Analytical Performances of the Point-of-Care BIOSYNEX COVID-19 Ag BSS for the Detection of SARS ‐ CoV ‐ 2 Nucleocapsid Protein in Nasopharyngeal Swabs: A Prospective Field Evaluation During the COVID-19 Third Wave in France

Background: Evaluating the accuracy and reliability of rapid diagnostic testing kits is crucial for surveillance and diagnosis of SARS-CoV-2 infections in general population. The aim of the study was to assess the analytical performances of the antigen-rapid diagnosis test (Ag-RDT) BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Freiburg, Switzerland), targeting the SARS-CoV-2 N nucleocapsid protein, for the diagnosis of COVID-19, by reference to real-time RT-PCR (rtRT-PCR). Methods: A total 967 adults living in Paris region were prospectively included during the third wave of the COVID-19 epidemic in France. Paired nasopharyngeal ocked swabs were collected at the same timepoint from persons aged ≥ 18 years receiving testing for SARS-CoV-2, at two private laboratories. Results: Overall, the Ag-RDT showed high sensitivity, specicity, PPV and NPV of 81.8%, 99.6%, 96.6% and 97.5%, respectively, as well as high or almost perfect agreement (97.0%), reliability assessed by Cohen’s κ coecient (0.87), and accuracy assessed by Youden’s J index (81.6%) to detect SARS-CoV-2. The analytical performances of the Ag-RDT remained high in the event of signicant viral excretion (i.e., N gene C t values ≤ 33 by reference rtRT-PCR), while the sensitivity of the Ag-RDT dropped to 55.2% with low or very low viral shedding (C t > 33). Conclusions: The Ag-RDT BIOSYNEX COVID-19 Ag BSS showed high specicity and sucient sensitivity for the detection of SARS-CoV-2. This test is a promising potential easy diagnostic tool, especially in situations of symptomatic COVID-19 and/or proven contagiousness. nucleocapsid protein in nasopharyngeal secretions sample. The test includes a reaction membrane, and three buffers (sample, reagent and absorbent). The reagent buffer contains colloidal gold particles conjugated to monoclonal antibodies directed against the SARS-CoV-2 N protein; the reaction membrane also contains secondary antibodies directed against the N protein. The test strip is placed inside a plastic cassette. The test was performed according to manufacturer instruction by mixing nasopharyngeal secretions with 300 µL of dilution buffer in a tube. After, one minute, 4 drops were added in the appropriate well. When the nasopharyngeal secretions are added to the sample well, crossing the strip, the dry conjugates of the reagent buffer are solubilized, allowing the conjugate to migrate with the sample and to react with the anti-SARS-CoV-2 antibodies immobilized on the membrane. If SARS-CoV-2 antigens are present in the sample, the complexes between the anti-SARS-CoV-2 conjugate and the virus are captured by anti-SARS-CoV-2 monoclonal antibodies specic to the test line area (T). The absence of a T line suggests a negative result. To serve as a procedural control, a red line appears in the control line area (C), indicating that the correct volume of sample has been added and that the membrane has played its role. Visual interpretation of results is performed 15 min after. A questionnaire capturing demographic information (sex and age), reasons for testing and current and past–14-day symptoms for symptomatic patients was administered to all participants. Suggestive symptoms of COVID-19 were headache, fatigue, fever, or upper or lower respiratory symptoms. Asymptomatic individuals were dened as those not reporting any of these symptoms. Inclusion criteria were aged more than 18 years and agreement to undergo two concurrent nasopharyngeal swabs for rtRT-PCR and Ag-RDT. Thus, at both sites, a health care professional rst collected nasopharyngeal secretions in one nostril, using the swab provided in the BIOSYNEX COVID-19 Ag BSS kit, immediately followed by a nasopharyngeal swab for rtRT-PCR. Covid-19 antigen rapid testing was immediately carried out on-site using the Ag-RDT according to the manufacturer's instructions. The other nasopharyngeal swabs were stored in physiological serum (NaCl 0.9%) (1000 µL) at + 4°C and analyzed within 24–48 hours by the reference rtRT-PCR. of rtRT-PCR concordance The reliability between multiplex estimated κ coecient [11], ranked Landlis Youden’s index (J = sensitivity specicity Bayes’s formulae, prevalence in on April e.g. around peak of third (Santé https://www.santepubliquefrance.fr/


Introduction
The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. The effective isolation and early treatment of patients infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require rapid, simple and accurate diagnostic tools.
