Methods to evaluate the impact of SARS-CoV-2 nucleocapsid mutations on antigen detection by rapid diagnostic tests.

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance.

Avoiding False-Positive SARS-CoV-2 Rapid Antigen Test Results with Point-of-Care Molecular Testing on Residual Test Buffer.

Antigen-based rapid diagnostic tests (Ag-RDTs) have been widely used for the detection of SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic. In settings of low disease prevalence, such as asymptomatic community testing, national guidelines recommend confirmation of positive Ag-RDT results with a nucleic acid amplification test (NAAT). This often requires patients to be recalled for repeat specimen recollection and subsequent testing in reference laboratories. This project assessed the use of a point-of-care molecular NAAT for SARS-CoV-2 detection (i.e., ID NOW), which was performed on-site at a volunteer-led asymptomatic community testing site on the residual test buffer (RTB) from positive Ag-RDTs. The ID NOW NAAT assay was performed on RTB from two Ag-RDTs: the Abbott Panbio and BTNX Rapid Response assays. Results of ID NOW were compared to real-time RT-PCR at a reference laboratory. Along with investigations into the clinical performance of ID NOW on RTB, analytical specificity was assessed with a panel of various respiratory organisms. Of the Ag-RDTs results evaluated, all 354 Ag-RDTs results characterized as true positives by RT-PCR were accurately identified with ID NOW testing of RTB. No SARS-CoV-2 detections by ID NOW were observed from 10 specimens characterized as false-positive Ag-RDTs, or from contrived specimens with various respiratory organisms. The use of on-site molecular testing on RTB provides a suitable option for rapid confirmatory testing of positive Ag-RDTs, thereby obviating the need for specimen recollection for molecular testing at local reference laboratories. IMPORTANCE During the COVID-19 pandemic, rapid antigen tests have been widely used for the detection of SARS-CoV-2. These simple devices allow rapid test results. However, false-positive results may occur. As such, individuals with positive rapid tests often must return to testing centers to have a second swab collected, which is then transported to a specialized laboratory for confirmation using molecular tests. As an alternative to requiring a repeat visit and a prolonged turn-around time for result confirmation, this project evaluated whether the leftover material from rapid antigen tests could be confirmed directly on a portable point-of-care molecular instrument. Using this approach, molecular confirmation of positive antigen tests could be performed in less than 15 min, and the results were equivalent to laboratory-based confirmation. This procedure eliminates the need for individuals to return to testing centers following a positive rapid antigen test and ensures accurate antigen test results through on-site confirmation.

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.

Utility of SARS-CoV-2 rapid antigen testing for patient triage in the emergency department: A clinical implementation study in Melbourne, Australia.

Background: Early, rapid detection of SARS-CoV-2 is essential in healthcare settings in order to implement appropriate infection control precautions and rapidly assign patients to care pathways. Rapid testing methods, such as SARS-CoV-2 rapid antigen testing (RAT) may improve patient care, despite a lower sensitivity than real-time PCR (RT-PCR) testing. Methods: Patients presenting to an Emergency Department (ED) in Melbourne, Australia, were risk-stratified for their likelihood of active COVID-19 infection, and a non-randomised cohort of patients were tested by both Abbott Panbio™ COVID-19 Ag test (RAT) and SARS-CoV-2 RT-PCR. Patients with a positive RAT in the 'At or High Risk' COVID-19 group were moved immediately to a COVID-19 ward rather than waiting for a RT-PCR result. Clinical and laboratory data were assessed to determine test performance characteristics; and length of stay in the ED was compared for the different patient cohorts. Findings: Analysis of 1762 paired RAT/RT-PCR samples demonstrated an overall sensitivity of 75.5% (206/273; 95% CI: 69·9-80·4) for the Abbott Panbio™ COVID-12 Ag test, with specificity of 100% (1489/1489; 95% CI: 99·8-100). Sensitivity improved with increasing risk for COVID-19 infection, from 72·4% (95% CI: 52·8-87·3) in the 'No Risk' cohort to 100% (95% CI: 29·2-100) in the 'High Risk' group. Time in the ED for the 'At/High Risk' group decreased from 421 minutes (IQR: 281, 525) for those with a positive RAT result to 274 minutes (IQR:140, 425) for those with a negative RAT result, p = 0.02. Interpretation: The positive predictive value of a positive RAT in this setting was high, allowing more rapid instigation of COVID-19 care pathways and an improvement in patient flow within the ED. Funding: Royal Melbourne Hospital, Melbourne, Australia.

Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Evaluation of Panbio rapid antigen test for SARS-CoV-2 in symptomatic patients and their contacts: a multicenter study.

BACKGROUND: Point-of-care rapid tests to identify SARS-CoV-2 can have clinical benefits. METHODS: A cross-sectional study in adults visiting emergency services or screening sites of referral hospitals for COVID-19 to validate the diagnostic performance of a rapid antigen test for SARS-CoV-2 (Abbott's Panbio) compared with reverse transcription-polymerase chain reaction (RT-PCR) testing. Tests were performed by health personnel in a routine situation during a COVID-19 outbreak. RESULTS: A total of 1060 participants (mean age 47, 46% with a self-reported comorbidity) were recruited from 8 hospitals in Mexico. Participants provided 1060 valid Panbio rapid test-RT-PCR test pairs with 45% testing positive in the RT-PCR. Overall sensitivity of the Panbio test was 54.2% (95% CI 51%-57%), and 69.1% (95% CI 66%-73%) for patients during the first week of symptoms. Sensitivity depended on viral load (cycle threshold (Ct) of RT-PCR) and days of symptoms. With a Ct ≤25, sensitivity was 82% (95% CI, 76%-87%). Specificity of the Panbio test was >97.8% in all groups. CONCLUSIONS: The Panbio rapid antigen test for SARS-CoV-2 had good specificity but low sensitivity. A negative test requires confirmation with RT-PCR, especially for testing after the first week of symptoms.

SARS-CoV-2 rapid antigen test in comparison to RT-PCR targeting different genes: A real-life evaluation among unselected patients in a regional hospital of Italy.

We assessed the performance of the Panbio rapid antigen detection (RAD) test for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and we compared it with the routine reverse transcriptase-polymerase chain reaction (RT-PCR)-based molecular test in a population of 4167 unselected patients admitted to IRCCS Sacro Cuore Don Calabria Hospital. Analysis stratified by cycling threshold (Ct ) value of SARS-CoV-2 gene targets indicated that antigen (Ag)-positive Ct values were significantly lower compared to Ag-negative values (p < 0.0001). Overall, we found discordance in 140, tested negative by RAD and positive by RT-PCR, and in 4 resulted positive by RAD and negative by RT-PCR. RAD test achieved a sensitivity and specificity of 66.82% and 99.89%, respectively. The positive predictive value was shown to be 97.87% while the negative predictive value was shown to be 97.62%. In our context, the RAD test showed a reliable diagnostic response in subjects that displayed high Ct values, corresponding to high viral load, while low ability was displayed to identify positive cases with medium-low Ct values, thus presenting low viral load and where confirmatory RT-PCR was needed. Our finding supports the use of the RAD test in real-life settings where a high volume of swabs is being processed but with caution when interpreting a positive test result in a low prevalence setting.

Rapid COVID-19 antigenic tests: Usefulness of a modified method for diagnosis.

The current reliable recommended test for coronavirus disease 2019 (COVID-19) diagnosis is quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Rapid antigen test devices could be useful as they are less expensive, faster without the need of specialized laboratories to perform the test. We report the performances of two rapid immunochromatographic antigen testing devices compared with RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal samples. We carried out a lateral-flow tests study on 401 nasopharyngeal swab samples from nonduplicated suspected COVID-19 subjects. An equal volume of universal transport medium tubes-containing samples (dilution ratio = 1:15) were added to the manufacturer's extraction buffer solution (dilution ratio = 1:2) and analyzed on BioSpeedia COVID19Speed-Antigen Test and on Abbott Panbio™ COVID-19 Ag Rapid Test, devices. Qualitative results were compared to those obtained by the RT-qPCR (Allplex™ SARS-CoV-2 Assay Seegene). Based on our data, the overall sensitivity for BioSpeedia and Panbio devices was estimated at 65.5% and 75.0%, respectively. The sensitivity was greater for cycle threshold values less than 25 achieving 90.4 and 96.8 for BioSpeedia and Panbio devices, respectively. A perfect specificity of 100.0% was observed for both devices.

Comparative evaluation of Panbio and SD Biosensor antigen rapid diagnostic tests for COVID-19 diagnosis.

