Approval for the study was obtained from the Nationwide Children's Hospital Institutional Review Board. Written consent was obtained from the legal guardian, and written assent was obtained from any child 9 years of age or older. We recruited patients younger than 18 years old who presented to the ED with symptoms of influenza meeting enrollment criteria. These criteria included a fever of >100.4°F in the ED and/or a reported fever of >100.4°F in the last 48 h at home in conjunction with two or more of the following symptoms: chills/sweats, cough, dyspnea, fatigue, headache, myalgia, nasal congestion, rhinorrhea, and/or sore throat. Exclusion criteria included the administration of any antiviral agents in the previous 7 days. Candidates with qualifying symptoms had study procedures performed irrespective of physician order for a rapid influenza test. For the uniformity of the collection technique, specimens were collected by two prespecified research personnel: a nurse (E.S.) and a physician (K.A.S.). If the treating physician did not order a rapid influenza test, the test was still collected by the research nurse or physician, but the results were utilized only for study purposes and not for management. Subjects were enrolled during two separate influenza seasons: either from January 2007 through March 2007 or from January 2008 through February 2008. Fifty subjects were enrolled for each of the study periods.
The study was designed as a prospective cohort study with a head-to-head comparison of two types of swab devices used for AN collections. One swab device was a traditional polyurethane foam-tipped swab (catalog number 20171; manufactured by Puritan Medical Products, Guilford, ME, for Quidel Corporation, San Diego, CA) in a dry, hard-plastic transport tube. Quidel established the performance characteristics of the rapid influenza test using polyurethane swabs, and these are recommended for collection of secretions from AN. The Quidel test kit is packaged with polyurethane swabs in paper wrappers; the same swab type in a dry, hard-plastic tube suitable for transport and as used in this study is available as a separate item. The other swab device was a flocked, nylon fiber-tipped swab (catalog number 552C; Copan Diagnostics, Murrieta, CA) in a dry plastic transport tube. Quidel makes no claims regarding the performance of nylon swabs in the rapid influenza test; thus, testing with these swabs is considered off-label use.
When the respiratory specimens were collected, the AN swabs were obtained first, with one swab placed into each naris. The swab was inserted just inside the opening of one naris, rotated for several seconds, removed, and placed directly into the swab collection sheath. The swabs were placed directly into separate transport tubes rather than into viral transport medium. Following the collection of the two AN specimens, a single posterior NP swab was then collected by using a Dacron polyester minitipped aluminum shaft swab (catalog number 25-800D; Puritan Medical). The Dacron swab was inserted into one naris and extended past the turbinates until resistance was met at the level of the posterior nasopharynx. The swab was rotated for several seconds and then removed with further rotation. The swab tip was then cut off and immediately placed into 2 ml of M4 viral transport medium (catalog number 12520; Remel, Lenexa, KS). The AN swabs in the transport tubes and the NP swab in a vial of M4 medium were immediately transported to the clinical virology laboratory to arrive within 30 min for testing.
The Quidel QuickVue Influenza A+B test (catalog number 20183) is an FDA-cleared test for the detection and differentiation of nucleoprotein antigens of the influenza A or B virus. It was performed directly on the AN swab samples according to instructions provided on the package insert. Testing requires 10 to 15 min to complete, with no more than 5 min of hands-on time.
For DFA testing, up to 1.0 ml of the remaining M4 sample was washed with 10 ml of phosphate-buffered saline (PBS) in a 15-ml sterile centrifuge tube and centrifuged, and the cells were resuspended in 1.5 ml of PBS. Double-well cytospin slides were prepared by using 200 μl of the cell suspension per well, fixed with acetone, and stained. One well was stained with SimulFluor respiratory screening reagent (catalog number 3296; Millipore/Chemicon, Temecula, CA), and the other well was stained with Flu A/B direct FA reagent (catalog number 3121; Millipore/Chemicon) according to the manufacturer's recommendations.
For the viral culture, 0.2 ml of the posterior NP swab specimen in M4 medium was inoculated into each of two R-Mix vials (catalog number 96-0102; Diagnostic Hybrids, Athena, OH) and incubated at 36°C according to the manufacturer's recommendations. One vial was stained approximately 42 h later with the SimulFluor respiratory screening reagent, and if positive, the companion vial was stained with SimulFluor Flu A/B direct FA reagent to differentiate between the two influenza virus types.
For RT-PCR, total nucleic acids were extracted from 0.2 ml of the NP swab specimen in M4 medium by use of an easyMag extractor (bioMerieux Inc., Durham, NC) and eluted into 55 μl. RT-PCR was performed by a method developed and validated in our laboratory and utilized the Prodesse (Waukesha, WI) Pro Flu-1 analyte-specific reagent (ASR) (catalog number HSM58). This reagent is no longer available and has been replaced by the Prodesse ProFlu Plus FDA-cleared assay (detection kit catalog number H44VK00 and control kit catalog number H44VK55). The laboratory-developed RT-PCR method was shown to be 100% sensitive for the detection of influenza A and B virus RNAs in clinical specimens that were positive by culture, DFA, or both. The RT-PCR method was also subsequently shown to be equivalent to RT-PCR using the ProFlu Plus FDA-cleared assay (our unpublished data). Amplification was performed by use of a 7500 sequence detection system (Applied Biosystems, Foster City, CA) using 5 μl eluate and 20 μl mastermix for a total reaction volume of 25 μl and the following amplification profile: 30 min at 42°C (1 cycle), 5 min at 95°C (1 cycle), and a cycle program consisting of 5 s at 95°C and 60 s at 55°C (40 cycles). Results of each run were determined by comparison of amplification curves with the positive- and negative-control curves and the cycle threshold.
Paired samples were analyzed with a marginal model for repeated binary outcomes (SAS PROC GENMOD) so that correlations between diagnoses of the same subject were taken into account. We used two separate gold standards against which the performances of rapid antigen testing of AN swabs were compared: the result of RT-PCR alone with a posterior NP swab sample (26
) and the result of DFA and/or culture. A positive result by either DFA or culture was indicative of a patient with an influenza virus infection. Statistical significance was set at an α value of <0.05.