While currently-recommended nucleic acid ampli cation tests (NAAT), such as real-time reverse transcription polymerase chain reaction (rtRT-PCR) assays, remain the gold standard cornerstone for the diagnosis of SARS-CoV-2 infection [1,2], immunological methods can also be used to detect viral antigens [2][3][4]. Indeed, performing rtRT-PCR is expensive, time-consuming, and requires special equipment and quali ed operators. Faster, cheaper, and easier to use alternative tools could be represented by novel point-of-care antigen-detecting rapid diagnostic tests (Ag-RDT) [3]. Ag-RDT relies on direct detection of SARS-CoV-2 viral proteins produced by replicating virus in nasal swabs and other respiratory secretions, often the virus N nucleocapsid protein, preferred because of its relative abundance and conserved structure, or other viral proteins such as the spike protein [4]. Most Ag-RDTs use sandwich catching by anti-SARS-CoV-2 monoclonal antibodies to detect viral antigens in the simple-to-use lateral ow immunoassay format allowing results in < 30 minutes. Around 180 Ag-RDTs for SARS-CoV-2 infection are currently commercially available or in development [5]. However, there is signi cant variability reported with respect to their diagnostic performances and a lack of external validation for many of the available tests, which still require clinical validation [6][7][8][9].
Our study aimed to evaluate in the eld the Ag-RDT BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Freiburg, Switzerland; reference SW40006) for the diagnosis of COVID-19 using prospectively collected samples from adults living in the Paris region during the third wave of the COVID-19 epidemic in France.
The results of this test were compared with qualitative and quantitative results obtained in parallel using rtRT-PCR as a reference test.

Material And Methods
Rapid antigen test. The Ag-RDT BIOSYNEX COVID-19 Ag BSS is a rapid qualitative membrane-based immunochromatographic test that uses highly sensitive monoclonal antibodies to detect the SARS-CoV-2 N nucleocapsid protein in nasopharyngeal secretions sample. The test includes a reaction membrane, and three buffers (sample, reagent and absorbent). The reagent buffer contains colloidal gold particles conjugated to monoclonal antibodies directed against the SARS-CoV-2 N protein; the reaction membrane also contains secondary antibodies directed against the N protein. The test strip is placed inside a plastic cassette. The test was performed according to manufacturer instruction by mixing nasopharyngeal secretions with 300 µL of dilution buffer in a tube. After, one minute, 4 drops were added in the appropriate well. When the nasopharyngeal secretions are added to the sample well, crossing the strip, the dry conjugates of the reagent buffer are solubilized, allowing the conjugate to migrate with the sample and to react with the anti-SARS-CoV-2 antibodies immobilized on the membrane. If SARS-CoV-2 antigens are present in the sample, the complexes between the anti-SARS-CoV-2 conjugate and the virus are captured by anti-SARS-CoV-2 monoclonal antibodies speci c to the test line area (T). The absence of a T line suggests a negative result. To serve as a procedural control, a red line appears in the control line area (C), indicating that the correct volume of sample has been added and that the membrane has played its role. Visual interpretation of results is performed 15 min after.
Study population and procedures. Paired nasopharyngeal ocked swabs were collected at the same timepoint from persons aged ≥ 18 years receiving testing for SARS-CoV-2, at two private laboratories (site A: Centre Cardiologique du Nord, Saint-Denis, France; site B: Laboratoire Paris XV, Paris, France) during the third wave of the COVID-19 epidemic (March and April 2021). The sites offered SARS-CoV-2 testing to anyone in the community who wanted testing for suspected COVID-19, travel, as a pre-operative assessment, as a contact-case exposure of an individual infected with SARS-CoV-2 or as control of SARS-CoV-2 infection in the 30 days preceding. A questionnaire capturing demographic information (sex and age), reasons for testing and current and past-14-day symptoms for symptomatic patients was administered to all participants. Suggestive symptoms of COVID-19 were headache, fatigue, fever, or upper or lower respiratory symptoms. Asymptomatic individuals were de ned as those not reporting any of these symptoms. Inclusion criteria were aged more than 18 years and agreement to undergo two concurrent nasopharyngeal swabs for rtRT-PCR and Ag-RDT. Thus, at both sites, a health care professional rst collected nasopharyngeal secretions in one nostril, using the swab provided in the BIOSYNEX COVID-19 Ag BSS kit, immediately followed by a nasopharyngeal swab for rtRT-PCR. Covid-19 antigen rapid testing was immediately carried out on-site using the Ag-RDT according to the manufacturer's instructions. The other nasopharyngeal swabs were stored in physiological serum (NaCl 0.9%) (1000 µL) at + 4°C and analyzed within 24-48 hours by the reference rtRT-PCR.
