Evaluation of the Panbio™ COVID-19 ag rapid test at an emergency room in a hospital in São Paulo, Brazil

Background The performance characteristics of the Panbio™ COVID-19 Ag test was evaluated at an emergency room setting against RT-PCR, considered the gold-standard for the detection of SARS-CoV-2, in São Paulo, Brazil. The study aimed to determine the sensitivity, specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA) as compared to RT-PCR. Methods Specimens from 127 suspected patients were tested by both the Panbio™ COVID-19 Ag test and by RT-PCR. Results In relation to RT-PCR using Ct values ≤ 40 as the upper limit for positivity, the Panbio™ COVID-19 Ag test showed an overall sensitivity of 84.3% (95% CI 75‒93.8%) and 98.2% (95% CI 96‒98.8%) overall specificity. For Ct values ≤ 25 (n = 37), the Panbio™ COVID-19 Ag test showed 97% sensitivity. Discussion The concordance between the Panbio™ COVID-19 Ag test and RT-PCR was 97% at Ct values below 25 but decreased at higher Ct values. For disease control, it is very important to identify infected individuals who present COVID-19 symptoms and also those who are suspected of infection due to contact with infected individuals. Conclusion The Panbio™ COVID-19 Ag test is suitable for use as a diagnostic test for rapid screening of patients presenting COVID-19 symptoms, or those suspected of being infected, prior to being admitted to hospital.


Background
In late 2019, an outbreak of respiratory illness of unknown etiology was reported in Wuhan City, Hubei Province, China. The International Committee for Taxonomy of Viruses (ICTV) named the virus SARS-CoV-2 and is the cause of the global COVID-19 pandemic which, as of 18th February 2022, has claimed more than 5.8 million lives (https://covid19.who.int/). The preferred diagnostic test for COVID-19 is Nucleic Acid Amplification Test (NAAT) using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) performed in fluid from the nasal or nasopharyngeal cavities. The turn-around time can vary from same one day to one week. 1-3 Alternatively, a COVID-19 antigen test using nasal or nasopharyngeal samples can produce results in minutes. 4 A positive antigen test result is considered accurate, but low viral load in the sample can produce false-negative results. 5,6 The Abbott Panbio TM COVID-19 Ag Rapid Test device is an in vitro rapid diagnostic test intended to aid in the diagnosis of COVID-19. The Panbio TM COVID-19 Ag Rapid Test device is a lateral flow immunochromatographic test used for the qualitative detection of SARS-CoV-2 antigens in human tissue fluids obtained from nasal or nasopharyngeal swabs. The Panbio TM COVID-19 Ag platform uses a cassette containing a lateral flow test strip and is intended for use by trained healthcare professionals in point-of-care and hospital settings. The product may be used in both laboratory and nonlaboratory environments that meet the requirements specified in the product's Instructions For Use (IFU).
The Panbio TM COVID-19 Ag test can be used for rapid screening in patients presenting COVID-19 symptoms, or in those who had contact with infected persons, prior to being admitted to hospital. In this study, the diagnostic performance, namely sensitivity, specificity, positive and negative predictive values, of the Panbio TM COVID-19 Ag Rapid test device using fluids obtained from nasopharyngeal swabs was assessed having RT-PCR test as gold-standard.

Study population
This cross-sectional study was conducted at São Paulo Hospital, São Paulo, Brazil. Patients (≥18 years), treated at the emergency room and admitted for at least 24 hours were included if they met one of the following criteria: (1) symptoms suspected to be related to SARS-CoV-2 and/or contact with COVID-19 infected persons, (2) decompensation of underlying disease, or (3) suggestive tomographic alteration (ground glass opacities).

Testing scheme
Nasopharyngeal swab samples were simultaneously tested using the Panbio TM COVID-19 Ag test and by RT-PCR. The Panbio TM COVID-19 Ag test was performed according to the product Instructions For Use (IFU). 7 Results were obtained and interpreted at 15 minutes after test initiation according to the IFU. The RT-PCR testing was performed with GeneFinder TM COVID-19 Plus RealAmp Kit (OSANG Healthcare Co., Ltd.) according to manufacturer's instructions targeting the RdRp (RNA-dependent RNA Polymerase), E (Envelope), and N (Nucleocapsid) SARS-CoV-2 genes. 8 Samples with inconclusive GeneFinder TM test results were tested using a second RT-PCR test − Mobius XGEN MASTER COVID-19 test − targeting the ORF1ab and N SARS-CoV-2 genes, per the IFU. 9 Test results for both RT-PCR methods were available within 6-24 hours.

