Quantitative and qualitative test results were presented as median with interquartile range (IQR) or percentage, respectively. Correlations between the Ct values of each SARS-CoV-2 gene were compared using Spearman’s rank correlation coefficient.
The diagnostic efficiency of Roche SARS-CoV-2 Rapid Antigen Test for diagnosing SARS-CoV-2 infection was assessed by calculating the diagnostic accuracy, sensitivity and specificity, both cumulative and stratified according to NAAT Ct values. The statistical analysis was carried out using Analyse-it (Analyse-it Software Ltd, Leeds, UK) and MedCalc (MedCalc Software Ltd, Ostend, Belgium). The investigation was based on pre-existing specimens, already collected for routine SARS-CoV-2 diagnostic testing in the local facility, and thereby no patient’s informed consent, nor Ethical Committee approval were necessary. This study was conducted in accordance with the Declaration of Helsinki, under the terms of relevant local legislation.
The ongoing coronavirus disease 2019 (COVID-19) pandemic outbreak is causing dramatic clinical, societal, and economic consequences all around the world.
While this strategy has been endorsed by many international and national organizations, evidence suggests that the current testing policy for diagnosing SARS-CoV-2 infections, mostly based on nucleic acid amplification tests (NAATs), appears to be failing. It is not readily apparent that the ideal SARS-CoV-2 testing volume cannot be reached in most countries worldwide, including the most industrialized nations such as the United Kingdom .
This generates a kaleidoscope of unfavourable consequences, including delayed diagnosis and late clinical management, which would then be associated with unfavourable disease progression, insufficient contact tracing and delayed isolation of SARS-CoV-2 positive cases, which in turn further contributes to propagate onward viral transmission . Moreover, insufficient testing of individuals with high-risk exposures, most importantly, front-line healthcare workers, can lead to prolonged self-quarantine, jeopardizing health care delivery in conventional, urgent, and intensive care settings .
Due to the practical limitations of NAATs, encompassing a long turnaround time, low throughput and the need for specific instrumentation and skilled employees to conduct the test, rapid antigen immunoassay are emerging as a viable prospective for mass testing, as recently endorsed by the World Health Organization (WHO) . Despite a reportedly lower diagnostic sensitivity compared to molecular testing, which would hence limit infection identification to subsets of patients with higher SARS-CoV-2 viral load, their widespread usage under well-defined circumstances may enable ample population screenings . Nonetheless, analytical and clinical validation of these rapid immunoassays is a necessary preamble before their implementation within locally defined diagnostic algorithms. To this end, the purpose of this study was the clinical assessment of the new SARS-CoV-2 Rapid Antigen Test.
Materials and methods
- The Roche SARS-CoV-2 Rapid Antigen Test is an immunochromatographic assay for rapid qualitative detection of SARS-CoV-2 infection in nose and/or throat swabs. Briefly, the clinical specimen is collected and then deposited with twisting motion in a pre-filled extraction buffer container. After removing the swab, three drops of sample material are applied to the reagent tray (Figure 1).
- The presence of viral antigens in sufficient concentration enables their binding to specific mouse monoclonal anti-SARS-CoV-2 antibodies, then reflected by the appearance of a visual indication (i.e., a coloured line) in the lower section of the result window of the test strip, along with another “control” coloured line, which appears in the top section of the result window, when the device has been properly employed (Figure 1).
- The entire procedure can be completed within 15–30 min and does not require a dedicated environment (e.g., clinical laboratories), nor highly trained personnel to perform the test. According to manufacturer’s information, the limit of detection (LoD) per viral strain and the lowest concentration with uniform positivity per parameter are both 3.12×102.2 TCID50/mL, whilst the diagnostic sensitivity and specificity in nasopharyngeal swabs collected from both symptomatic and asymptomatic patients are reported to be 96.5 and 99.7%, respectively.
- The study population consisted of all consecutive patients referred for SARS-CoV-2 diagnostic testing to the Pederzoli Hospital (Peschiera del Garda, Verona, Italy), over a 2-week period (16–30 November, 2020). Upper respiratory specimens were collected in agreement with WHO recommendations .
- A single swab (Virus swab UTM™, Copan, Brescia, Italy) was collected from each patient and concomitantly used for both Roche SARS-CoV-2 Rapid Antigen testing and molecular testing, which was performed using a commercial revere-transcription polymerase chain reaction (RT-PCR) assay (Seegene AllplexTM2019-nCoV Assay, Seegene, Seoul, South Korea). This method uses a volume of 350 µL and enables SARS-CoV-2 RNA identification by targeting three viral genes (N, E and RdRP), thus fulfilling internationally validated testing protocols . Real-time PCR was interpreted using Seegene’s Viewer software.
- The viral load was finally expressed as cycle threshold (Ct), and test results with Ct values <37 for all three SARS-CoV-2 gene targets were considered “reactive” for SARS-CoV-2 RNA, thus specifically aimed at increasing the specificity of RNA viral detection, in accordance with current recommendations , and avoiding potential false negative test results due to emerging variants such as the VUI-202012/01 recently identified in the UK.
The final study population consisted of 321 consecutive patients (mean age, 46 years and IQR, 32–56 years; 181 women, 56.4%), who underwent SARS-CoV-2 diagnostic testing at the Pederzoli Hospital of Peschiera del Garda, Verona.
Overall, 149/321 (46.4%) samples tested positive for SARS-CoV-2 RNA with the Seegene AllplexTM2019-nCoV Assay and 109/321 (34.0%) samples tested positive with the Roche SARS-CoV-2 Rapid Antigen Test, respectively. The median Ct values of positive samples were 25 (IQR, 21–32) for the E gene, 26 (IQR, 21–31) for the RdRP gene and 22 (IQR, 18–29) for the N gene, respectively. A high Spearman’s correlation was found between the Ct values of three gene targets, as follows: E vs. RdRP gene, r=0.98 (95% CI, 0.97–0.98; p<0.001); E vs. N gene, r=0.98 (95% CI, 0.97–0.98; p<0.001); RdRP vs. N gene, r=0.97 (95% CI, 0.96–0.97; p<0.001).
The distribution of Ct values is summarized .The overall accuracy of the Roche SARS-CoV-2 Rapid Antigen Test was 86.9%, with 72.5% sensitivity and 99.4% specificity. The best performance was found by comparison antigen test results with RdRP gene (86.0% accuracy, 71.0% sensitivity), followed by comparison with E gene (84.4% accuracy, 68.8% sensitivity) and N gene (80.7% accuracy, 63.9% sensitivity).
A progressive decline in performance could be observed as the Ct values for the different SARS-CoV-2 gene targets increased. The sensitivity was found to range between 97–100% in clinical samples with Ct values <25, between 50–81% in those with Ct values between 25 and <30, but was as low as 12–18% in samples with Ct values between 30 and <37