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%). Specificity (> 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 significant viral excretion (i.e., N gene Ct 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 specificity (> 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 (Ct> 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 beneficial 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 Ct ≤ 33. Twelve of 14 (85.7%) false-negative subjects with significant viral excretion (Ct ≤ 33) were asymptomatic, although conflicting evidence exists regarding the relationship between symptom severity and viral shedding . In the present large series, false-positive test results were rarely observed, providing 99.6%-specificity in our study, which exceeded the performance recommended by the World Health Organization (WHO) . Some false-positive results have been reported in other antigen tests [21–23]. While definitive proof is lacking, possible causes for the false-positives include the high viscosity of specimens and interference of human antibodies .
Finally, the Ag-RDT RDT BIOSYNEX COVID-19 Ag BSS fulfilled the current WHO’s recommendations for a screening Ag-RTD stating that, at minimum, Ag-RDTs would need to correctly identify significantly more cases than they would miss (sensitivity ≥ 80%) and would have very high specificity (≥ 97–100%) . Furthermore, analytical performances of comparable order as those of our study Ag-RDT were previously reported for some Ag-RDTs in lateral flow immunoassay format [7, 9, 21, 25–35], while several studies have reported much lower sensitivity levels contrasting with always high specificity [3, 36–41]. For example, a comparable Ag-RDT such as the novel COVID-VIRO® from AAZ (Boulogne Billancourt, France) showed a sensitivity of 96.7% and a specificity of 100% in a real-life community setting . In addition, the Ag-RDT RDT BIOSYNEX COVID-19 Ag BSS fulfilled also the current recommendations of the French High Authority of Health (Haute Autorité de santé, Saint-Denis, France) for a screening Ag-RTD stating that, at minimum, Ag-RDTs would need to correctly identify significant proportions of symptomatic patients (sensitivity ≥ 80%) as well as asymptomatic individuals (sensitivity ≥ 50%) and would have very high specificity (≥ 90%) .
We analyzed our results according to the estimated viral load in SARS-CoV-2 in the samples. There is an ongoing debate regarding the Ct value corresponding to the threshold of infectivity (i.e., patient considered as contagious) . 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 . La Scola et al. found that patients with Ct value > 33 are not contagious because of the low number of positive cultures . This is consistent with the Centers for Disease Control and Prevention (CDC) recommendations, which propose a Ct value of 33 as a surrogate of contagiousness , with Ct values ≤ 20 indicating very high viral shedding [16–18]. In our series, we have stratified the nasopharyngeal samples according to the level of viral excretion, indirectly evaluated by the value of the Ct 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 significant 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 Ct-values with medium and low Ct-values dominating. This allowed the calculation of sensitivity and specificity values with higher relevance for clinical practice. The Ct-dependent evaluation showed very good sensitivity for highly and moderately SARS-CoV-2 positive samples (Ct ≤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 Ct-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 Ct thresholds around 25–33 were previously reported [7, 38, 45, 46], confirming 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., Ct 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 . Compounding this uncertainty, we have largely exceeded the minimum sample size of 30 positive cases that is required to apply for evaluation , which made it possible to restrict the confidence intervals of the evaluated variables.
Our study has several strengths. All samples were collected from one nasopharynx with flocked 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 findings in this investigation are also subject to limitations. Participants might have inadvertently reported common nonspecific 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.