key: cord-0306476-phfwetg6 authors: Lunca, C.; Cojocaru, C.; Gurzu, I. L.; Petrariu, F. D.; Cojocaru, E. title: Performance of antigenic detection of SARS-CoV-2 in nasopharyngeal samples date: 2021-07-16 journal: nan DOI: 10.1101/2021.07.12.21260263 sha: e64b7c2369ed61b7ad408c4fa006285d251dc992 doc_id: 306476 cord_uid: phfwetg6 Objectives: SARS-CoV-2 virus detection on nasopharyngeal specimens to infected individuals has become a challenge for the COVID-19 pandemic outbreak. We aim at comparing the performance of antigenic detection of SARS-CoV-2 in nasopharyngeal samples via an immunochromatographic method to molecular detection via qRT-PCR. Materials and Methods: 47 nasopharyngeal exudates were collected from suspicious COVID-19 cases. The samples were performed both via the qualitative immuno-chromatographic method for S protein detection in the SARS-CoV-2 structure, using fluorescent labelled anti-protein S antibodies and via qRT-PCR test for the qualitative detection of the screening gene E and the specific ORF1ab region of the RNA-SARS-CoV-2. Results: There was a fair correlation between the positive antigen tests and the positive PCR assays measured through threshold cycle ORF1ab region (Ct orf). A better correlation was obtained between the antigen test results and the Ct orf when including patients with Ct orf below 25. Conclusions: Using antigen tests as screening tests is useful on symptomatic persons during the viral replication period, therefore during the contagious period. A positive test shows a high predictive value for infection, while a negative antigen test result via immuno-chromatography must be confirmed by a qRT-PCR test. Since reporting the first human infection case caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019, humanity experienced a new alert bout. On January 30th 2020, World Health Organization (WHO) declared that the infectious disease called Coronavirus disease 2019 (COVID- 19) had become an international public health emergency and introduced the COVID-19 pandemic term on March 11, 2020 [1] . SARS-CoV-2 virus belongs to the Coronaviridae family, within the Sarbecovirus subgenus of the Betacoronavirus genus [2] . It is coated and has a non-segmented, single-stranded, positivesense ribonucleic acid (ssRNA+) as nuclear material, being pathogenic in vertebrates [3] . The virus is capable of causing severe forms of infection, sometimes life-threatening disease, being different from its two predecessors SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which in turn produced severe forms of respiratory damage starting with 2002-2003, and 2012 respectively [4] . Current data suggest that the infection transmission and the disease severity (extending from asymptomatic to fatal forms) are different from what was known for SARS-CoV [5] . These aspects are important for differentiating the different types of coronavirus by the diagnostic tests used. Thus, the specificity of diagnostic tests becomes as important as their sensitivity. So far, the available diagnostic methods have been related to a) specific viral gene regions through nucleic acid amplification techniques Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and isothermal nucleic acid amplification, b) the antibodies produced by the immune system in response to the viral infection (serology/Immunoglobulin M (IgM)/Immunoglobulin G (IgG) tests), and c) the test detection of antigen (TDA). Among these, the most reliable method is the viral gene detection via qRT-PCR [6] . The "gold standard" for diagnostic detection of SARS-CoV-2 is qRT-PCR technique which identifies nucleotide sequences of SARS-CoV-2 RNA present in the respiratory samples. Results are provided within 24 -48 hours and require reagents and laboratories with special equipment as well as highly qualified personnel [7] . All these methods must be entirely operated in designated testing laboratories by trained personnel, under specified experimental and Biological Safety Level (BSL) conditions. To reduce qRT-PCR assay processing time and test costs, several antigen detection laboratory techniques have been introduced [8] . Technical features, result communication delays and qRT-PCR high costs in countries with less access require the implementation of containment measures, diagnosis and treatment of COVID-19. All these are required to reduce the high rate of disease transmission within the community. Therefore, rapid antigen tests were developed, being easy-to-use, offering rapid results at low costs [9] . The U.S. Food and Drug Administration (FDA) approved an emergency use authorization (EUA) for antigen tests able to identify SARS-CoV-2, publishing them on a list [10] . The current recommendations support antigen tests usage as screening tests on asymptomatic persons or to detect infection in previously diagnosed COVID-19 persons [11] . The study aims at comparing the performance of antigenic detection of SARS-CoV-2 in nasopharyngeal samples via an immunochromatographic method to molecular detection via qRT-PCR assay. All nasopharyngeal samples used in this prospective study, obtained from suspect COVID-19 cases, were tested by SARS-CoV-2 RT-PCR and antigen test. In order to evaluate antigenic test utility, we compared and analysed results of the antigenic test and of the qRT-PCR, based on positive and negative values and Ct orf, respectively. We used Ct orf value given that literature data show its good correlation with the viral load [12] . The antigenic test was done using immuno-chromatographic technique. Nowadays, the gold standard for detecting SARS-CoV-2 is the molecular detection via qRT-PCR assay. The study was conducted in the Netconsult Medical Center, Iasi City, Romania, between April and November 2020 on 47 nasopharyngeal exudates collected from suspicious COVID-19 cases. Written