The 2019 coronavirus disease (COVID-19) is a current pandemic respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1). In Chile, more than 1.6 million infections and 37 thousand deaths have been reported from this disease (2). Currently, the most effective way to prevent and control its spread is the timely detection and isolation of infected people. The reverse transcription quantitative PCR (RT-qPCR) is the recommended and widely used technique for diagnosis due to its high sensitivity and precision (3). However, new and faster methodologies for detecting and diagnosing have recently been implemented, including the rapid antigen test (RAT) for SARS-CoV-2. The technique's principle consists of a rapid chromatographic immunoassay for the qualitative detection of specific SARS-CoV-2 antigens from nasopharyngeal swab samples (NPSs) (4). This strategy has begun to be used in several countries, and its effectiveness compared to the standard RT-qPCR method has been the target of study and analysis (5). In Chile, it was implemented in Health centers by the Ministry of Health in March 2021 as an alternative to RT-qPCR. More than 20 million RT-qPCR tests and more than 750 thousand RATs have been carried out in Chile. Although its effectiveness has been verified in various studies showing differences between manufacturers, currently, there are no studies on the effectiveness of the antigenic tests used in Chile, nor on their performance for detecting the outbreak of new SARS-CoV-2 variants. In this study, we compare the efficacy of the results of 55 NPSs obtained by the rapid antigen test for SARS-CoV-2 (SD Biosensor, South Korea), a chromatographic assay using a monoclonal antibody against the nucleocapsid (N) protein of the SARS-CoV-2 virus, and the standard RT-qPCR detection method. We observed differences in the detection of positive COVID-19 cases for the different ranges of amplification assessed. Interestingly, we determined that this antigen test loses sensitivity for detecting SARS-CoV-2 variants carrying the K417N/T, E484K and N501Y single nucleotide polymorphisms (SNPs).

To determine the detection sensitivity of the rapid antigen test (RAT), we evaluated 55 samples in different ranges of the cycle of quantification (Cq) for ORF1ab, in a similar way to studies previously reported (6, 7). The sensitivity of RAT was performed only for those samples containing the ancestral strain SARS-CoV-2 from Wuhan. Using NPSs, we determined that the detection sensitivity of RAT for the RT-qPCR Cq range 20 ≤ Cq <25 was 90% (Figure 1), while a 10% sensitivity was observed for samples comprised within the range of 25 ≤ Cq <30 (Figure 1). On the other hand, at higher Cq values, which include lower viral loads, the detection sensitivity was 0% for both Cq value ranges, 30 ≤ Cq <35 and Cq≥35 (Figure 1). We made semi-quantitative analysis of the RAT band intensity, where the SARS-CoV-2 positive samples for the Cq range 20 ≤ Cq <25 and 25 ≤ Cq <30 showed a high band intensity (Table 1). Negative NPSs samples were also determined. Altogether, data showed a significantly lower sensitivity of RAT for the detection of SARS-CoV-2 in comparison to RT-qPCR technique.

On the other hand, we determined the ability of RAT to detect variants of the SARS-CoV-2 virus. We select NPSs from patients diagnosed as SARS-CoV-2 positive by RT-qPCR, containing variants according to the RT-qPCR SNP detection. When we evaluated these NPSs containing SARS-CoV-2 variants, we observed that the sensitivity of RAT decreased in the variants that carry K417N/T and E484K amino acid substitutions, even in samples with the lowest ranges of Cq (20 ≤ Cq <25). As a result, detection sensitivity decreases to 42% detection, obtaining 58% false-negative samples when the virus presents these mutations (Figure 2). Interestingly, from the three SARS-CoV-2 (K417N/T, E484K, and N501Y) positive samples for RAT, two of them presented a band of low intensity (Table 2) compared to SARS-CoV-2 samples with no mutations identified, which present a high-intensity band in the range 20≥Cq <25 (Table 1). Furthermore, for Cq over 25 no SARS-CoV-2 variant (K417N/T, E484K, and N501Y) sample was detected by RAT. A flow chart of the results obtained are described on Figure 3.

Taking together, these data suggest that RAT loses its full ability to detect SARS-CoV-2 against these variants.

RT-qPCR test is the gold standard technique for COVID-19 diagnosis (3). However, despite its high sensitivity, the extent of analysis and the associated costs have encouraged the use of faster and low-cost SARS-CoV-2 detection assays for better control of the pandemic (8). Previous studies have documented the efficiency of different brands of rapid antigen tests for SARS-CoV-2 detection as a diagnostic alternative compared to standard RT-qPCR. For example, Chaimayo et al. compared the sensitivity and specificity of the Standard Q COVID-19 Ag rapid test (SD Biosensor) against Allplex ™ 2019-nCoV Assay RT-qPCR assay (Seegene) in the diagnosis of COVID-19 in a Thailand population (9). They showed a 98% of comparable sensitivity with the real-time RT-PCR assay. Other rapid detection kits have shown, for example, a sensitivity of 85% (Shenzhen Bioeasy Biotech-2019-nCoV Ag) (10) or 72% (Abbott-Panbio COVID-19 Ag) (11), while the minimum sensitivity accepted in the rapid test by the World Health Organization is 80% for its diagnostic use (12). Dinnes et al. (5), after an analysis of 58 rapid tests, concluded that sensitivity of antigen assays significantly decrease in asymptomatic patients or after the second week of infection and, indicated that the sensitivity of these tests increases with the Cq ≤ 25. For example, Linares et al. (13) reported that patients with less than seven days of symptoms showed a high viral load and a sensitivity of 86.5%, while the sensitivity dropped to 53.8% in asymptomatic patients or lower viral loads using the Panbio COVID-19 Ag Rapid Test Device (Abbot). The same effect was observed using the SD Biosensor-STANDARD Q COVID-19 test, where a sensitivity of 65.3% was observed in symptomatic patients and 44% in asymptomatic patients (14). Krüttgen et al. (6) using the rapid SARS-CoV-2 antigen test from the Roche manufacturer, indicated that the sensitivity of the assay in 75 NPSs is 100% (Cq ≤ 25), 95% (Cq ≤ 30), 44.8% (Cq ≤ 35), and 22.2% (Cq>35) compared to RT-qPCR. Such sensitivity is directly related to the patient's viral load. In the same line, Schildgen et al. (15) reported a sensitivity of 100% in symptomatic patients and 84.6% in asymptomatic patients using the rapid test from Roche.

