Field evaluation of a rapid antigen test (Panbio COVID-19 Ag Rapid Test Device)

Abstract

Objectives: To our knowledge no previous study has assessed the performance of a rapid antigen diagnostic immunoassay (RAD) conducted at the point of care (POC). We evaluated the Panbio™ COVID-19 Ag Rapid Test Device for diagnosis of coronavirus 2019 disease (COVID-19) in symptomatic patients (n = 412) attending primary healthcare centres.

Methods: RAD was performed immediately after sampling following the manufacturer’s instructions (reading at 15 min). RT-PCRs were carried out within 24 h of specimen collection. Samples displaying discordant results were processed for culture in Vero E6 cells. Presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cell cultures was confirmed by RT-PCR.

Results: Out of 412 patients, 43 (10.4%) tested positive by RT-PCR and RAD, and 358 (86.9%) tested negative by both methods; discordant results (RT-PCR+/RAD-) were obtained in 11 patients (2.7%). Overall specificity and sensitivity of rapid antigen detection (RAD) was 100% (95%CI 98.7-100%) and 79.6% (95%CI 67.0-88.8%), respectively, taking RT-PCR as the reference. Overall RAD negative predictive value for an estimated prevalence of 5% and 10% was 99% (95%CI 97.4-99.6%) and 97.9% (95%CI 95.9-98.9), respectively. SARS-CoV-2 could not be cultured from specimens yielding RT-PCR+/RAD- results (n = 11).

Conclusion: The Panbio™ COVID-19 Ag Rapid Test Device performed well as a POC test for early diagnosis of COVID-19 in primary healthcare centres. More crucially, the data suggested that patients with RT-PCR-proven COVID-19 testing negative by RAD are unlikely to be infectious.

Keywords: COVID-19; Early diagnosis; Primary healthcare centre; Rapid antigen detection test (RAD); SARS-CoV-2.

Background

RT-qPCR is the reference test for identification of active SARS-CoV-2 infection, but is associated with diagnostic delay. Antigen detection assays can generate results within 20 min and outside of laboratory settings. Yet, their diagnostic test performance in real life settings has not been determined.

Methods

The diagnostic value of the Panbio™ COVID-19 Ag Rapid Test (Abbott), was determined in  comparison to RT-qPCR (Seegene Allplex) in community-dwelling mildly symptomatic subjects in a medium (Utrecht, the Netherlands) and high endemic area (Aruba), using two concurrently obtained nasopharyngeal swabs.
Findings: 1367 and 208 subjects were enrolled in Utrecht and Aruba, respectively. SARS-CoV-2 prevalence, based on RT-qPCR, was 10.2% (n = 139) and 30.3% (n = 63) in Utrecht and Aruba respectively. Specificity of the Panbio™ COVID-19 Ag Rapid Test was 100% (95%CI: 99.7–100%) in both settings. Test sensitivity was 72.6% (95%CI: 64.5–79.9%) in the Netherlands and 81.0% (95% CI: 69.0–89.8%) in Aruba. Probability of false negative results was associated with RT-qPCR Ct-values, but not with duration of symptoms. Restricting RT-qPCR test positivity to Ct-values <32 yielded test sensitivities of 95.2% (95%CI: 89.3–98.5%) in Utrecht and 98.0% (95%CI: 89.2–99.95%) in Aruba.

Interpretation

In community-dwelling subjects with mild respiratory symptoms the Panbio™ COVID-19 Ag Rapid Test had 100% specificity, and a sensitivity above 95% for nasopharyngeal samples when using Ct-values <32 cycles as cut-off for RT-qPCR test positivity. Considering short turnaround times, user friendliness, low costs and opportunities for decentralized testing, this test can improve our efforts to control transmission of SARS-CoV-2.

Introduction

The SARS-CoV-2 pandemic has extensive impact on healthcare globally, with over 37 million confirmed cases and currently more than one million deaths . Rapid diagnosis of SARS-CoV-2 infection and subsequent contact tracing are essential in the containment of transmission .The reference test for detection of acute SARS-CoV-2 infection is reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RT-qPCR requires the use of expensive laboratory instrumentation as well as dedicated lab supplies and trained personnel, which causes significant challenges to generate sufficient testing capacity and short turnaround times.
Lateral Flow Assay (LFA)-based point of care tests (POCT) for rapid antigen detection using antibodies are cheap, simple to perform, do not require laboratory instrumentation and generate results within 20 min. Several different rapid antigen tests have been developed with usually high specificity but varying sensitivity. These tests have the potential to alter testing strategies worldwide. Recently the WHO approved the first rapid diagnostic POCT and initiated a global partnership to pledge 120 million tests to low- and middle-income countries. However, the diagnostic performance of individual POCTs in real-life community settings is unknown.
We evaluated the Abbott Panbio™ COVID-19 Ag rapid test in community testing locations in both a medium- and high endemic population and compared results to RT-qPCR and determined associations with duration of symptoms and risk of exposure.

Materials and methods

Populations and study period

All individuals visiting COVID-19 community testing centers, located at the University Medical Center Utrecht (UMCU) in the Netherlands (September 22nd to October 6th 2020) and the Horacio Oduber Hospital on Aruba (September 23rd to October 9th 2020), aged 16 and over were asked to participate in this prospective evaluation. These settings were chosen based on the different SARS-CoV-2 prevalence at onset of the study (approximately 4% in Utrecht compared to approximately 30% in Aruba), while using the same platform for the reference RT-qPCR test.
In both study sites, subjects were first sampled for routine RT-qPCR testing, using a combined throat/nasopharyngeal swab. Study participants received an additional nasopharyngeal swab. Participants at the Utrecht study site were asked to fill out a questionnaire regarding (onset of) symptoms and risk of exposure to SARS-CoV-2. Inclusion of participants was continued until the target of 100 positive LFA results, as recommended by the National Institute for Public Health and the Environment (RIVM), was obtained.
Abbott
Abbott

 RT-qPCR

PCR was conducted in a certified clinical laboratory and all procedures were validated according to the ISO 15,189 standard. After collection, swabs were transferred into 3 ml Universal transport medium until further processing. Nucleic acid extraction, RT-PCR and results interpretation were performed according the instructions of the manufacturer (Seegene, South-Korea).
In short, RNA was isolated and purified using the MagC extraction kit (Seegene, South-Korea) on an automatic nucleic acid extractor Hamilton MicroLAB StartLET (Bonaduz, Switzerland). Subsequently, cDNA was generated and amplification was performed in a single tube assay using the Allplex 19-nCoV multiplex platform for detection of SARS-CoV-2 (Seegene, South-Korea), and results were interpreted with Seegene Viewer data analysis software.
The assay uses fluorescent Taqman probes for three SARS-CoV-2 genes (E [Envelope], N [Nucleocapsid]-, and RdRP [RNA dependent RNA Polymerase]genes). Amplification and detection were performed for 45 cycles on a Biorad CFX96 thermocycler (Biorad Laboratories, the Netherlands), the threshold Cycle (Ct) was automatically determined by the manufacturer’s software.
A positive result was defined as amplification of any of the three SARS-CoV-2 genes. If not all targets showed a positive result, this always corresponded with high Ct-values, suggesting low levels of SARS-CoV-2 RNA. If viral RNA levels are very low and around the limit of detection of the assay, amplification of these targets is more subject to stochasticity which can result in positive results in only one or two targets. Based on our experience within clinical practice and results from viral culture studies,[ we use a cut-off Ct-value of 32 to determine clinically relevant levels of SARS-CoV-2 RNA.

 LFA

The Panbio COVID-19 Ag rapid test device by Abbott (Lake Country, IL, U.S.A) is a membrane-based immunochromatography assay which detects the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples. Collected swabs were transferred into dedicated sample collection tubes containing a sampling buffer and transported to the laboratory.
The laboratory is located within 5 min of walking distance from the sampling location. All samples were analysed within a maximum of 2 h after collection, and in practice the delay was much shorter (between 30 and 45 min on average), during which time the samples were kept at ambient temperature, which should have very little effect on relatively stable viral proteins. The PCR samples are processed similarly with no perceivable negative effect. Collected samples were subsequently processed in a level 2 biosafety cabinet in accordance with the manufacturer’s protocol. Test results were recorded after 15 min of assay initiation, by two independent observers (blinded to each other and to the PCR results). Single lot LFA testing devices were used: lot 41ADF011A.

Leave a Comment