Nasopharyngeal aspirates were collected from 289 patients with a suspected influenza virus infection. Twenty-one cases 7.3% were excluded from the study due to nonrepresentative samples (<50 nasopharyngeal epithelial cells per preparation). Specimens from 268 patients remained for analysis, and from 79 of these patients nasopharyngeal swabs were also collected. The patients were between 2 months and 83 years old.
Nasopharyngeal aspirates were collected by using a baby-feeding tube and an aspiration trap. After suction the feeding tube was rinsed with approximately 2 ml of sterile saline (17
). Samples were transported to the lab immediately.
Nasopharyngeal swabs were collected in parallel with nasopharyngeal aspirates from 79 patients by using rayon-tipped swabs (Copan Italia, Brescia, Italy). The swabs were rubbed against the mucosal surface of the nasopharynx. They were then transported in sterile empty tubes to the lab and were processed within 6 h for use in the optical immunoassay (OIA).
The nasopharyngeal aspirates were centrifuged at 1,000 × g
for 10 min. The supernatant was used for inoculation of two tubes with MDCK cells, as has been described previously (17
). The cell cultures were observed for cytopathic effect (CPE) for a total of 2 weeks. If no CPE was seen, hemadsorption with guinea pig red blood cells was performed. CPE or positive hemadsorption was confirmed by IF staining with influenza A and B monoclonal antibodies (Dako AS, Glostrup, Denmark).
Antigen detection by IF assay.
Cells from centrifuged nasopharyngeal aspirates were washed and resuspended in phosphate-buffered saline and spotted onto multiwell slides (20 μl/well). The cells were air-dried and fixed in acetone, and multiple wells were incubated with 20 μl of fluorescein-labeled influenza A and B monoclonal antibodies (Imagen, Dako AS) as previously described (17
). The slides were incubated in a moist chamber at 37°C for 30 min and washed three times with phosphate-buffered saline. Examination was performed in a fluorescence microscope at 200 to 400 × magnification. A specimen was considered representative if more than 50 nasopharyngeal epithelial cells per preparation was present. The presence of at least two cells (or cell nuclei) with a typical distinct staining was regarded as a positive result.
Antigen detection by the OIA test.
For the OIA test, the influenza virus antigens were extracted from two types of specimens according to specifications from the manufacturer: (i) rayon-tipped swabs dipped into concentrated cells obtained from centrifuged nasopharyngeal aspirates (the same preparations as were used for IF slide preparation) and (ii) rayon-tipped swabs rubbed against the mucosal surface of the nasopharynx.
The OIA test uses a mirrorlike surface of a silicon wafer coated with optical molecular thin film and a capture antibody specific for influenza A and B. White light reflected through this surface appears as a gold-colored background.
The extracted material is incubated with a horseradish peroxidase-conjugated antibody specific for influenza A and B virus. The resulting antigen-antibody complex binds to the solid phase with influenza-specific antibodies and is thereafter visualized with horseradish peroxidase substrate. The obtained mass enhancement of the antigen-capturing optical surface of the silicon wafer develops a purple spot, indicating detection of influenza virus without differentiation between types A and B. If no binding occurs, the original molecular thickness remains unchanged, and the test surface retains the gold color, indicating a negative result. The test procedure for one sample is completed within 15 to 20 min. In our study positive test results were scored from 1+ to 4+.
Nucleic acid extraction.
RNA in 140 μl of the crude nasopharyngeal aspirate was prepared by binding to a silica matrix followed by a spin column purification with a final eluate of 60 μl according to the instructions of the manufacturer of the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany). To monitor for cross-contamination during the RNA preparation and the subsequent PCR, every fourth RNA extraction was a negative water control.
Design of RT-PCR.
Target sequences were from the hemagglutinin genes of influenza virus type A subtype H1 and subtype H3 and influenza virus type B. Primers were selected after analysis with OLIGO primer analysis software (version 5.0; National Biosciences Inc.) and compared with known sequences in both GenBank (http://www.ncbi.nlm.nih.org/BLAST
) and results from a previous study (23
; see Table ).
Characterization of primers in RT-PCR for detection of influenza viruses A and B
Optimization of a thermal amplification profile and concentration of deoxynucleotide triphosphates (dNTPs) and Mn(OAc)2 (step one) or MgCl2 (step two) were performed for each primer set.
For the analysis of clinical samples, 10 μl of the eluted influenza RNA was converted into complementary DNA (cDNA) and subsequently amplified by PCR using 5 U of rTth DNA polymerase in EZ-buffer (50 mM bicine, 115 mM potassium acetate, 8% glycerol [pH 8.2]), purchased from PE Biosystems, with 2.5 mM Mn(OAc)2, 0.1 mM dNTP, and 0.3 μM each primer (Table ). The reaction volume was 50 μl, and the optimized profile in the thermal cycler (Biometra T3; Biometra, Göttingen, Germany) in step one was 37°C for 2 min, 58°C for 30 min, and 94°C for 2 min during the RT reaction, followed by 40 amplification cycles (denaturation at 94°C for 30 s, annealing at 62 to 58°C for 40 s, and synthesis at 72°C for 40 s). To enhance specificity the annealing temperature was 62°C for the initial five cycles, 60°C for the next five cycles, and 58°C for the remaining 30 cycles. Amplification was completed with a prolonged synthesis at 72°C for 5 min.
In the nested PCR step, 3 μl of the initial reaction product was added to a second PCR reaction mixture of 50 μl containing 1.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Uppsala, Sweden) in 1.5 mM MgCl2–50 mM KCl–10 mM Tris-HCl (pH 8.3)–0.2 mM dNTP–0.4 μM each primer (Table ). The optimized cycle profile comprised an initial denaturation at 95°C for 1 min followed by 40 cycles of amplification and final synthesis as in step one above. Obtained PCR products were detected by electrophoresis on 1% ethidium bromide-stained agarose gels.
Cloning of plasmids for sensitivity determination of RT-PCR.
Products obtained after primary RT-PCR reactions for influenza A/Stockholm/01/96 H1N1, influenza A/Wuhan 359/95 H3N2, and influenza B/Sweden/2724/99 were inserted into plasmid pCR 2.1-TOPO and propagated in Escherichia coli strain TOP 10 according to the directions of the manufacturer (TOPO TA cloning kit; Invitrogen, Carlsbad, Calif.). Selected plasmid clones were purified by using a Wizard Plus Minipreps DNA Purification System (Promega, Madison, Wis.), and the DNA concentration was determined by using luciferase measurement of restriction enzyme-cleaved plasmid (DNAquant; Promega, Madison, Wis.). The calculated plasmid copy number was used in end-point titration of the sensitivity for each primer pair. A stock solution of each plasmid was diluted in TE buffer (10 mM Tris-HCl [pH 7.6], 1 mM EDTA) in half-log dilution steps at each run. For each dilution, one measure point was recorded, and three sensitivity determinations were performed for each primer pair.