informed consent and free choice were complied with, for both RT-PCR and antigen test in the same sample, as well as for this comparative study data collection. Patient's privacy and confidentiality were ensured, complete and understandable information about the tests were provided. The subjects were encouraged to ask questions and answers were provided during the consent process. Patients were not charged. All patients had symptoms suggesting the SARS-CoV-2 infection and were tested within 24 hours after onset. According to staging of the COVID-19 all study patients were in stage 2 [13] . After collection, the exudate buffers were immersed in 3 mL virus transport medium (VTM) and refrigerated at 4°C until processing. The equipment used in qRT-PCR testing was protocol [14] . The method has a detection limit of 4.1 copies/rxn for E genes and 12.9 copies/rxn for the ORF1ab region. Samples that displayed an exponential growth curve and a cycle threshold value (Ct) <35 were considered positive, while no Ct value indicated a negative result (non-numeric value). For each sample with positive result in the qRT-PCR test, the Ct value was marked down. In addition, the antigenic detection was performed via a qualitative immuno-chromatographic method using Fluorecare® SARS-CoV-2 Spike Protein Test Kit [15] . The test detected S All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. Out of 47 analysed samples, 40 were positive via qRT-PCR testing for SARS-CoV-2 RNA (85%). The median Ct value of qRT-PCR-positive samples was 21.75 (mean 22.8±6.1) for All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 85.18% to 100.0%). According to WHO data, antigenic tests have a superior utility in high viral load cases (Ct ≤ 25 or > 10 6 genomic virus copies/mL) [12] . In this study, by reporting 66.3% to 95.8%), a specificity of 100.0% (95%CI; 75.3% to 100.00%), a positive predictive All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. There is a moderate negative relationship between antigen level and Ct orf, as presented in figure 1. In order to stop SARS-CoV-2 from spreading and prevent the capacity of SARS-CoV-2 molecular detection from overloading, WHO recommended testing prioritization for early All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 16, 2021. ; https://doi.org/10.1101/2021.07.12.21260263 doi: medRxiv preprint diagnosis and protection of vulnerable people [6] . WHO also developed a user's guide for TDA SARS-CoV-2 in respiratory secretions in order to optimize patient management and epidemiological surveillance measures. The guide pleads for the use of TDAs with sensitivity 80% and specificity 97%, respectively, when qRT-PCR is not available or when late results lower their clinical utility. TDA could be useful the first 5-7 days after infection onset, characterized by high viral load: Ct 25 and infectivity). TDA can also be used to prioritize qRT-PCR testing of negative TDA patients in confirmed COVID-19 outbreaks and of asymptomatic non-quarantined contacts in case of negative results [12] [16] . In our study, the TDA of SARS-CoV-2 S protein had high specificity and sensitivity in samples with high viral load (Ct ≤ 25). Our data have values comparable to those in the study conducted by Lambert-Niclot et al. [17] Although the group in our study was about three times smaller, the value of the sensitivity of the global antigen detection as well as that obtained in the samples for which the PCR test provided Ct ≤ 25 values are similar. Also, our results are comparable to those mentioned in other studies, even though the overall diagnosis sensitivity was lower, as shown in Table 5 [17] [18] [19] . A recently published study used the same method of identifying SARS-CoV-2 antigens, as in the present study, in patients either symptomatic or in close contact with COVID-19 cases, revealed a lower sensitivity, with a similar specificity [20] . The differences obtained can be generated by both different RT-PCR technique (in terms of equipment but also Ct cut-off) as well as by the studied population. The table 5 listed studies aimed at evaluating rapid test performance to detect SARS-CoV-2 antigens, via immunochromatographic method, compared to qRT-PCR. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In medical practice, antigen tests could be successfully used for their high specificity, for being relatively inexpensive and for largely, currently offering results within approximately 15 minutes. This study results support using Fluorecare® SARS-CoV-2 Spike Protein Test All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This manuscript is not an in Interventional clinical study and a registration number is not necessary. Data are available from the corresponding author upon request. The authors declare that there is no conflict of interest regarding the publication of this paper. This research received no external funding. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Scientific Council/Ethics Board of the Netconsult SRL, Iasi, Romania (No 2/23 March 2020). All rights reserved. No reuse allowed without permission. 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No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Predicting infectious SARS-CoV-2 from diagnostic samples Evaluation of a rapid diagnostic assay for detection of Sars-CoV-2 antigen in nasopharyngeal swabs Evaluation of a novel antigen-based rapid detection test for the diagnosis of SARS-CoV-2 in respiratory samples Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis CoV-2 Spike Protein Test Kit Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load on Risk of Intubation and Mortality Among Hospitalized Patients With Coronavirus Disease 2019 Clinical Infectious Diseases The authors would like to thank to all participants who volunteered to participate in this study.