In this study, we observed that the rapid antigen test (SD Biosensor) has a detection efficiency depending on the Cq values, being 90, 10, 0, and 0% within the ranges of 20 ≤ Cq <25; 25 ≤ Cq <30; 30 ≤ Cq <35, and Cq≥35, respectively. In this regard, the manufacturer and distributor indicate that 95.5% of effectiveness at Cq <30. Although our study shows lower sensitivity as previously described, it is also important to mention that we used a smaller number of samples in each Cq range compared to previous evidence (16). Furthermore, we observed that RAT sensitivity for detecting ancestral strain of SARS-CoV-2 decreased from 90 to 42.8% with the SARS-CoV-2 variants that carry K417N/T, E484K and N501Y amino acid substitutions, even in the lower Cq range of 20 ≤ Cq <25. The low intensity of bands showed for two of the three SARS-CoV-2 variants identified by RAT suggests a decreased specificity for samples with the K417N/T, E484K, and N501Y mutations. By contrast, the virus with no mutations showed a high intensity band of detection by RAT in the 90% of the samples evaluated. Because the amino acid substitutions E484K and N501Y are present in the beta (B.1.351) and gamma (P.1) variants, and the substitution K417N is found in the beta variant and K417T in the gamma variant (17), we hypothesized that RAT might not detect both or at least one of these two virus variants. In this line, Frediani et al. (7) reported that the Abbott BinaxNOW COVID-19 Ag Card rapid test detects the P.1 variant, even between the Cq ranges of 20-22. However, this data corresponds only to one sample analyzed (7). Other variant studies using rapid antigen tests, including analysis of the alpha (B.1.1.7) and beta (B.1.351) variants (18), showed no detection at Cq values between 29–35 (7) like this study.

The rapid antigen test analyzed in this study detects the nucleocapsid (N) of the original SARS-CoV-2 virus using a monoclonal antibody. This kind of detection can explain the low capacity of RAT to detect the variants, which has the substitutions P80R and R203K in the N protein. These data further suggest that this rapid antigen test could also fail to detect other variants, such as delta (B.1.617.2), because, in addition of Spike protein mutations (19), the delta variant also has mutations in the N protein (different to R203K). This data suggests a lack of specificity of RAT to detect specific variants, which agrees with the fact that RAT initially manufactured to detect the ancestral strain of SARS-CoV-2. In addition, this takes on real relevance when the RAT is used as an active search or mass testing in public places in infected patients with a high Cq (low viral load). According to the results shown in our work, the diagnosis by RAT will be negative, and the patient will be able to infect their contacts and, consequently, may generate important local outbreaks. Therefore, detecting positive patients with low viral load, who will probably be asymptomatic, should be done only by the RT-qPCR assay, one of the safest ways to be massively tested in the population to control the pandemic. Further studies are needed for evaluating by RAT a higher number of samples and SARS-CoV-2 variants. Although we found significant differences in each condition (ancestral strain of SARS-CoV-2; SARS-CoV-2 variants), it is still necessary to increase the number of samples to obtain more accurate detection percentages of SARS-CoV-2 in each range of Cq analyzed. We suggest restricting the use of RAT only to confirm COVID-19 symptomatic positive cases but not as a massive strategy for traceability and identification of cases in the population.

The sensitivity of the rapid antigen test (SD Biosensor, South Korea) is determined by the viral load of the sample. While high viral loads (20 ≤ Cq <25) present 90% sensitivity, 25 ≤ Cq <30 and >30 present 10 and 0% respectively. On the other hand, the rapid test reduces its detection sensitivity compared to samples positive for SARS-CoV-2, but that present the mutations K417N/T, E484K, and N501Y observing a detection capacity of 42% in ranges of 20 ≤ Cq <25.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Ethical Committee of the University of Santiago of Chile (No. 226/2021), and Scientific Ethical Committee of the Central Metropolitan Health Service, Ministry of Health, Government of Chile (No. 370/2021). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

CB-A, RL, MI, CA-C, FER-L, and AMS: conceptualization. CB-A, RL, and FER-L: data curation. CB-A, RL, FER-L, and AMS: formal analysis. MI, CA-C, FER-L, and AMS: funding acquisition. CB-A, AM-T, FH, MF, and CR: investigation. CB-A, EV-V, FER-L, and AMS: methodology. DV, FER-L, and AMS: supervision. DV, MI, CA-C, FER-L, and AMS: validation. CB-A, RL, CA-C, and FER-L: writing—original draft. CB-A, RL, EV-V, CA-C, MI, FER-L, and AMS: writing—review and editing. All authors have read and agreed to the published version of the manuscript.

The Laboratory of Virology supported the COVID-19 diagnosis in the University laboratories network (Ministry of Sciences, Ministry of Health, Government of Chile) for diagnosis tasks. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.