In recent years, advances in point of care tests for influenza have provided new options for better patient care. As a result of the recent flu pandemic, it became generally recognized that many of the traditional, visually read immunoassays did not perform as well as their package inserts suggested.1 This recognition coincided with a broadening use of DNA-based detection technologies.2 While DNA-based technology assays continue to become more common, their complexity rating and long time-to-result have limited their use in physician office settings. According to third-party data, visually read “rapids” still represent a majority of the total number of influenza tests performed each year.3
Recently, however, new options for point of care testing have emerged. These digital immunoassay (DIA) systems combine new technological advances in detection particles, optical image recognition, fluorescent detection, and/or interpretation algorithms to provide improved reliability and performance. Ongoing concerns regarding the performance of the earlier rapid immunoassays have culminated in a recent FDA proposal to reclassify these tests, requiring them to meet significantly higher performance standards.4 This proposal, already approved by the FDA’s advisory committee, will require manufacturers with assays which do not meet these standards to improve the performance of these products, perform a new clinical trial, and resubmit data to demonstrate compliance with the new requirement. In the absence of completing such studies, manufacturers will have to suspend sale of these lower-performing test kits.
The new, CLIA-waived DIA-based systems have performance characteristics which conform to the new proposed FDA standard. The currently available DIAs utilize different detection methodologies, each having advantages over the standard visual tests. One system uses fluorescence detection, and the other relies on the use of modified colloidal metal detector particles.
Fluorescence detection is intrinsically a sensitive method, but the instrumentation required is complex, and assay specificity can be impacted by the inherent fluorescent properties of many biological materials including blood, mucous, and other bodily fluids.5 These issues can be mitigated to a degree by careful selection of the fluorescent label used, with a focus on labels with large stoke shifts or fluorescence emission spectra outside those exhibited by substances in biological samples.
The other DIA system, which uses modified metal colloids, incorporates technology which results in enhanced reflectance properties, thereby increasing the detectability of these particles. The modified metal colloids also incorporate the means to covalently attach antibodies or other biological molecules to the particles. Use of these advanced colloidal particles enhances the sensitivity of resultant assays, while the covalent attachment of biologicals produces greater consistency and overall stability.
The use of instrumented readers in these DIA systems represents another key technological advance that produces improvements in overall assay performance. Using a reader for test interpretation eliminates the subjectivity associated with the scoring of visually read tests. Environmental effects and variations in visual acuity of test operators can be eliminated by the objective interpretation of assays in DIA systems. A recent study published in the Journal of the Japanese Society of Clinical Microbiology examined the visual reading capabilities of people (N=84) in various age groups.6 While operators in all age groups had relatively high overall agreement for interpretation of moderate-level positives (85% to 99% correct), interpretations of low-level positives were incorrectly scored in up to 56% of cases with lower agreement noted as a function of operator age. Studies such as this suggest that in real world testing situations, where operator skill and environmental factors can vary greatly, removal of subjective interpretation alone can be expected to significantly affect overall test performance.
Instrumented readers utilize proprietary assay algorithms to determine positivity and also incorporate “adaptive-read” methods to evaluate non-specific binding events which can affect overall assay specificity. For example, incorporating a negative control feature which can be analyzed by the reader on each individual test device can provide a means to identify and adjust for non-specific signal generation. It is well known in the diagnostics industry that substances present in certain biological samples can facilitate non-specific protein-protein interactions, potentially resulting in false signal generation in immunoassays. DIA test systems compare a negative control signal to each test line signal to determine if the signal produced is truly target-specific. This same technology is employed in most laboratory-based immunoassay instruments, but use of this approach is not feasible in visually read point of care tests. Both of the available DIA systems use this approach as a means to optimize overall assay specificity. For this approach to be most effective, appropriate decisions regarding the biological material used as the negative control are essential.
Finally, a focus on workflow and ancillary test kit materials is also essential for maximizing the overall clinical performance of point of care tests. The new, CLIA-waived DIA-based systems supply specimen collection/delivery devices that are determined to be optimal for collecting biological samples and releasing high amounts of target antigen, if present. For example, disposable collection swabs or volumetric pipettes (for liquid specimens) are provided to allow easy collection and transfer of specimens for assay processing. Assay processing steps are minimized and materials are ergonomically designed to reduce operator fatigue, especially when high numbers of tests may be run, as is typically seen in an influenza epidemic. Instrumented test systems include fail-safe mechanisms to ensure that assays are run according to the manufacturer’s instructions and that instruments are operating properly to deliver accurate results.
One DIA-based system eliminates workflow steps that are determined to have little or no value. This includes removing timed specimen extraction steps, mixing steps when not required, and keeping the number of pipetting steps to a minimum. Employnig appropriate mnemonic devices such as defining a common number of drops dispensed per assay device regardless of assay type, or instructions that provide directional consistency—e.g., “rotate swab in a circular direction three times, swirl in reagent three times, and add three drops to the test cassette”—help to improve compliance to instructions in the package insert and ultimately ensure the product is used the way the manufacturer intended.
Available options and platform improvements for influenza testing continue to evolve. New rapid DIA test systems demonstrate enhanced performance, allowing a comparison to new standard reference methods such as the polymerase chain reaction (PCR). As a result of this increase in performance, the new class of DIA influenza tests has many laboratorians reconsidering the role of traditional immunoassay testing. The perceived gap in relative performance between DIAs and nucleic acid amplification assays such as PCR has clearly narrowed, challenging prior decisions to trade off time-to-result for overall performance. Given the enhanced performance and ease of use of this new generation of DIA systems, many hospital systems have now added point of care testing back to their test menu. Additionally, facilities that have continued to rely on standard visually read immunoassays now have new digital immunoassay options that will comply with the new FDA-proposed performance standards.
- CDC. Guidance for Clinicians on the Use of Rapid Influenza Diagnostic Tests. http://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm. Accessed September 28, 2013.
- CAP surveys 2009-2013. ID2
- Global Health Exchange market share data
- CDRH Microbiology Devices Advisory Committee Meeting. Executive Summary: Proposed Reclassification of the Rapid Influenza Detection Tests http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/ MedicalDevices/MedicalDevicesAdvisoryCommittee/MicrobiologyDevicesPanel/UCM356185.pdf. Accessed September 28. 2013.
- Hallsall DJ. Antibody interference in immunoassay: know your enemy. Ann Clin Biochem. 2013;50:397-399.
- Yamaguchi I, Aoyama T, Yamamoto M, et al. Evaluation of the sensitivity of a densitometry system, in judging the result of influenza antigen-detection kit using immunochromatography. J Jpn Soc Clin Microbiol. 2013;23(3):213.