The aim of our study was to evaluate the diagnostic performance of two antigen rapid diagnostic tests (Ag-RDTs) to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We evaluated Panbio and SD-Biosensor Ag-RDTs. We employed 186 polymerase chain reaction (PCR) negative samples to evaluate the specificity and 170 PCR positive samples to assess the sensitivity. We evaluated their sensitivity according to Cycle threshold (C t ) values and days post onset of symptoms (d.p.o.). Tests were compared using the McNemar's test. Agreement was evaluated using the kappa score. Specificity was 100% for Panbio and 97.3% for SD-Biosensor. Sensitivity for samples with C t ≤ 20 was 100% for both assays and for samples with C t = 20-25 was 93.0% (Panbio) and 95.3% (SD-Biosensor) (p = 1.000). Sensitivity decreased for samples wit C t = 25-30 (Panbio: 41.3%, SD-Biosensor: 52.2%, p = 0.125) and samples with C t ≥ 30 (Panbio: 5.0%, SD-Biosensor: 17.5%, p = 0.063). Sensitivity within seven d.p.o. was 87.7% for Panbio and 90.4% for SD-Biosensor and notably decreased after seven d.p.o. Agreement with PCR was excellent for high viral load samples (C t ≤ 25): Panbio, 98.9%, kappa = 0.974; SD-Biosensor, 97.4%, kappa = 0.940. Agreement between Ag-RDTs was excellent (94.9%, kappa = 0.882). Panbio and SD-Biosensor Ag-RDTs showed excellent agreement and diagnostic performance results for samples with high viral loads (C t ≤ 25) or samples within seven d.p.o.

Validation and implementation of the Panbio COVID-19 Ag rapid test for the diagnosis of SARS-CoV-2 infection in symptomatic hospital healthcare workers.

BACKGROUND: RT-PCR is the current recommended laboratory method to diagnose SARS-CoV-2 in healthcare workers (HCW). As RT-PCR is not widely available and is time-consuming, it limits decision making on removal from and return to work of possibly contagious HCW. AIM: In this study we evaluated the Panbio™ COVID-19 Ag rapid test (PanbioCAgRT) in 825 hospital HCW. METHODS AND FINDING: This study consisted of two phases. In the validation phase, we tested hospital HCW with mild symptoms (three days or less) in parallel using the PanbioCAgRT and the RT-qPCR test. The PanbioCAgRT demonstrated 86.7% sensitivity, 100% specificity, 100% PPV and 98.5% NPV with regard to RT-qPCR. For HCW with PanbioCAgRT-/RT-qPCR+, the median Ct value was 30.9, whereas for the HCW with PanbioCAgRT+/RT-qPCR+ the median Ct value was 19.3 (P<0.001). In the second phase, we implemented an on-site antigen test-based strategy for symptomatic hospital HCW: HCW that tested positive with the PanbioCAgRT on-site were considered SARS-CoV-2 positive and were sent home. HCW that tested negative with the PanbioCAgRT on-site were allowed to work with PPE pending RT-qPCR test results from the laboratory. Sensitivity of the antigen test-based strategy was 72.5% and NPV was 97%. For HCW with PanbioCAgRT-/RT-qPCR+ median Ct values were 27.8. CONCLUSION: The PanbioCAgRTt validated in this study showed a high sensitivity and specificity in samples obtained from HCW with high viral loads. The antigen-based testing strategy proposed in this study seems to be effective, safe and easy to implement in a wide range of occupational healthcare settings.

Clinical performance of the Abbott Panbio with nasopharyngeal, throat, and saliva swabs among symptomatic individuals with COVID-19.

SARS-CoV-2 antigen tests used at the point-of-care, such as the Abbott Panbio, have great potential to help combat the COVID-19 pandemic. The Panbio is Health Canada approved for the detection of SARS-CoV-2 in symptomatic individuals within the first 7 days of COVID-19 symptom onset(s). Symptomatic adults recently diagnosed with COVID-19 in the community were recruited into the study. Paired nasopharyngeal (NP), throat, and saliva swabs were collected, with one paired swab tested immediately with the Panbio, and the other transported in universal transport media and tested using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We also prospectively evaluated results from assessment centers within the community. For those individuals, an NP swab was collected for Panbio testing and paired with RT-PCR results from parallel NP or throat swabs. One hundred and forty-five individuals were included in the study. Collection of throat and saliva was stopped early due to poorer performance (throat sensitivity 57.7%, n=61, and saliva sensitivity 2.6%, n=41). NP swab sensitivity was 87.7% [n=145, 95% confidence interval (CI) 81.0-92.7%]. There were 1641 symptomatic individuals tested by Panbio in assessment centers with 268/1641 (16.3%) positive for SARS-CoV-2. There were 37 false negatives and 2 false positives, corresponding to a sensitivity and specificity of 86.1% [95% CI 81.3-90.0%] and 99.9% [95% CI 99.5-100.0%], respectively. The Panbio test reliably detects most cases of SARS-CoV-2 from adults in the community setting presenting within 7 days of symptom onset using nasopharyngeal swabs. Throat and saliva swabs are not reliable specimens for the Panbio.

