key: cord-0875628-370o1rxa authors: García-Fernández, Sergio; Pablo-Marcos, Daniel; de la Fuente, Silvia Velasco; Rodríguez, María José Reina; Gozalo, Mónica; Rodríguez-Lozano, Jesús; Méndez-Legaza, José Manuel; Calvo, Jorge title: Evaluation of the rapid antigen detection test STANDARD F COVID-19 Ag FIA for diagnosing SARS-CoV-2: Experience from an Emergency Department date: 2022-03-18 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2022.115683 sha: fef995d0a2b22ae8dc9cd1b0a10dd86dd54d13bd doc_id: 875628 cord_uid: 370o1rxa The purpose of this study was to assess the clinical performance of STANDARD F COVID-19 Ag FIA (SD Biosensor Inc., Gyeonggi-do, Republic of Korea), a rapid antigen detection test (RADT) for diagnosing SARS-CoV-2, in patients attended at the Emergency Department with signs or symptoms compatible with COVID-19 that had started in the last 5 days. The clinical performance of the antigen test was compared with RT-PCR, the reference standard. We included 663 specimens from non-repetitive patients. Clinical sensitivity and specificity were 84.0% (95% CI 76.1-89.7) and 99.6% (95% CI 98.5-99.9), respectively. The positive and negative predictive values were 98.1% (95% CI 92.7-99.7) and 96.4% (95% CI 94.4-97.7), respectively. The kappa index agreement between RT-PCR and the RADT was 0.89 (95% CI 0.84-0.93). We concluded that STANDARD F COVID-19 Ag FIA is an excellent first-line RADT method to diagnose symptomatic patients in the emergency department. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide becoming a great diagnostic challenge for microbiology laboratories (Wiersinga et al., 2020 ). An early and rapid SARS-CoV-2 detection has become mandatory for preventing the virus spread (Kevadiya et al., 2021) . Although the RT-PCR is the gold-standard method to diagnose the infection, point of care (POC) methods remain a highly useful tool to speed up the diagnostic process (Corman et al., 2020; Nelson et al., 2020) . Although there are POC systems based on RT-PCR with final results in less than 1 hour (Basu et al., 2020; Loeffelholz et al., 2020) , its use to perform large screenings represent huge economical costs. On the contrary, rapid antigen detection tests (RADT) represent both, economic and fast POC methods to accelerate diagnosis. Nevertheless, these RADT have lower sensitivity and specificity than molecular-based diagnostics (Albert et al., 2021; Scohy et al., 2020) . This loss in accuracy forces an evaluation in different real-life scenarios in order to assess their accuracy and utility. The purpose of this study was to assess the clinical performance of STANDARD F COVID-19 Ag FIA (SD Biosensor Inc., Gyeonggi-do, Republic of Korea), an automated RADT based on immunofluorescence detection for diagnosing SARS-CoV-2. The evaluation was performed using nasopharyngeal swabs from patients attended in the Emergency Department with signs or symptoms compatible with COVID-19 that had started in the last 5 days. This prospective study was conducted from October 14 th 2020 to November 18 th 2020 in a single tertiary university hospital, Hospital Universitario Marqués de Valdecilla (HUMV), in Santander (Spain). Patients attended in the Emergency Department of HUMV with signs or symptoms compatible with COVID-19 that had started in the last 5 days were recruited. Demographic data were no collected. Left-over nasopharyngeal swab specimens used firstly in routine RT-PCR-based diagnosis in the hospital were used in antigen test evaluation within 24 hours after collection. Specimens were kept at 4°C until testing. Virus transport and preservation media used for nasopharyngeal testing included Biocomma (Biocomma Limited, ShenZen, China) and DeltaSwab (Deltalab, Rubí, Spain). Both systems are nasopharyngeal flocked swabs with non-inactivated preservation virus transport medium and were employed interchangeably. The clinical performance of the RADT STANDARD F COVID-19 Ag FIA was compared with RT-PCR, the reference method for SARS-CoV-2 diagnosis. The RT-PCR reagent used was VIASURE SARS-CoV-2 Real Time PCR Detection Kit (Certest Biotec, Zaragoza, Spain), based on amplification and detection of ORF1ab and N genes. The limit of detection of the RT-PCR was ≥ 10 copies of viral RNA for both genes. Nucleic acid extraction was performed using automated systems as Biocomma Nucleic Acid Purification Kit (Biocomma Limited, ShenZen, China) and IndiMag Pathogen IM48 Cartridge (Indical Bioscience, Leipzig, Germany). Amplification was performed on a BioRad CFX96 thermocycler (BioRad Laboratories, the Netherlands). The cycle threshold (Ct) was automatically determined by the manufacturer's software. A positive RT-PCR result was defined as amplification of any of the two SARS-CoV-2 target genes, following manufacturer's instructions. RADT was performed following manufacturer's instructions. Reading of the results was performed using a F200 or F2400 analyser (SD Biosensor Inc., Gyeonggi-do, Republic of Korea), which automatically reads the intensity of fluorescence following antibody-antigen complex formation. A sample is considered positive if cutoff index (COI) value is ≥ 1. The results obtained with the antigen test in this study did not impacted patient management and informed consent was not needed. A total of 663 specimens from non-repetitive patients were included in the study. In 125 specimens (18.9%) the RT-PCR result was positive, while the RADT detected 105 (15.8%). A negative result was obtained in 538 specimens (81.1%) by RT-PCR and in 558 (84.2%) by the RADT. Clinical sensitivity and specificity of the RADT compared with RT-PCR were 84.0% (95% CI 76.1-89.7) and 99.6% (95% CI 98.5-99.9), respectively. The positive and negative predictive values were 98.1% (95% CI 92.7-99.7) and 96.4% (95% CI 94.4-97.7), respectively. The kappa index agreement between RT-PCR and the RADT was 0.89 (95% CI 0.84-0.93), which means an almost perfect agreement (Table 1) . Two false positive results (FP, 0.3%) were obtained with the RADT. Cutoff index values (COI) of FP were 1.22 and 1.69. Twenty false negatives results (FN, 3.0%) were found with the antigen test. In 16 out of 20 of FN the Ct obtained with the RT-PCR was ≥ 30 ( Table 2) . Sensitivity of RADT according to Ct results of RT-PCR has also been analysed ( Table 3) . Sensitivity of the RADT when testing positive samples for SARS-CoV-2 with Ct ≤ 30, was 92.9%. In samples with Ct  31, sensitivity of the RADT was 7.7%. Rapid detection of infected patients with SARS-CoV-2 is mandatory to interrupt transmission of the virus (Kevadiya et al., 2021) . Rapid molecular methods based on RT-PCR offer results in less than one hour, however the price is not feasible for large-scale screening. For this reason, RADT represent an efficient alternative as first-line diagnostic method combining both advantages: low price and fast results. Notwithstanding, performance of RADT is highly dependent on the time elapsed since the onset of symptoms, since this accuracy is closely linked to the viral load (Dinnes et al., 2021) . Clinical evaluations in different scenarios are imperative in order to use the correct diagnostics in the correct time. In this evaluation the antigen test STANDARD F COVID-19 Ag FIA presented an excellent clinical performance (84.0% sensitivity and 99.6% specificity) and an almost perfect agreement compared with the reference method, the RT-PCR, when used in patients with signs or symptoms compatible with COVID-19 that had started in the last 5 days. This finding is consistent with other RADT evaluations performed in symptomatic patients with similar clinical courses (onset of symptoms ≤ 5 days) (Albert et al., 2021; Merino et al., 2021) . The antigen test STANDARD F COVID-19 Ag FIA shows good clinical sensitivity (92.9%) in symptomatic patients with high viral loads (Ct ≤ 30). Meaning that this RADT is effective in cases of active viral replication, i.e. in the early acute phases of infection. As expected, sensitivity of the RADT decreases as the viral load in patients becomes lower (7.7% sensitivity; Ct  31). Similar findings have been reported previously (Fenollar et al., 2021; Liotti et al., 2021) . The main reason of FN of RADT is a lack of sensitivity in cases with high Ct. In this evaluation; in the 80% of FN the Ct was ≥ 30. showed 100% sensitivity in samples with Ct < 26 (Baccani et al., 2021; Orsi et al., 2021) . As seen in our study, time from the onset of symptoms is determinant for a correct use of RADT. This evaluation has been done in a high pre-test probability scenario. During the study period (October 14 th -November 18 th , 2020) the cumulative incidence rate per 100,000 inhabitants in the last 14 days, rise from 118 to 547 in the region of Cantabria. Together with a final high positive predictive value of the test, no confirmation of positive samples with RT-PCR was needed. However, in our experience, because of the two false positive results with antigen test (COI: 1.22 and 1.69), we decided to include in our internal diagnostic algorithm a confirmatory RT-PCR in samples with antigen test result with COI values between 1 -2 in order to obtain 100% of specificity. This is consistent with a previous work showing a median COI value of 1.4 in samples performed with the same RADT and not confirmed to be positive by RT-PCR (Colavita et al., 2021) . An actual matter of concern is how SARS-CoV-2 new variants and mutations could affect the performance of COVID assays. As most RADT capture the viral nucleocapsid protein, and typically mutations affect in the Spike protein, mutations are not likely to limit the performance of RADT (Harvey et al., 2021) . Nevertheless, since mutations in the nucleocapsid protein affecting performance of RADT have already been reported (Bourassa et al., 2021) , continuous control of accuracy of diagnostic tests is mandatory. A limitation of the study is that not information about cellularity of nasopharyngeal swabs was provided. We aware some of discrepant results could be explain with this data. Based on these data, the antigen test STANDARD F COVID-19 Ag FIA is an excellent first-line method to diagnose symptomatic patients in both emergency departments and primary healthcare centres. 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Silvia Velasco de la Fuente: Investigation, data curation, validation, review and editing of draft. María José Reina Rodríguez: Investigation, data curation, validation, review and editing of draft. Mónica Gozalo: Investigation, data curation, validation, review and editing of draft. Jesús Rodríguez-Lozano: Investigation, data curation, validation Jorge Calvo: Supervision, conceptualization, Investigation, data curation, validation, review and editing of draft We would like to acknowledge routine laboratory technicians for their assistance in the production of the data. The authors have no conflict of interests.