliva 
had greater detection. 
Ct < 18.57 = high viral 
load. 

Scohy(5) 
 
CORIS 
rtPCR 
Belgium, 
hospital 
samples 
30.4% 
asymptomatic, 
mean 
symptomatic 
time 4 days 
Sens (overall) – 
30.2% 
Ct < 25, sens 100% 
Ct < 30, sens 70.6% 
Ct <35, sens 46.9% 
Mean Ct = 33 
Porte(6) 
 
Bioeasy 
Biotech Ag 
Test 
(NP+OP 
samples) 
rtPCR 
Respiratory 
emergency 
room at 
private 
hospital, 
Chile 
Resp 
symptoms 
and/or fever 
with epi risk 
factor 
Overall – sens 
93.9%, spec 100% 
0-7 d sxs – sens 
94.7%, spec 100% 
8-12 d sxs – sens 
80.0%, spec 100% 
Ct <= 26 sens 100% 
Ct > 26 sens 72% 
 
Blairon(7) 
 
CORIS 
Respi-Strip 
(from NP 
swabs) 
rtPCR 
General 
hospital 
Not clear 
Sensitivity 23.9% 
Sent antigen samples 
from hospital to 
university center for PCR 
testing. 
No Ct-based analysis 
performed 
No symptoms or time 
from symptom onset 
reported. 
Mertens(8) 
(clinical 
study) 
 
CORIS 
respi-strip 
(from NPA, 
NPS, BAL 
samples) 
rtPCR 
(several) 
Multi-site 
retrospective 
sample 
study 
yes 
Overall Sens 57.6%, 
spec 99.5% 
Ct<25 sens 73.9%, 
spec 99.5% 
HCW overall sens 
68%, spec 100% 
Ct<25 sens 92.9%, 
spec 100% 
 
Castro(9) 
Meta-
analysis 
 
Eco 
diagnostics 
rtPCR 
Unclear 
Yes, unclear 
time since 
symptoms 
Overall sens 89-
91%, spec 87-93% 
Specificities here lower 
than in any other study (?) 

(2 different 
tests) 
Restricted to tests 
available in Brazil