Real-world assessment of Fluorecare SARS-CoV-2 Spike Protein Test Kit

Introduction

Due to the huge pressure imposed on routine diagnostic laboratories by the ongoing coronavirus disease 2019 (COVID-19) pandemic outbreak, rapid antigen tests hold premise as suitable alternatives for widespread, rapid and efficient identification of subjects with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [1]. Nonetheless, as recently endorsed by both the World Health Organization (WHO) [2], and by the Task Force on COVID-19 of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) [3], the clinical performance of each SARS-CoV-2 antigen rapid detection test (Ag-RDT) must be thoroughly evaluated before its introduction – at whatever stage – into clinical practice. Therefore, this work was aimed to assess the clinical performance of Fluorecare SARS-CoV-2 Spike Protein Test Kit in a real-life scenario.

Materials and methods

Results

The final study population consisted of 354 patients (mean age, 47 ± 20 years; 195 women, 55.1%), 223 of whom (65.8%) were positive at NAAT (i.e., Ct values of both SARS-CoV-2 S and E genes <45). The Ct values of positive samples were 29.8 ± 7.1 and 30.3 ± 7.0 for the SARS-CoV-2 S and E genes, respectively. A highly significant Spearman’s correlation was found between values of Fluorecare SARS-CoV-2 Spike Protein Test Kit and measurable Ct values of S (r=−0.75; 95% CI, −0.80 to −0.69; p<0.001) and E (r=−0.75; 95% CI, −0.80 to −0.68; p<0.001) genes.

The overall diagnostic performance of Fluorecare SARS-CoV-2 Spike Protein Test Kit, as well as performance stratified according to Altona Ct values, is summarized in Table 1. The cumulative area under the curve (AUC) in all nasopharyngeal samples was 0.68. At the ≥1.0 S/CO manufacturer’s cut-off, the sensitivity, specificity, NPV and PPV values were 27.5, 99.2, 41.5 and 98.5%, respectively. The best cut-off calculated from the ROC curve was 0.18 S/CO, associated with 42.9% (95% CI, 36.5–49.5%) sensitivity, 92.6% (95% CI, 86.4–96.5%) specificity, 45.7% (95% CI, 42.7–48.8%) NPV and 91.7% (95% CI, 85.4–95.5%) PPV. Nonetheless, considerable improvement of diagnostic sensitivity was observed as the Ct values decreased, increasing to 66.7% in samples with mean Ct values <30, 90.5% in those with mean Ct values <25, up to 100% in those with very high viral load (i.e., mean Ct values <20).

The distribution of Fluorecare SARS-CoV-2 Spike Protein Test Kit values according to the Altona thresholds for sample positivity (i.e., Ct values of both SARS-COV-2 S and E genes <45) or higher risk of infectivity (i.e., Ct values of both SARS-COV-2 S and E genes <26.3) is shown in Figure 1. The mean Fluorecare SARS-CoV-2 Spike Protein Test Kit values were found to be significantly higher in samples with Ct values <45 (n=233; 2.1 ± 3.6 S/CO) than in those with higher Ct values (n=121; 0.1 ± 0.1 S/CO; p<0.001), as well as in those with Ct values <26.3 (n=68; 6.7 ± 4.0 S/CO), than in those Ct values over this threshold (n=286; 0.2 ± 0.2 S/CO; p<0.001).

Figure 1:

Distribution of Fluorecare SARS-CoV-2 Spike Protein Test Kit values according to the Altona thresholds for sample positivity (i.e., Ct values of both SARS-COV-2 S and E genes <45) and higher risk of infectivity (i.e., Ct values of both SARS-COV-2 S and E genes <26.3).

Ct, cycle threshold; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S/CO, signal to cut-off.

Discussion

SARS-CoV-2 antigen testing, now mostly represented by Ag-RDT, is emerging as a possible alternative to overcome the dramatic burden posed on clinical laboratories by COVID-19 diagnostics [1, 4, 5]. A recent analysis of the Cochrane COVID-19 Diagnostic Test Accuracy Group has highlighted the theoretical advantages and possible drawbacks of these techniques [6]. As concerns the benefits, these tests are indeed simple, can be used easily by quickly trained staff or even by the patients themselves, do not necessitate centralized laboratory instrumentation and provide manageable results in a very short time. On the other hand, the clinical performance of Ag-RDTs is still considerably lower than that of molecular assay, and their cumulative diagnostic sensitivity remains limited at around 60–75% [6]. Especially modest performance has been reported in samples with low viral load (i.e., Ct values ≥25), with diagnostic sensitivity comprised between 30 and 50%, but increasing to 91–97% in samples with higher viral load (i.e., Ct values <25). Nevertheless, a rather broad heterogeneity of diagnostic performance of the different kits has been noted by the Cochrane COVID-19 Diagnostic Test Accuracy Group, with sensitivities ranging between dramatically low estimates (i.e., 12%), up to exceptionally high values (i.e., 90%, or even higher). This uncertain scenario has persuaded both the WHO and the IFCC Task Force on COVID-19 to recommend that each Ag-RDT should undergo a local validation process before being introduced into clinical practice [2], [3].

The results of our work have two major clinical implications. First, the relatively modest overall sensitivity and NPV of Fluorecare SARS-CoV-2 Spike Protein Test Kit would make it unadvisable to use this assay as alternative or even surrogate of conventional NAAT for precisely diagnosing SARS-CoV-2 infections. Nonetheless, its satisfactory sensitivity at higher nasopharyngeal viral load could make it a suitable test for screening patients with higher infective potential. A recent study, published by Gniazdowski et al. [7], found that the likelihood of obtaining a positive SARS-CoV-2 viral culture was <3% in nasopharyngeal samples with Altona Ct values <26.2. At a similar Ct threshold, Fluorecare SARS-CoV-2 Spike Protein Test Kit displayed a diagnostic sensitivity of around 90% in our cohort of unselected patients undergoing routine COVID-19 diagnostics, which would have permitted to identify the vast majority of individuals with high viral in the upper respiratory tract, who are those at the largest risk of spreading the virus in the community (i.e., the so-called super-spreaders), especially during mass gathering events [8].

Another important aspect to be considered, is that the nasopharyngeal viral load not only influence the likelihood of inter-individual SARS-CoV-2 transmission, but is also positively associated with the risk of progression towards severe/critical COVID-19 illness and/or death, as already demonstrated by several previous studies [9], [10], [11]. Therefore, rapid and efficient identification and/or monitoring of COVID-19 patients bearing high nasopharyngeal SARS-CoV-2 load during triage (even outside healthcare facilities), as well as during hospital stay or intensive care unit admission, would enable to establish a more timely and aggressive treatment, thus enhancing the likelihood of a positive outcome of this life-threatening disease.

In conclusion, our findings would hence suggest reserving the use of this Ag-RDT for identifying patients with higher viral load.