Reference multiplex molecular detection of SARS-CoV-2. Nucleic acid extraction was performed from 300 µL elution volume of nasopharyngeal ocked swab sample, using EX3600 extractor (Liferiver & Shanghai ZJ Bio-Tech Co.), according to the manufacturer's instructions, and nally eluted in 50 µL ( nal volume). SARS CoV-2 was detected in 5 µL of extracted RNA using the multiplex real-time PCR Novel Coronavirus (2019-nCoV) Real-Time Multiplex RT-PCR Kit (Detection for 3 Genes) (Liferiver & Shanghai ZJ Bio-Tech Co., Ltd, Shanghai, China), which constituted the reference multiplex rtRT-PCR for SARS-CoV-2 RNA detection. This assay can simultaneously detect 3 coronavirus target genes, including the SARS-like (including SARS-CoV-2, SARS-CoV, bat SARS-like coronavirus) conserved region of envelope protein gene (E), RNA-dependent RNA polymerase gene (ORF1ab of RdRP gene) and nucleocapsid protein gene (N), using reverse transcription followed by real-time PCR, providing individual cycle threshold (C t ) values for each target gene. Real-time PCR was carried out with CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. The experiment and result interpretation were carried out according to the manufacturer's protocol. According to the manufacturer's instructions, samples showing an exponential growth curve and Ct value ≤ 41 were considered positive. A unique C t value > 41 was considered negative.
Statistical analyses. Data were entered into an Excel database and analyzed using IBM® SPSS® Statistics 20 software (IBM, SPSS Inc, Armonk, New York, USA). Medians were calculated for quantitative variables. The results were presented along with their 95% con dence interval (CI) using the Wilson score bounds for categorical variables [10]. Comparisons of frequencies between positive and negative results of Ag-RDT and rtRT-PCR testing between sites and all other variables were computed using the Pearson's Chi-square test or Fisher's exact test according to their validity conditions. The results of SARS-CoV-2 RNA detection by the multiplex rtRT-PCR were used as the reference standard to estimate the sensitivity and speci city of the study Ag-RDT, with corresponding 95% CI. The concordance between study Ag-RDT and multiplex molecular detection of SARS-CoV-2 RNA was assessed by percent agreement corresponding to the observed proportion of identical results between Ag-RDT compared to rtRT-PCR detection. The reliability between the study Ag-RDT and the multiplex molecular detection of SARS-CoV-2 RNA was estimated by Cohen's κ coe cient [11], and the degree of agreement was determined as ranked by Landlis and Koch [12]. The accuracy of the study Ag-RDT to correctly diagnose SARS-CoV-2 infection was estimated by Youden's J index (J = sensitivity + speci city − 1) [13]. Positive predive values (PPV) and negative predictive values (NPV) were calculated according to Bayes's formulae, by considering the o cial reported prevalence of SARS-CoV-2-RNA positivity in symptomatic patients in Paris's area, France, on 12th April 2021, e.g. around the peak of the third wave epidemic in France (Santé publique France 2021; https://www.santepubliquefrance.fr/ ). Ethics statement. The study was used as a clinical evaluation of the continuous quality improvement program and COVID-19 management measures performance evaluation, according to the national law on the accreditation of medical biology laboratories [14]. The dataset was completely anonymous and did not contain any identi able personal health information.
