Rapid Antigen Influenza A/B Test

Across about 1,550 evaluable samples (about 1,200 prospective, about 300 banked), influenza A sensitivity was about 90% and specificity about 96%. On discrepant testing with a second molecular assay, influenza A was detected in more than half of the device false positives and not detected in a minority of the false negatives. All banked samples were influenza A negative.

Influenza B sensitivity was about 88% and specificity about 99%. Most influenza B false positives were prospective samples, while most false negatives came from the banked set. Invalid results were extremely rare (an invalid rate of about 0%).

In the reproducibility study, agreement with expected results was 100% for every panel member except influenza A low positive, at about 98%. This showed that untrained intended users could perform and interpret the test accurately at and below the limit of detection.

FDA found the device substantially equivalent under 21 CFR 866.3328 (Class II) through the dual-track pathway, and FDA's CLIA database lists the test as waived.

Against the special-control criteria for a molecular comparator (agreement with positives of at least 80% with a lower bound of at least 70%; agreement with negatives of at least 95% with a lower bound of at least 90%), both influenza types pass with room to spare, including the sensitivity lower bounds for both types. Without the banked specimens, the influenza B estimate would have rested on very few prospective positives and its confidence interval would have been too wide to show the 70% floor was met. The supplement worked because it added positives to the denominator.

Two points help in reading the tables. First, the performance estimates pool prospective and banked samples and pool nasal and nasopharyngeal swabs as reported. The public summary does not break results out by specimen type. Second, the discrepant-analysis footnotes are informative but are not folded into the headline numbers. A second molecular test detected influenza A in more than half of the device false positives, which suggests that some apparent specificity loss reflects comparator misses at low viral load rather than device error.

The test is a 10-minute, visually read test stick intended to aid the rapid differential diagnosis of influenza A and B in patients with signs and symptoms of respiratory infection, using nasal or nasopharyngeal swabs tested directly. The intended use locations were clinical laboratories and point of care. Because the result is read by eye rather than by an instrument, the clinical evidence had to show that people with no laboratory training could perform the extraction, run the stick and interpret faint lines correctly in CLIA-waived settings.

Accuracy had to meet the influenza antigen special controls against an FDA-cleared molecular comparator, for both influenza types. The study season did not cooperate. The FDA summary describes an atypically low prevalence of influenza B: the device's prospective positivity rate was about 33% for influenza A and about 1% for influenza B. Prospective enrollment alone could not give enough influenza B positives to bound sensitivity, so the study needed a way to add positives without leaving the intended-use setting or the untrained-operator design.

Reproducibility at and below the limit of detection also had to be shown in the hands of the intended users, at sites that took part in the clinical study.

Rapid influenza antigen tests are regulated under 21 CFR 866.3328, influenza virus antigen detection test system (devices detecting influenza A, B, and C virus antigens). A final order placed these tests in Class II with special controls. The regulation's own identification explains why: the virus mutates, new strains emerge that may affect performance, and because influenza can cause severe illness, the accuracy of these devices has serious public health implications.

The special controls set published minimum clinical performance criteria that apply to each specimen type claimed. Against an FDA-cleared nucleic acid-based test or another FDA-accepted comparator other than viral culture, positive percent agreement for influenza A and B must have a point estimate of at least 80 percent with a 95% confidence interval lower bound of at least 70 percent. Negative percent agreement must reach at least 95 percent with a lower bound of at least 90 percent. Against correctly performed viral culture, influenza A sensitivity must reach at least 90 percent (lower bound at least 80 percent), influenza B sensitivity at least 80 percent (lower bound at least 70 percent), and specificity at least 95 percent (lower bound at least 90 percent). The special controls also require annual analytical reactivity testing with contemporary strains identified by FDA in consultation with CDC, with the last three years of results in labeling by July 31 each year, and expedited testing when an emergency involving an influenza strain is declared.

Many of these tests are also meant for physician offices, urgent care and other CLIA-waived settings, so sponsors often use FDA's dual 510(k) and CLIA waiver by application pathway (described in final guidance). That pathway lets one set of comparison and reproducibility studies, run by untrained operators at intended-use sites, support both substantial equivalence and waiver. The companion CLIA waiver guidance recommends at least three intended-use sites, one to three untrained operators per site and at least nine overall. Reproducibility should be run at a minimum of three of the same sites with panels near the detection limit. What makes the category hard is that the clinical lower bounds must be met during a single respiratory season whose strain mix and prevalence the sponsor cannot control.

• About twenty point-of-care sites is well above the three-site minimum recommended for waiver studies. The number was driven mainly by the need to accumulate positives in one season, and it also widened the range of operators and patient populations in the estimate.
• Masking banked influenza B specimens as patient samples and running them in the daily workflow at CLIA-waived sites added influenza B positives without moving testing to a laboratory or to trained staff. This keeps the waiver-relevant conditions intact while working around an unusually weak influenza B season.
• Paired swabs from the same nostril, with the device swab tested immediately, mirror real use and limit sampling differences between the device and comparator arms. Eluting the comparator swab in VTM and testing it at a central laboratory kept the reference arm uniform across all sites.
• A pre-specified second molecular test for discordant specimens gives context for false positives and negatives without changing the primary performance calculation.
• The reproducibility panel used high-negative and low-positive levels around the LoD and was masked into routine work. For a visually read test, this is where operator interpretation of faint lines is most likely to fail.