Abstract

There is limited information on the performance of rapid antigen detection kits used in Mongolia to detect COVID-19 infection. In this study, we evaluated twenty-two rapid SARS-CoV-2 antigen detection kits used in the COVID-19 testing centers and in the laboratory of the National Center for Communicable Diseases in Mongolia by using sample dilution method. We found that the sensitivity was ≥90% in 9 kits, ≥80% in 11 kits, and ≤70% in 9 kits according to the detection limit for the kits (Ct value ≤25), while the sensitivity was ≥80% in 7 kits and ≤79% in the others based on the total number of tests. Our study suggests the basic recommendation for selecting rapid antigen detection kits for COVID-19 infection in our country.

Keywords: COVID-19; Antigen; Rapid test; RT-PCR; CT value

Introduction                                                                     

The Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was first reported in Wuhan city, China in December 2019 [1]. Early diagnosis of COVID-19 infection is important for detecting the close contacts, reducing the transmission, and preventing from severe illness by starting treatment early. The nucleic acid amplification test (NAAT) -based assays such as real-time polymerase chain reaction (RT-PCR) is considered

as the golden standard to detect the COVID-19 infection [2, 3]. However, RT-PCR is a costly and timely method that requires trained personnel and laboratory environment. The Rapid SARSCoV-2 antigen detection test allows us to detect the virus within 30 minutes without requiring laboratory environment and trained personnel [4]. However, sensitivity of rapid antigen test for SARSCoV-2 is lower than the RT-PCR [5]. The different types of rapid antigen detection kits were developed, and its performance is variable depending on many factors. Therefore, the World Health Organization recommends that a rapid antigen test kit needs to meet a minimum performance requirement of at least 80% sensitivity and 97% specificity compared with a NAAT reference assay to be used [2]. The first case of COVID-19 was reported in Mongolia on March 10, 2020. According to the statistics, there have been 181646 COVID-19 cases reported in Mongolia by August 9, 2021 [6] and rapid SARS-CoV-2 antigen detection tests have been performed in the entrance of each province, countryside, family medicine hospitals, and pharmacies. Therefore, the evaluation on sensitivity and specificity of these rapid antigen detection kits used in Mongolia needs to be conducted to provide healthcare workers with valuable information.  

Materials and methods

We conducted a prospective, multi-center, point of care evaluation of twenty-two rapid antigen detection kits in comparison to RT-PCR on nasopharyngeal swabs. Human samples were collected by following protocols approved by the Research Ethics Review Committee of the National Center for Communicable Diseases of Mongolia. Signed informed consent was obtained from all participants.

Sample collection

Twenty-two rapid SARS-CoV-2 antigen detection kits used in the following 5 public testing centers in 3 districts of Ulaanbaatar from January 7, 2021, to August 9, 2021. These testing centers include family medicine hospital in 26-r khoroo and #147 kindergarten in Bayanzurkh district; #143 kindergarten in Sukhbaatar district; Nomin plaza center in Songinokhairkhan district; and a testing center at the National Center for Communicable Diseases of Mongolia.

Two nasopharyngeal swabs were collected from each person with suspected symptoms for COVID19 infection at the abovementioned testing centers by trained health care professionals: One for rapid antigen test and one for RT-PCR. Specimens for RT-PCR were collected in UTM (Universal Transport Medium). Thirty-one to fifty-four samples were collected for each kit and 810 samples were collected in total.

RT-PCR

Viral RNA was extracted from the 810 nasopharyngeal swab using ExiPrep^TM 96 Viral DNA/RNA kit and EP96L-BXD035 fully automated machine. RT-PCR was performed using SARS-CoV-2 viral E gene (Roche, Berlin, Germany), internal control EAV (LightMix® SarbecoV E-gene plus EAV) multiplex primer/probe and Applied Biosystems AgPath-ID™ One-Step RT-PCR (Thermo Fisher Scientific) according to the previously described protocol at the reference laboratory of the National Center for Communicable Diseases of Mongolia.

Rapid Antigen Detection Test

A total of 22 rapid antigen detection kits  including twentyone for nasopharyngeal swabs (SD BioSensor “STANDART Q covid-19 Ag”, Coretests “Covid-19 Ag test”, JD Biotech “SarsCov-2 (Covid-19) antigen Rapid test”, CTK Biotech “Onsite Covid-19 Ag Rapid Test”, GCMS “Genedia W Covid-19 Ag”, Safecare BIO-TECH “One step rapid test”, Genbody “COVID19Ag Detection of SARS-Cov-2 antigen”,Sansure Biotech “Sars-Cov-2 rapid antigen test”, Genesis “Kailibi Covid-19 Antigen”,Wantai “SARS-Cov-2 Ag rapid test (Colloidal Gold)”,PCL “PCL Covid-19 Ag Gold”, Watmind “SARS-Cov-2 Diagnostic test kit”, Won-med “Won-Med Covid-19 Ag test”, Healgen “Coronavirus Ag rapid test”, Lifotronic “Sars-Cov-2 Antigen”, Sugentech “SGTi-Flex Covid-19 Ag”, Lepu medical “Sars-Cov-2 Antigenrapid test, Abbot “Panbio Covid-19 Ag Rapid test device”, GP-Getein biotech” One step Test for Sars-Cov-2 Antigen, Rapigen “Biocredit Covod-19 Ag One step rapid test”) and one for saliva specimen (Coretests “COVID-19 Saliva Ag test”) were used to detect the SARS-CoV-2.  Coretests “COVID-19 Saliva Ag test” was used to detect COVID-19 infection for kids at the above-mentioned testing centers.  Thirty-one to fifty-four samples were tested with each rapid antigen detection kit to detect COVID-19 by medical professionals at the above-mentioned testing centers according to the manufacturer’s instruction of each kit. The results were reported as positive or negative. Two to three professionals independently interpreted the results of rapid antigen tests. Then, the samples were verified by RT-PCR at the Department of Virology of the National Center for Communicable Diseases of Mongolia.