The analytical results according to the level of viral excretion assessed by the N gene C t values by the reference rtRT-PCR are shown in Table 3. Overall, the Ag-RDT BIOSYNEX COVID-19 Ag BSS showed high or almost perfect agreement (97.0%), reliability assessed by Cohen's κ coe cient (0.87), and accuracy assessed by Youden's J index (81.6%) to detect SARS-CoV-2. These analytical performances were further strati ed according to the cycle threshold (C t ) values of the N gene detected by reference rtRT-PCR considering C t -related criteria of very high (C t ≤20) and high (C t ≤33) SARS-CoV-2 RNA excretion. Indeed, viral loads with C t > 33 are considered to be low and correspond to moderate or very low viral excretion [15][16][17][18]. Conversely, samples with C t ≤33 have a signi cant SARS-CoV-2 viral load, as in individuals symptomatic for COVID-19 or contagious. C t values ≤ 20 indicate very high viral shedding [16][17][18]. There were two distinct situations. In the event of signi cant viral loads (high or very high) in real-time PCR (C t ≤33), the Ag-RDT S BIOSYNEX COVID-19 Ag BSS showed excellent analytical performances, with sensitivities between 83.3% and 100.0%, speci cities of 99.8%, PPV between 98.3% and 98.6% and NPV between 97.7% and 100.0%. In the event of low or very low viral loads (C t > 33), the sensitivity of the Ag-RDT BIOSYNEX COVID-19 Ag BSS showed reduced analytical performances with 55.2% sensitivity, while its speci city remained high (98.8%).

Discussion
We herein evaluated the analytical performances of the novel point-of-care Ag-RDT BIOSYNEX COVID-19 Ag BSS by reference to multiplex rtRT-PCR for SARS-CoV-2 RNA detection as the gold standard in a real-life community setting. In this evaluation, the sensitivity of the Ag-RDT BIOSYNEX COVID-19 Ag BSS was lower among specimens from asymptomatic persons (79.4%) than among specimens from symptomatic persons (95.0%). Speci city (> 99.0%) was high in specimens from both asymptomatic and symptomatic groups. The prevalence of having SARS-CoV-2 RNA-positive rt-RT-PCR results in this population was relatively high (12.8% overall; 6.5% for asymptomatic participants and 31.4% for symptomatic participants), and the estimated PPVs and NPVs of the Ag-RDT BIOSYNEX COVID-19 Ag BSS were elevated in all groups of participants. However, administering the Ag-RDT in lower prevalence settings will likely result in lower predictive values. In the event of signi cant viral excretion (i.e., N gene C t values below 33 by reference rtRT-PCR), the Ag-RDT RDT BIOSYNEX COVID-19 Ag BSS showed high sensitivity (from 83.3-100.0%) and speci city (> 99.0%) for SARS-CoV-2 RNA detection, with excellent concordance, reliability and accuracy with the reference multiplex rtRT-PCR, and PPVs and NPVs above 97.0%. The sensitivity of the study Ag-RDT dropped however to 55.2% with low or very low viral shedding (C t > 33). Taken together, these observations demonstrate that the Ag-RDT BIOSYNEX COVID-19 Ag BSS harbored high analytical performances, which makes it suitable to be used as point-of-care Ag-RDT in various hospital and non-hospital settings where a rapid diagnosis of SARS-CoV-2 is necessary. Although less sensitive than RT-PCR, the Ag-RDT BIOSYNEX COVID-19 Ag BSS could be bene cial due to its rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase its diagnostic value.
In the present series, the sensitivity of the Ag-RDT BIOSYNEX COVID-19 Ag BSS was 81.8% overall, and the positive detection rate was comparable to the rtRT-PCR in the majority (88.2%) of patients with C t ≤ 33. Twelve of 14 (85.7%) false-negative subjects with signi cant viral excretion (C t ≤ 33) were asymptomatic, although con icting evidence exists regarding the relationship between symptom severity and viral shedding [19]. In the present large series, false-positive test results were rarely observed, providing 99.6%-speci city in our study, which exceeded the performance recommended by the World Health Organization (WHO) [20]. Some false-positive results have been reported in other antigen tests [21][22][23]. While de nitive proof is lacking, possible causes for the false-positives include the high viscosity of specimens and interference of human antibodies [24].
We analyzed our results according to the estimated viral load in SARS-CoV-2 in the samples. There is an ongoing debate regarding the C t value corresponding to the threshold of infectivity (i.e., patient considered as contagious) [16]. Indeed, there is a trend to a natural gradual decrease of the SARS-CoV-2 RNA load in the nasopharyngeal samples overtime during the course of infection, at the origin of varying levels of contagiousness [43]. La Scola et al. found that patients with C t value > 33 are not contagious because of the low number of positive cultures [44]. This is consistent with the Centers for Disease Control and Prevention (CDC) recommendations, which propose a C t value of 33 as a surrogate of contagiousness [15], with C t values ≤ 20 indicating very high viral shedding [16][17][18]. In our series, we have strati ed the nasopharyngeal samples according to the level of viral excretion, indirectly evaluated by the value of the C t of the N gene according to the reference rtRT-PCR, in order to calculate the performance of the study Ag-RDT at different proposed cut-offs for contagiousness.