Diagnostic performance of CerTest and Panbio antigen rapid diagnostic tests to diagnose SARS-CoV-2 infection.

OBJECTIVES: Antigen rapid diagnostic tests (Ag-RDT) have been developed as reliable tools to control the SARS-CoV-2 pandemic. The objective of our study was to evaluate the diagnostic performance of two Ag-RDTs. METHODS: We evaluated CerTest SARS-CoV-2 Ag One Step Card Test and Panbio COVID-19 Ag Rapid Test Device Ag-RDTs. We included 320 nasopharyngeal samples: 150 PCR negative samples to assess the specificity and 170 PCR positive samples to evaluate the sensitivity. We also evaluated their sensitivity according to cycle threshold (Ct) values and the time from the onset of symptoms. Tests were compared using the McNemar's test and agreement was evaluated using the kappa score (k). RESULTS: Both Ag-RDTs showed a specificity of 100 %. Overall sensitivity was 53.5 % for CerTest and 60.0 % for Panbio. For samples with Ct≤ 25, sensitivity was 94.0 % for CerTest and 96.4 % for Panbio (p = 0.500). Regarding samples with Ct>25, sensitivity was 14.0 % for CerTest and 24.4 % for Panbio (p = 0.004). Sensitivity for samples within the first 5 days after the onset of symptoms were 84.8 % for CerTest and 91.3 % for Panbio (p = 0.250) and notably decreased for samples taken after the fifth day. Both Ag-RDTs showed an excellent agreement between them (agreement = 96.7 %, k = 0.920). Agreement with PCR was also excellent for high viral load samples (Ct<25) for CerTest (98.0 %, k = 0.954) and Panbio (98.8 %, k = 0.973). CONCLUSIONS: CerTest SARS-CoV-2 and Panbio COVID-19 Ag showed excellent performance and agreement results for samples with high viral loads (Ct ≤ 25) or samples taken within the first 5 days after the onset of symptoms.

Nasopharyngeal Panbio COVID-19 Antigen Performed at Point-of-Care Has a High Sensitivity in Symptomatic and Asymptomatic Patients With Higher Risk for Transmission and Older Age.

BACKGROUND: Performance of point-of-care tests in different clinical scenarios and on different samples remains undetermined. We comprehensively evaluated the performance of the nasopharyngeal Panbio COVID-19 Ag Rapid Test Device. METHODS: This is a prospective study that includes consecutive patients attending 3 primary care centers (PCCs) and an emergency department. The antigen test was performed at point-of-care in nasopharyngeal and nasal swabs and in saliva. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated with the reverse-transcription polymerase chain reaction (RT-PCR) assay as reference standard. RESULTS: Of 913 patients included, 296 (32.3%) were asymptomatic and 690 (75.6%) came from the PCC. Nasopharyngeal swabs were collected from 913 patients, nasal swabs were collected from 659 patients, and saliva was collected from 611 patients. The RT-PCR was positive in 196 (21.5%) nasopharyngeal samples (NPS). Overall, PPA (95% CI) in NPS was 60.5% (53.3-67.4), and it was lower in nasal swabs (44.7%) and saliva (23.1%). Test performance in NPS was largely dependent on the cycle threshold (Ct) in RT-PCR, with PPA of 94% for Ct ≤25 and 80% for Ct <30. In symptomatic patients, the PPA was 95% for Ct ≤25, 85% for Ct <30, and 89% for the symptom triad of fever, cough, and malaise. Performance was also dependent on age, with a PPA of 100% in symptomatic patients >50 years with Ct <25. In asymptomatic patients, the PPA was 86% for Ct <25. In all cases, NPA was 100%. CONCLUSIONS: The nasopharyngeal Panbio COVID-19 Ag test performed at point-of-care has a good sensitivity in symptomatic patients with Ct <30 and older age. The test was useful to identify asymptomatic patients with lower Ct values.