Validation of Rapid Influenza Diagnostic Test Nasal Swab Specimens for Influenza RT-PCR

BACKGROUND: Rapid influenza detections tests (RIDTs) using nasal swabs (NS) allow point-of-care testing with results available within meaningful timeframes for clinical decision-making.  In some settings, such as outbreak investigations, confirmation of RIDT results with RT-PCR is essential.  We conducted a validation study of residual or “waste NS” specimens for confirmatory testing using a population from which NS and nasopharyngeal (NP) or oropharyngeal (OP) specimens were routinely and systematically collected for primary care influenza surveillance. 

METHODS: Patients of all ages with acute respiratory tract infections were evaluated at 5 Wisconsin Influenza Incidence Surveillance Project clinics from July 1, 2014 through June 30, 2015.  Both NS and NP or upper OP specimens were collected.  The NS was tested following Quidel’s Sofia Influenza A+B Fluorescent Immunoassay package insert.  The “waste NS” and any unused lysis buffer were placed into Remel MicroTest™ M4RT® Viral Transport Media (VTM).  The NP and OP swabs were placed in VTM, sent to the Wisconsin State Laboratory of Hygiene, and tested for influenza A and B viruses using the CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel.  We identified 199 “waste NS” for which companion NP/OP specimens were positive for influenza A (n=79) or B (n=60) or negative (n=60), and tested using RT-PCR.  Outcome measures included sensitivity and specificity of influenza detection using “waste NS” compared to NP and OP swabs and comparison of mean cycle-threshold (Ct) values using a paired t-test. 

RESULTS: The sensitivity of “waste NS” was 81.3% overall, 77.2% for influenza A and 86.7% for influenza B.  The specificity ranged from 96.7% to 100% by influenza group.  Similar results were obtained using only NP or OP comparators. The mean difference in Ct value between NP and OP specimens and “waste NS” was 2.44 units (NP/OP = 25.53±4.85 [mean ± standard deviation]; NS = 27.97±4.40; t =-5.10; p<0.001). 

CONCLUSIONS: Discarded NS specimens can be valuable sources of material for confirmatory testing by RT-PCR.  A higher Ct value for influenza detection using “waste NS” specimens may indicate less available nucleic acid due to less cellular material being available, or alternatively, loss of viral titer due to freeze/thaw of the “waste NS” specimens, however a moderate sensitivity and high specificity was maintained.  Our results provide performance characteristics and limitations of using “waste NS” specimens for secondary testing with RT-PCR, and can guide protocols for use of residual RIDT specimens for possible future testing when confirmation of test results is necessary.