Patients were enrolled in the study from January 1998 to April 1998. Patients came from three cities in the Midwest, Southwest, and Rocky Mountain regions of the United States. Patient enrollment was conducted at emergency rooms, physician offices, employee clinics, and urgent-care facilities. Patients were enrolled in the study based on the following clinical criteria: the onset of illness within the past 36 h, a temperature of ≥100°F, and at least two influenza-like symptoms, including, but not limited to, cough, eye or ear pain, headache, sore throat, myalgia, congestion, malaise, and chills. Patients who had previously received an influenza vaccine were not excluded from this study.
Any combination of throat swab, nasopharyngeal swab, and nasal aspirate and/or sputum specimens were collected from each patient. Specimens were collected by the following techniques.
(i) Throat swabs.
Two sterile rayon swabs (Hardwood Products LP, Guilford, Maine) were vigorously rubbed on both tonsillar surfaces and the posterior pharynx. One swab was then inserted, tip down, into the original paper wrapper for the OIA test; the second swab was inserted into 1.5 ml of Multi-Microbe Medium (M4; MicroTest, Inc., Lilburn, Ga.) for culture.
(ii) Nasopharyngeal swabs.
Two Dacron nasopharyngeal swabs (Hardwood Products LP) were inserted beneath the inferior turbinate of either nare and vigorously rubbed and rolled against the mucosal surface. One swab was then inserted, tip down, into the original paper wrapper for the OIA test; the second swab was inserted into 1.5 ml of M4 transport medium for culture.
(iii) Nasal aspirates.
A depressed bulb syringe (Bard, Atlanta, Ga., or Owen & Minor, Denver, Colo.) was deeply inserted into either nare and suctioned while being withdrawn. The specimen was expelled into a sterile cup and thoroughly mixed with a rayon throat swab. The swab was subsequently used for testing by the FLU OIA test. M4 medium (1.5 ml) was then added to the remaining nasal aspirate specimen for culture.
(iv) Sputum specimens.
Sputum specimens were obtained after either a spontaneous deep cough or a deep cough by mechanical induction with a throat swab. The specimen was collected in a sterile cup and thoroughly mixed with a rayon throat swab. The swab was subsequently used for testing by the FLU OIA method. M4 medium (1.5 ml) was then added to the remaining sputum specimen for culture.
All specimens were labeled with the patient identification data, date, and exact time of specimen collection. Specimens were stored at 2 to 8°C for up to 24 h until testing by the FLU OIA method or culture could be performed.
FLU OIA test.
OIA technology enables the direct visual detection of a physical change in the optical thickness of molecular thin films (5
). This change is a result of macromolecular binding on an optical surface (silicon wafer). When extracted specimen is placed directly on the optical surface, the immobilized specific capture of the target analyte increases the thickness of the film. This change in thickness alters the reflected light path and is visually perceived as a color change. Slight changes in optical thickness produce a distinct visible color change. A positive result appears as a blue to purple spot on the predominant gold background. When analyte is not present in the specimen, no binding takes place; therefore, the optical thickness remains unchanged and the surface retains the original gold color, indicating a negative result. Internal procedural control dots are visible in a valid test result. If a procedural control dot is not visible, the test was not performed correctly and the result is considered invalid.
The FLU OIA test is a 15-min antibody-based assay for the detection of influenza virus types A and B nucleoprotein antigen from clinical specimens. The FLU OIA test was performed according to the manufacturer's instructions provided in the package insert. Reagents were removed from refrigerated storage and allowed to warm to room temperature (18 to 30°C). All extraction tubes and test devices were labeled with a patient identification number.
For the antigen extraction procedure, 3 drops of sample diluent and 2 drops of extraction reagent were added to an extraction tube. A throat swab (used for pharyngeal specimens or to absorb nasal aspirate and sputum specimens) or a nasopharyngeal swab was added to the extraction tube, thoroughly mixed in the solution, and incubated for 3 to 5 min. One drop of conjugate was then added to the extraction tube, and the solution was thoroughly mixed with the swab.
For the assay procedure, 1 drop of the solution containing extracted antigen and conjugate was added to the test surface and incubated for 6 to 7 min. After the test surface was then washed and blotted, 1 drop of substrate was added to the center of the test surface and incubated for an additional 6 to 7 min. The test surface was then again washed and blotted, and the results were interpreted. Results were interpreted as positive for influenza virus A or B antigen if a blue or purple reaction zone was visible in the center of the test surface. Results were interpreted as negative for influenza virus A or B antigen if no color change was visible. The upper blotters of the test device were removed to retain the permanent result of the test.
Specimens were vigorously vortexed and centrifuged at 400 × g for 10 min. The supernatant was used for cell culture inoculation. Supernatant was filtered through a 0.45-μm-pore-size filter, and 0.2 ml of filtrate was inoculated onto each of two pRMK tubes (Viromed Laboratories, Minneapolis, Minn.) containing serum-free Eagle's minimal essential medium. All inoculated tubes were incubated at 33 to 35°C and examined for cytopathic effects (CPE) on alternating days for a period of up to 14 days. CPE-negative tubes were tested by hemadsorption on day 3 and day 7 with 0.25% guinea pig erythrocytes. Cells from tubes demonstrating CPE or positive for hemadsorption were harvested, and slides for influenza virus A and B direct fluorescent antibody staining (Imagen; Dako Diagnostics Ltd.) were prepared. Cells from tubes still negative on day 14 were stained to confirm the absence of influenza virus A or B antigen.
Direct fluorescent antibody staining.
Cells harvested from culture tubes were spotted onto two glass microscope slides. Specimens were air dried and acetone fixed for 10 min. Staining was performed according to the manufacturer's directions. A 25-μl drop of reagent containing fluorescein-conjugated influenza virus A and B monoclonal antibodies was used to cover cell spots. Slides were incubated for 15 min at 37°C, washed for 5 minutes in phosphate-buffered saline, air dried, covered with a coverslip, and read with a fluorescence microscope.
If excess specimen was available after culture and the OIA test were performed, the sample was frozen for reverse transcriptase PCR-enzyme hybridization assay (RT-PCR-EHA) detection of influenza virus A and B nucleic acids in OIA test-positive, culture-negative samples. All samples tested by the RT-PCR-EHA were sent off-site (Prodesse, Inc., Waukesha, Wisc.) and tested by Hexaplex (Prodesse, Inc.) technology as previously described (8
The percentage of specimens positive by the FLU OIA test or by viral culture was calculated by using one of the following formulae: (number of positive samples detected by the FLU OIA test for a specific specimen type/total number of samples for that specimen type, positive or negative, from influenza virus-positive patients) × 100 and (number of positive samples detected by viral culture for a specific specimen type/total number of samples for that specimen type, positive or negative, from influenza virus-positive patients) × 100. Fisher's exact test was used to compare the results of the two tests for each specimen type.