Performance of rapid tests for detection of avian influenza A virus types H5N1 and H9N2.

Among the avian influenza A virus subtypes, the H5N1 and H9N2 viruses have the potential to cause an influenza pandemic because they are widely prevalent in avian species in Asia and have demonstrated the ability to infect humans (8). Currently, human infections with wild-type (wt) strains of these viruses could occur in the United States in poultry and turkey farm workers and in travelers returning from countries in which avian influenza viruses are prevalent in birds, such as Thailand, Vietnam, and China. Laboratory-acquired infections could also occur in vaccine researchers working with wt or candidate vaccine viruses, including cold-adapted (ca) viruses (2, 3, 11). Published reports indicate that the Directigen Flu A antigen capture enzyme immunoassay (Becton Dickinson, Sparks, MD) can detect H5N1, H7N2, and H7N3 avian influenza viruses and that a DAKO direct fluorescent antibody reagent (DAKO, Cambridgeshire, United Kingdom) can detect H5N1 avian influenza virus, although these commercial rapid antigen tests are insensitive (1, 4, 6, 13, 14). We sought to determine if rapid assays routinely used in clinical microbiology laboratories, i.e., the shell vial assay and two commercial antigen capture enzyme immunoassays, could detect wt and ca avian influenza A virus types H5N1 and H9N2. wt and ca influenza A viruses derived from A/Vietnam/1203/ 2004 (H5N1) and A/chicken/Hong Kong/G9/97 (H9N2) were used to determine if the shell vial assay, Directigen Flu A B (Becton Dickinson), and x/pect Flu A&B (Remel Lenexa, KS) could detect avian influenza A viruses (2, 10). The Directigen Flu A B and x/pect Flu A&B kits were used to test serial 10-fold dilutions (10 to 10 50% tissue culture infective doses [TCID50]) of the A/Hong Kong/491/1997 wt and A/Indonesia/ 05/05 wt strains and of the A/Vietnam/1203/2004 wt strain of H5N1 influenza A virus. The shell vial assay was performed as previously described but without centrifugation of the inoculated shell vials, because the appropriate holders for the centrifuge in the biosafety level-3 (BSL-3) facility were not available (5). We used A549 cells and RhMK cells (Diagnostic Hybrids, Inc., Athens, OH) and Bartels influenza A monoclonal antibody (Trinity Biotech, Wicklow, Ireland). Shell vial coverslips were fixed for staining after 1, 2, and 5 days of incubation at 32°C. The shell vials inoculated with the wt and ca viruses were incubated at 32°C, a temperature that is permissive for the cold-adapted viruses and for wt viruses as well. The Directigen Flu A B and x/pect Flu A&B assays were performed by following the manufacturers’ instructions. Shell vial culture assays and the enzyme immunoassays were performed in duplicate. The wt and ca influenza viruses were propagated in the allantoic cavity of embryonated eggs. All manipulations with live virus were performed in a USDAapproved BSL-3 facility. At concentrations of 20 and 2 10 TCID50, H5N1 ca virus was detected after 1, 2, and 5 days of incubation equally well in both A549 and RhMK cells. The H5N1 wt virus at 2 10 TCID50 was also detected in both cell lines, but at 20 TCID50 the wt virus was detected only at 5 days in A549 cells and at 2 and 5 days in RhMK cells. The H9N2 wt virus was detected at both concentrations after 1, 2, and 5 days of incubation equally well in both cell lines. The H9N2 ca virus was also detected in both cell lines at 2 10 TCID50, but at 20 TCID50 the ca virus was detected only at 5 days in A549 cells and at 2 and 5 days in RhMK cells. Although this experiment was carried out at an incubation temperature of 32°C, we have previous unpublished experimental data showing that the wt H5N1 viruses replicate equally well at 32°C and 37°C. Table 1 shows the results obtained with the two antigen capture enzyme immunoassays with both concentrations of wt and ca H5N1 and H9N2 influenza A viruses. Neither assay detected virus at 50 TCID50, but H9N2 wt and ca viruses were detected by both assays at 5 10 TCID50. H5N1 ca virus was detected at 5 10 4