Our results clearly show that the analytical performances of the Ag-RDT BIOSYNEX COVID-19 Ag BSS were much better in the event of a high viral load, i.e., in the case of signi cant viral excretion. These observations demonstrate the interest of the Ag-RDT BIOSYNEX COVID-19 Ag BSS as a rapid rule-in test for COVID-19 with samples at high viral load, in symptomatic patients for example, and point caution with its use as a singular rule-out test especially in the setting of samples with lower viral loads.
The SARS-CoV-2 RNA positive subpopulation of our clinical samples collection was characterized by a wide range of C t -values with medium and low C t -values dominating. This allowed the calculation of sensitivity and speci city values with higher relevance for clinical practice. The C t -dependent evaluation showed very good sensitivity for highly and moderately SARS-CoV-2 positive samples (C t ≤33). In contrast, the sensitivity of the assay with specimens containing only a limited viral load was lower. Thus, COVID-19 infection would not be detected in patients in the very early or late phase of the infection typically associated with a low viral load. However, differentiation between contagious and non-contagious individuals may be possible with this assay. Samples with C t -values > 33 usually do not allow culturing of the virus indicating low infectivity [16,44]. Such individuals may be regarded as non-contagious despite carrying low virus loads. This differentiation of individuals may be of particular importance for the decision on access to susceptible individuals, for example in nursing homes or in many other medical circumstances. Similar observations of dramatic decrease of sensitivity of Ag-RDT for SARS-CoV-2 antigen detection at C t thresholds around 25-33 were previously reported [7,38,45,46], con rming that Ag-RDTs were most effective to identify RT-PCR positive symptomatic patients or asymptomatic subjects with high viral loads in their respiratory secretions (i.e., C t values ≤ 33).
In our study, the accuracy of the Ag-RDT BIOSYNEX COVID-19 Ag BSS was estimated by the percent positive agreement and not sensitivity. Since the agreement is measured relative to an RT-PCR test, which may be imperfect itself [47]. Compounding this uncertainty, we have largely exceeded the minimum sample size of 30 positive cases that is required to apply for evaluation [8], which made it possible to restrict the con dence intervals of the evaluated variables.
Our study has several strengths. All samples were collected from one nasopharynx with ocked swabs, which is optimal for the evaluation of Ag-RDT clinical performances in our study. The Ag-RDT was performed in parallel to RT-PCR. The study population included a variety of situations outside the hospital setting with a majority of young adults without comorbidities, who mostly had typical and mild COVID-19 symptoms when being symptomatic. This currently describes the majority of SARS-CoV-2 infected individuals, and an important group for limiting community transmission. The ndings in this investigation are also subject to limitations. Participants might have inadvertently reported common nonspeci c symptoms as COVID-19-compatible symptoms. This investigation evaluated the BIOSYNEX COVID-19 Ag BSS antigen test, and the results presented here cannot be generalized to other agencies-authorized SARS-CoV-2 antigen tests. Finally, the BIOSYNEX COVID-19 Ag BSS antigen test characteristics might be different depending on whether an individual had been previously tested positive.

Conclusion
The Ag-RDT BIOSYNEX COVID-19 Ag BSS showed very high speci city and su cient sensitivity for the detection of SARS-CoV-2. Given the simple procedures and shorter turnaround time involved with this test, it is a promising option as an alternative diagnostic modality especially in situations of symptomatic COVID-19 and/or proven contagiousness. The test can also be offered to test asymptomatic individuals in many situations of potential exposure to SARS-CoV-2 and in mass screening at the population level.

Declarations
Ethics approval and consent to participate. The study has been approved by the local scienti c committee of Parc de l'Innovation, Strasbourg, France, and informed participants consent was obtained.
Consent for publication. All authors approved the submission of the manuscript for publication.
Availability of data and materials. The data is available and can be used for the academic or research purposes.
Competing interest. The authors report no con icts of interest. The authors alone are responsible for the content and the writing of the paper.