Determination of the detection limits based on the Ct value

The high titer COVID-19 viral stock was prepared by collecting nasopharyngeal swabs from twenty COVID-19 positive patients who stayed at the National Center for Communicable Diseases for 1 to 2 in 4 mL UTM and serial dilution was done. Each diluted sample was tested by RT-PCR and CT value was determined. 28 samples with CT value of 15 to 35 were tested with SD BioSensor “STANDART Q covid-19 Ag” rapid antigen detection kit. According to the WHO guidance for detecting COVID-19 virus using rapid antigen detection test (possible limit for detecting virus is Ct value ≤25 or >10⁶ genomic virus copies/ mL) approved on September 11, 2022 was used as a reference [7]. Fifteen samples with 15-27 Ct value were chosen and tested by diluting the samples with lysis buffer of each kit.

Methods for determining sensitivity and specificity of the rapid antigen detection kits

The following 2x2 table was used to determine the specificity and sensitivity of the rapid tests.Sensitivity=(A/A+C)*100;

Specificity=D/(D+B)*100; Negative predictive value=D/ (D+C)*100; Positive predictive value=A/(A+B)*100

A-Positive; B-False positive; C-False negative; D-negative

Statistical analysis

Sensitivity and specificity of each rapid antigen detection kit compared to results from SARS-CoV-2 specific RT-PCR were assessed, including overall accuracy and 95% confidence intervals (CI). P<0.05 is considered as statistically significant.

Results

Sensitivity and specificity of rapid antigen detection kits

Twenty-two rapid SARS-CoV-2 antigen detection kits were used in our study. 21 of all the rapid antigen detection  kits were for detecting virus in nasopharyngeal swab and one was for detecting virus in saliva. Twenty-two rapid SARS-CoV-2 antigen detection kits were used to detect COVID-19 virus in 810 samples. Two to three professionals independently interpreted the results of rapid antigen tests by confirming negative or positive. The interpretation of the results by those professionals were same. Out of 810 samples, 135(16.7%) were positive; 662(81.7%) were negative; and 10 (1.2%) were false positive by rapid antigen detection test, respectively. 3(0.4%) of them were not qualified for the viral detection. However, 216 (26.7%) of total samples were RT-PCR positive whereas 594(73.3%) were negative. Seven rapid antigen detection kits showed the greater than 80% detection limit whereas fifteen rapid antigen detection kits showed less than 80% detection rate as compared to the results of RT-PCR (Table 1). In our study, 33 participants were tested with Coretests “COVID-19 Saliva Ag test” and 0 of them were positive while 8(24.2%) were confirmed with RT-PCR. It indicated that SARS-CoV-2 viral load in the saliva was not stable as compared to the viral load in the nasopharyngeal swab.

Table 1: Performance of point of care testing for rapid SARS-CoV-2 antigen detection kits.

CT value-dependent specificity and sensitivity of rapid antigen detection kits

Viral load is presented as Ct values and the higher the Ct value, the lower the viral load is [8,9]. The high titer COVID-19 viral stock was prepared, and then serial dilution was done to determine the CT value-dependent detection limit for rapid antigen detection test. Ct value was identified at each dilution and 15 samples with Ct value of 15-27 were tested again by rapid antigen detection kits. One out of 22 rapid antigen detection kit detected virus in samples with Ct value ≤25; 11 of them detected virus in samples with Ct value ≤24; 6 of them detected virus in samples with Ct value ≤23; one of them detected virus in samples with Ct value ≤22; one of them detected virus in samples with Ct value ≤21, one of them detected virus in samples with Ct value ≤20, and one of them detected virus in samples with Ct value ≤18.5, respectively. Out of 22 rapid antigen detection kits, GCMS «Genedia W Covid-19 Ag» rapid antigen detection kit had the lowest detection limit and detected the virus at Ct≤18.5 whereas SD BioSensor «STANDART Q covid-19 Ag» rapid antigen detection kit had the highest detection limit and detected the virus at higher Ct value or Ct≤25 (Figure 1).

Figure 1: Ct value-dependent detection limit for rapid SARS-CoV-2 antigen detection kits.

Sensitivity, specificity, positive and negative predictive values of the rapid antigen tests are evaluated according to the abovementioned method. There were 2 rapid antigen detection kits with greater than 90% sensitivity in samples with Ct value ≤25. There were 11 rapid antigen detection kits with greater than 80% sensitivity in samples with Ct value ≤25. There were 9 rapid antigen detection kits with more than 70% sensitivity in samples with Ct value ≤25. The higher sensitivity and specificity of these rapid antigen detection kits might be related to the limited number of specimens or symptoms of the patients. Overall, seven rapid antigen detection kits had more than 80% sensitivity while fifteen tests had less than 79% sensitivity (Table 2, Supplementary table.1). The saliva SARS-CoV-2 antigen detection kit showed 0% detection rate of SARS-CoV-2 virus at the testing centers, but at the laboratory, it detected the virus at Ct≤23.

Table 2: Sensitivity, specificity, PPV and NPV of the rapid antigen detection kits.

Supplementary Table 1: 95% CI and P value of sensitivity of rapid antigen detection kits.