Avoiding false positive SARS-CoV-2 rapid antigen test results with point-of-care molecular testing on residual test buffer (preprint)

Objectives: Antigen-based rapid diagnostic tests (Ag-RDTs) have been widely used for the detection of SARS-CoV-2 during the Covid-19 pandemic. In settings of low disease prevalence, such as asymptomatic community testing, national guidelines recommend molecular confirmation of positive Ag-RDT results. 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 method for SARS-CoV-2 detection on-site at a volunteer-led asymptomatic community testing site, using the residual test buffer (RTB) from positive Ag-RDTs. Methods: The Abbott COVID-19 ID NOW assay was performed on RTB from two Ag-RDTs: the Abbott Panbio COVID-19 Ag Rapid Test Device and the BTNX Rapid Response COVID-19 Antigen Rapid Test Device. All RTBs were tested using real-time RT-PCR at a reference laboratory using the ThermoFisher TaqPath COVID-19 Combo kit which was used to assign positive Ag-RDTs results as true or false positives. Analytical specificity of the ID NOW was assessed with a panel of various respiratory organisms. Results: Of 419 positive Ag-RDTs from 5148 tests performed, ID NOW testing of the RTB was positive in 100% of the samples characterized as true positives by RT-PCR. 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. Conclusions: 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.

Combined oropharyngeal/nares and nasopharyngeal swab sampling remain effective for molecular detection of SARS-CoV-2 Omicron variant (preprint)

The world has experienced several waves of SARS-CoV-2 variants of concern (VoCs) throughout the COVID-19 pandemic since the first cases in December 2019. The Omicron VoC has increased transmission, compared to its predecessors, and can present with sore throat and other cold-like symptoms. Given the predominance of throat symptoms, and previous work demonstrating better sensitivity using antigen-based rapid detection tests when a throat swab is included in the standard nasal sampling, this quality improvement project sought to ensure ongoing suitability of both combined oropharyngeal/nares (OPN) and nasopharyngeal (NP) swab sampling used throughout the pandemic. Consenting participants meeting Public Health testing criteria (mostly symptomatic or a close contact of a known case) were enrolled, and paired NP and OPN swabs collections were subjected to nucleic acid amplification testing (NAAT). Comparing paired specimens from 392 participants sensitivity of NP swabs was 89.1% (95% CI, 78.8-94.9), and that of OPN was 98.4% (95% CI: 90.9->99.9) (p-value 0.052). This project demonstrated that both NP and combined OPN swabs detected the Omicron variant with similar sensitivity by NAAT, supporting the continued use of either swab collection for SARS-CoV-2 molecular detection.

Investigating sensitivity of nasal or throat (ISNOT): A combination of both swabs increases sensitivity of SARS-CoV-2 rapid antigen tests (preprint)

The COVID-19 pandemic has been hallmarked by several waves of variants of concern (VoCs), each with novel challenges. Currently, the highly transmissible Omicron VOC is predominant worldwide, and sore throat is common among other cold-like symptoms. Anecdotes on social media suggested sampling ones throat can increase sensitivity for Omicron detection by antigen-based rapid testing devices (Ag-RDTs). This work determines whether the sensitivity of Ag-RDTs designed for nasal sampling is altered with use of self-administered throat swabs in self-perceived asymptomatic individuals. This investigation compared results of a common Ag-RDT (i.e. Abbott Panbio COVID-19 Ag Rapid Test Device) using three sampling sites: nasal swab; throat swab and; combined nasal/throat. All Ag-RDT results were confirmed with molecular testing. Compared to RT-PCR, samples from nasal or throat swabs each detected 64.5% of SARS-CoV-2 cases; however, combining the contributions of each swab increased sensitivity to 88.7%. This trend was also evident with the Rapid Response Ag-RDT (BTNX), which uses a more flexible swabs than Panbio. When nasal swab collection was compared to paired sampling of the nasal/throat using a single swab with the Panbio Ag-RDT, the sensitivity of each was 68.4% and 81.6%, respectively. No false-positive results were observed with nasal, throat, or combined nasal/throat sampling. Self-administered throat and nasal/throat swabs both had >90% acceptability. These findings support the use of self-collected combined nasal/throat sampling for Ag-RDT based SARS-CoV-2 detection in self perceived asymptomatic individuals.

Generation of false positive SARS-CoV-2 antigen results with testing conditions outside manufacturer recommendations: A scientific approach to pandemic misinformation (preprint)

Objectives: Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for SARS-CoV-2 detection. However, misleading demonstrations of the Abbott Panbio 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 of manufacturer recommendations. Methods: 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 temperatures (4{degrees}C, 20{degrees}C , and 45{degrees}C) and humidity (90%) on assay performance. Results: 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 non-specific 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 of manufacturer claims. Conclusions: 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.

Look before diving into pooling of SARS-CoV-2 samples on high throughput analyzers (preprint)

Given the unprecedented demand for SARS-CoV-2 testing during the COVID-19 pandemic, the benefits of specimen pooling have recently been explored. As previous studies were limited to mathematical modeling or testing on low throughput PCR instruments, this study aimed to assess pooling on high throughput analyzers. To assess the impact of pooling, SARS-CoV-2 dilutions were performed at varying pool depths (i.e. 1:2, 1:4, and 1:8) into test-negative nasopharyngeal or oropharynx/anterior nares swabs matrix. Testing was evaluated on the automated Roche Cobas 6800 system, or the Roche MagNApure LC 2.0 or MagNAPure 96 instruments paired with a laboratory-developed test using a 96-well PCR format. The frequency of detection in specimens with low viral loads was evaluated using archived specimens collected throughout the first pandemic wave. The proportion of detectable results per pool depths was used to estimate the potential impact. In addition, workflow at the analytical stage, and pre-and post-stages of testing were also considered. The current study estimated that pool depths of 1:2, 1:4, and 1:8 would have allowed the detection of 98.3%, 96.0%, and 92.6% of positive SARS-CoV-2 results identified in the first wave of the pandemic in Nova Scotia. Overall, this study demonstrated that pooling on high throughput instrumentation can dramatically increase the overall testing capacity to meet increased demands, with little compromising to sensitivity at low pool depths. However, the human resources required at the pre-analytical stage of testing is a particular challenging to achieve.