A range of human influenza type A and B viruses and animal influenza type A viruses were tested by the Directigen FluA+B assay to confirm its reaction with viruses of diverse subtypes (Table ). The viral infectivity titers of the preparations used for testing ranged from 104
per 100 μl. Eighteen contemporary human influenza isolates, 6 each of influenza virus type A H3N2, influenza virus type A H1N1, and influenza virus type B, and 21 other influenza viruses (Table ) isolated between 1934 and the present were studied. Influenza type A viruses of subtypes H5N1 and H9N2 were those isolated from humans in Hong Kong in 1997 and 1999, respectively (14
). In addition, animal influenza type A viruses spanning a range of subtypes (Table ) were investigated. All the human and animal influenza type A viruses gave positive reactions for influenza virus type A but were negative for influenza virus type B. Conversely, 12 human influenza type B viruses, 6 of them isolated between 1940 and 1972 (Table ) and 6 other recent isolates from Hong Kong, gave positive reactions for influenza virus type B and negative reactions for influenza virus type A.
The analytical sensitivity of the Directigen FluA+B test was established using seven human influenza virus type A (subtype H1N1 and H3N2) and six influenza virus type B strains. The limit of detection of the Directigen FluA+B test ranged between 6.5 × 101 and 3.3 × 104 CEID50/ml for influenza virus type A viruses. The detection limit for influenza virus type B viruses ranged between 4.6 × 101 and 2.5 × 103/ml for influenza virus type B, with one outlying result for B/Lee/40 (detection limit, 1.2 × 106/ml).
A wide range of other viruses, bacteria, and yeasts (see Materials and Methods) tested for cross-reactivity were negative by both influenza virus type A and B tests in the Directigen FluA+B test.
The patient group studied was predominantly children. The age distribution was as follows: 111 patients were <2 years old, 89 were 2 to 6 years old, 13 were between 7 and 11 years old, 6 were between 12 and 16 years old, 12 were between 16 and 55 years, 18 were over 55 years old, and for 1 patient, information on age and sex was unavailable. The male-female ratio was 149:100.
Fifty-four of the 250 clinical specimens examined were positive for influenza virus by culture, 22 being influenza virus type A and 32 influenza virus type B. Seventeen of the influenza virus type A isolates were of subtype H3N2 and 5 were H1N1. The performance characteristics of the Directigen FluA+B assay compared to culture are shown in Table . All 22 influenza virus type A specimens positive by culture and 28 of the 32 influenza virus type B-positive specimens reacted positively to influenza virus type A and B, respectively, in the Directigen FluA+B test. Three specimens that were influenza virus type A positive and seven that were influenza virus type B positive by the Directigen FluA+B assay were culture negative. Furthermore, four specimens that yielded influenza virus type B in culture were negative by the Directigen FluA+B test.
Performance characteristics of Directigen FluA+B compared to culture
In comparison to culture, the Directigen FluA+B had a sensitivity of 100%, specificity of 98.7%, positive predictive value of 88%, and negative predictive value of 100% for influenza virus type A. In comparison, the DFA test had sensitivity, specificity, and positive and negative predictive values of 100%, 95.6%, 88%, and 100%, respectively. Similarly, for influenza virus type B, the sensitivity, specificity, and positive and negative predictive values for the Directigen FluA+B tests were 87.5%, 96.8%, 80%, and 98.1%, respectively, and for DFA they were 65.6%, 97.2%, 100%, and 95.5%, respectively.
The spare aliquot of specimens that were culture negative but positive for either virus by the Directigen FluA+B or DFA test were tested by RT-PCR for the presence of influenza virus type A and B viral RNA. Of four culture-negative specimens that were influenza virus type A positive by one or both of the antigen detection tests, three were confirmed by RT-PR to be “resolved true-positive” results. Of these, the two that were Directigen FluA+B positive were scored as weakly positive reactions (intensity, 0.5). The one specimen that was not confirmed by RT-PCR was positive by Directigen FluA+B alone (intensity of reaction, 0.5) and was regarded as a false-positive result (Table ). In comparison, of the 24 Directigen FluA+B-positive results confirmed (by culture or RT-PCR) to be true positives, 21 had reaction intensity scores of 2 to 4, 1 had a reaction intensity score of 1, and only 2 had a reaction intensity score of 0.5.
