In accordance with local Infection Control policy, nasopharyngeal aspirates (NPAs) were collected in negative-pressure isolation rooms. Deep nasal swabs were obtained as an alternative in situation where an isolation facility was unavailable.19 These specimens were put immediately in viral transport medium and kept at 4°C during transportation. Both viral RNA and DNA were extracted on the same day of collection by PureLink Viral RNA/DNA Mini Kit (Invitrogen; Carlsbad, CA). RNA extracted was converted to cDNA by reverse transcriptase (Superscript III Reverse Transcriptase; