The NT was performed according to a previously described procedure (20) , in agreement with indications from the World Health Organization (21) and the US Department of Health and Human Services (22) . Specifi cally, 2fold serial dilutions of heat-inactivated (30 min at 56°C) human sera were performed in 50 μL DMEM without FCS in 96-well microplates. An equal volume of infl uenza virus (H5N1) (10 3 TCID 50 /mL) was then added to each well. Uninfected-cell wells, incubated with each test serum, were included in each plate as negative controls. After 1 h incubation at 37°C, the mixtures were transferred on MDCK cell monolayers and adsorbed at 37°C for 1 h. After washing, DMEM was added, and the plates were incubated for 2 days at 37°C in 5% CO 2 . NT titer was assessed as the highest serum dilution in which no cytopathic effect was observed by light microscope inspection. All serum specimens were tested in duplicate, and t0, and t1 samples from each patient were assayed in the same plate at the same time. The results were scored by persons blinded to the study participant's identifi cation. The test results were reproducible because random replication of the assays on independent occasions gave consistent results.