While nucleic acid amplification tests remain the gold‐standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections, immunological methods can also be used to detect viral antigens or specific antibodies.^1-9 Several rapid diagnostic tests employing antigen detection (Ag‐RDTs) are now commercially available. However, there remains limited evidence on the utility of Ag‐RDTs for coronavirus disease 2019 (COVID‐19) diagnosis and surveillance, with significant variability reported with respect to their diagnostic performance and a lack of external validation for many of the available tests.¹⁰ Thus, we initiated our study to evaluate an Ag‐RDT, the PCL COVID‐19 Ag rapid fluorescent immunoassay (FIA), for diagnosis of COVID‐19 in hospitalized patients with suspect SARS‐CoV‐2 infection. The results of this test were compared with qualitative and quantitative results of parallel reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) examinations.

Patients hospitalized with suspected COVID‐19 between May and September 2020 at a primary care hospital in Siedlce, Poland, were enrolled in this prospective observational study. Patients were simultaneously sampled by nasopharyngeal swabs for antigen detection and RT‐PCR. Swabs were collected four times at 2 days intervals from all but one patient, that was examined only three times. Blood samples for serology testing were taken two times at a 6‐day interval, together with the first and last nasopharyngeal sample. The antigenic assessment was performed using PCL COVID‐19 Ag Rapid FIA (PCL) as a point of care test in accordance with the manufacturer's instructions. The molecular assessment was performed within a few hours after collection at the central COVID‐19 laboratory in Warsaw using commercial RT‐PCR kits (Liferiver, DiaplexQ, or Vitassay). Serology testing for anti‐SARS‐CoV‐2 immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA) was performed using ELISA (Euroimmun). This study was performed in accordance with the Declaration of Helsinki and under patient informed consent with anonymization of data.

A total of 42 patients (21 male and 21 female) with a median age of 57.7 years (range, 21–78) were included resulting in collection of 167 nasopharyngeal swabs. The median time between onset of symptoms and the first sample taken was 8.3 days (range, 2–16).

A summary of results over the course of the study are presented in Figure 1. Among the 167 nasopharyngeal swabs, 95 (56.9%) were positive, 26 (15.6%) were inconclusive, and 46 (27.5%) were negative for SARS‐CoV‐2 by RT‐PCR, while only 25 (15.0%) of swabs were positive by Ag‐RDT. The overall percent agreement between Ag‐RDT and RT‐PCR was 48.9%. Positive results of RT‐PCR were obtained in 36 (85.7%) patients, while 1 (2.4%) patient had inconclusive RT‐PCR results, 1 (2.4%) patient had negative results, and 4 (9.5%) had mixed negative and inconclusive RT‐PCR results over their course of hospitalization. Ag‐RDT revealed antigen detection in 15 (35.7%) patients. In one case, Ag‐RDT was positive in a patient with a negative RT‐PCR results at all time points. Using RT‐PCR as reference standard, the specificity of the Ag‐RDT was 83.3%. Among the 36 RT‐PCR‐positive patients, the Ag‐RDT test was positive in only 14 cases, resulting in a sensitivity of 38.9%. Among the 15 Ag‐RDT positive patients, nine had SARS‐CoV‐2 antigen detected only in the first sample, two patients in two samples, and four cases in three consecutive samples. No patient had persistence of a positive Ag‐RDT result in all four samples obtained during the study. In patients with a positive Ag‐RDT, cycle threshold (CT) values for the Orf1ab gene in the RT‐PCR test ranged from 18.5 to 36.5 (mean 27.8). In all cases, Ag‐RDT positivity was observed no later than 10 days after symptom onset (Table S1). The results of serology testing revealed the presence anti‐SARS‐CoV‐2 antibodies in 41 out of 42 (97.6%) patients. Anti‐SARS‐CoV‐2 IgA (90.5%) and IgG (85.7%) were frequently observed, while anti‐SARS‐CoV‐2 IgM was detected in only 20 (47.6%) patients.

In our investigation, the Ag‐RDT PCL COVID‐19 Ag rapid FIA had a sensitivity of 38.9% and specificity 83.3%, with only 48.9% overall agreement between Ag‐RDT and RT‐PCR. The observed sensitivity of the PCL Ag‐RDT was significantly lower than that announced by the manufacturer, who reported a sensitivity of 100% (27/27) and a specificity of 97.8% (44/45). Nonetheless, the results of an Ag‐RDT depend on several factors, such as time from the symptom onset, the specimen viral content, and other preanalytical and analytical considerations.^{2, 4, 10, 11} In our study, the PCL COVID‐19 Ag rapid FIA had a sensitivity 92.9% for CT values under 25, which demonstrated that CT values were crucial to the results of Ag‐RDT. Thus, PCL COVID‐19 Ag Rapid FIA was more sensitive at high viral loads and therefore its utility may be limited to patients with a recent onset of symptoms, when viral load is very high.

Although robust and simple in performance, this Ag‐RDT presented insufficient capabilities to replace the RT‐PCR. Similar conclusions about different RDTs have been presented by others.^3-5 However, despite relatively low clinical sensitivity among Ag‐RDTs, they may be helpful at reducing the number of patients requiring RT‐PCR and the burden on laboratories, particularly at a time of rapidly increasing number of COVID‐19 cases worldwide. Nonetheless, the performance of this particular Ag‐RDT limits its applicability for both clinical and community testing. While viral loads in patients with presymptomatic or asymptomatic disease are lower as compared to symptomatic patients, thus leading to limited detection of SARS‐CoV‐2 by Ag‐RDTs in such individuals,¹⁰ this may be deemed acceptable, as such a low viral load seemingly correlates to the reported reduced transmission potential among asymptomatic or presymptomatic persons.¹² However, this assay failed to accurately detect SARS‐CoV‐2 in symptomatic hospitalized patients, raising substantial concerns over its utility. The low sensitivity could lead to missing a substantial number of symptomatic COVID‐19 cases with potentially high transmission potential or leading to delays in initiating self‐quarantine procedures and contract tracing while pending repeat testing. Further studies should evaluate this assay in a larger, more diverse cohorts.

In conclusion, we observed low sensitivity for the PCL COVID‐19 Ag rapid FIA in detecting active SARS‐CoV‐2 infection in hospitalized patients. Moreover, the overall agreement between Ag‐RDT and RT was poor. These results highlight the need for careful evaluation and external validation of novel Ag‐RDT assays before implementation in clinical practice or in community surveillance.