Reference testing
For this study, the performance of the Panbio TM COVID-19 Ag test was evaluated against the results of the gold standard test (RT-PCR). For RT-PCR, RNA was isolated from the subject's nasopharyngeal swab using the Quick-RNA Viral Kit (Zymo Research, Irvine, CA), according to the manufacturer's protocol. 10 After extraction, the RNA was used immediately, and the remaining RNA was stored at -80°C. In contrast to the GeneFinder TM kit, the Master Mixture in the Mobius TM kit was used directly without further preparation. The 96-well plate was prepared by adding 15 mL of the Master Mixture to the sample wells, followed by either (1) 5 mL of the subject's RNA, (2) 5 mL negative control (DEPC-treated water), or (3) 5 mL positive control (a synthetic cDNA of the ORF1ab and N SRS-CoV-2 genes). In addition, 1 mL of an internal control was added to all the subjects' RNA samples. It is important to highlight that the content of the internal control is not described by the manufacturer. The plate was sealed and centrifuged, as described above. The thermal cycling conditions were as follows: (1) 1 cycle at 45°C for 15 min, (2) 1 cycle at 95°C for 2 min, and (3) 45 cycles at 95°C for 10 sec and 60°C for 50 sec.

Statistical analysis
To assess differences between patient subgroups, continuous variables are expressed by mean (and standard deviation) or median (and interquartile range) values and compared using the Student t-test, ANOVA, Kruskal-Wallis test and/or the Mann-Whitney U test, as indicated. Categorical variables are presented as proportions and the respective 95% Confidence Intervals (95% CI) were compared using the Chi-Square test and the Fisher exact test.
The impact on sensitivity and specificity of the Panbio TM COVID-19 Ag test according to the Ct value used to define a positive RT-PCR test was assessed by a Receiver Operating Characteristic (ROC) curve. The Area Under the Curve (AUC) and 95% Confidence Interval (95% CI) were calculated for Ct cutoffs of 25, 30, 35 and 40. All analyses were performed using the SPSS 26 program. Differences were considered statistically significant at p-values lower than 0.05.
The agreement beyond chance between results of RT-PCR, defining positivity according to the above Ct cutoffs, and the Panbio TM COVID-19 Ag test was assessed by the kappa statistic.

Results
A total of 127 subjects were included. The median age was 63 years (22-69) and there were more males (54.3%) than females (vs. 45.7%). The characteristics of the study population are given in Table 1.
The Panbio TM COVID-19 Ag testing summary is shown in Table 2. Table 3 provides details of the 10 samples that were negative by the Panbio TM COVID-19 Ag test but were positive by RT-PCR using the GeneFinder TM COVID-19 Plus RealAmp Kit. The Ct values of the samples ranged from 21-34 and were collected from subjects 2-11 days post onset of symptoms. Table 4 shows the diagnostic parameters sensitivity, specificity and positive and negative predictive values as a function of Ct. The results indicate that the Panbio TM COVID-19 Ag test has high sensitivity (97.3%) when positivity was defined as a     The number of data points is less than 70 because some samples produced the same Ct values and had the same number of days since symptom onset. No PCR-negative samples are presented here. The red squares represent PCR-positive samples that were tested as negative by the Panbio TM COVID-19 Ag test. The highest viral loads were observed soon after symptom onset, which then gradually decreased but viral loads were very heterogeneous.

Discussion
This study was designed to evaluate the performance of the Panbio TM COVID-19 Ag test in comparison to RT-PCR, considered the gold-standard diagnostic test for COVID-19. The sensitivity was 84.3% (95% CI 75%-93.8%) and the specificity was 98.2% (95% CI 96%-98.8%), when the Ct cutoff used to define positivity was ≤40, similar to results seen in other recent studies. [11][12][13][14][15] In our study, the agreement beyond chance between the Panbio TM COVID-19 Ag test and RT-PCR decreases as Ct levels increase. In particular, the agreement was 78% for samples with Ct values of ≤ 25 but decreases at higher Cts. Additionally, sensitivity is not significantly different between subjects tested 0 to 11 days after symptom onset due to the high viral load observed in the patients even after 7 days. Following SARS-CoV-2 infection, the virus undergoes a period of incubation during with viral titers usually too low to detect, after which the virus undergoes exponential growth, leading to a peak in viral load and infectivity, followed by declining viral levels and clearance when infectivity is low. For disease control, it is very important to identify both infected individuals who present COVID-19 symptoms and also those who are suspected of infection due to contact with infected individuals.

Conclusion
The Panbio TM COVID-19 Ag test has sufficient sensitivity and specificity to be used as a tool for the primary screening of such individuals. This is especially true in point-of-care and in patient settings where more elaborate laboratory facilities for RT-PCR are not available.

Conflicts of interest
The authors declare no conflicts of interest.