Comparison of culture, Directigen FluA+B, immunofluorescence, and RT-PCR for influenza A virus
None of seven culture-negative, Directigen FluA+B influenza virus type B-positive specimens was RT-PR positive, and the seven were regarded as false-positive enzyme immunoassay (EIA) results (Table ). All of them had weak (0.5) reaction intensity. In comparison, of the 28 influenza virus type B-positive specimens that were reactive in the Directigen FluA+B assay, 24 had reaction intensities of 2 or greater, 2 had reaction intensities of 1, and 2 had a reaction intensity of 0.5.
Comparison of culture, Directigen FluA+B, immunofluorescence, and RT-PCR for influenza B virus
In addition, three randomly selected specimens that were culture and Directigen FluA+B positive for influenza virus types A and B and 6 of 185 specimens negative by culture and antigen detection tests for both viruses were also tested by RT-PCR. The RT-PCR result confirmed the Directigen FluA+B result in each instance.
After the resolution of discrepant results (Tables and ), the sensitivity, specificity, positive predictive value and negative predictive value for diagnosis of influenza virus type A of the Directigen FluA+B assay were 96%, 99.6%, 96%, and 99.6%, respectively, and they were 100% for DFA for all parameters. In contrast, the sensitivity of culture for influenza virus type A was only 88%. For influenza virus type B, the sensitivity, specificity, positive predictive value, and negative predictive value were 87.5%, 96.8%, 80%, and 98%, respectively, for the Directigen FluA+B and 65.6%, 100%, 100%, and 95.2%, respectively, for the DFA assay. Thus, the Directigen FluA+B assay had better sensitivity than DFA for detection of influenza virus type B but led to more false-positive results.
Performance characteristics of Directigen FluA+B after analysis of discrepant specimens by RT-PCRa
Performance of immunofluorescent antigen detection after analysis of discrepant specimens by RT-PCRa
Other viruses, including 28 respiratory syncytial viruses, 10 adenoviruses, and 4 parainfluenza type 3 viruses were identified in 51 of the nasopharyngeal specimens. They all gave negative results with the Directigen FluA+B assay, with the exception of two specimens that were reactive for influenza virus type B in the Directigen FluA+B test. One specimen had a culture-confirmed double infection with influenza virus type B and adenovirus type 7, while the other was one of the weak (intensity, 0.5) influenza virus type B false-positive reactions noted above.
Specimen stability was evaluated by testing replicate aliquots of specimens in virus transport medium on the day of collection and after storage for 48 and 72 h at 2 to 8°C and after freezing at −20°C for 1 week. The specimens selected for stability testing were among the 250 specimens recruited for the rest of the study. Five influenza virus type A-positive specimens remained unchanged after storage for up to 72 h at 2 to 8°C. All 10 negative specimens remained negative after storage for 48 h at 2 to 8°C. While 9 of 10 negative specimens remained negative when stored for 72 h at 2 to 8°C, 1 gave an uninterpretable result. Of 17 influenza virus type A-negative specimens retested after 1 week at −20°C, 16 remained negative but 1 gave a false-positive result, the intensity score being 0.5.
Three of four influenza virus type B-positive specimens remained positive after storage for 48 or 72 h at 2 to 8°C. One specimen gave a false-negative result after 48 h, and another gave an uninterpretable result at 72 h. Eleven influenza virus type B-negative specimens remained negative after storage for 48 or 72 h at 2 to 8°C. Of 11 influenza virus type B-positive specimens that were stored frozen for 1 week, 9 remained positive, 1 gave an uninterpretable result, and 1 was false negative. All 11 influenza virus type B-negative specimens remained negative after storage for 48 or 72 h at 2 to 8°C. Ten of 11 remained negative after 1 week of storage frozen, while one specimen gave an uninterpretable result.
The intensity of the Directigen FluA+B reaction in positive specimens correlated with the time taken for the appearance of CPE in the cell culture tubes. Influenza virus type B-positive specimens giving a Directigen FluA+B intensity of 4 resulted in CPE in cell culture at a median of 2 days, while specimens with weaker reactions (0.5 to 3) took longer (median, 5 days; P = 0.0001, Mann-Whitney test). With influenza virus type A, Directigen FluA+B scores of 4 were associated with CPE with a median of 5 days, compared to 10.5 days in those specimens giving a weaker reaction (P = 0.12). The time required for the appearance of CPE is probably related to virus load in the clinical specimen.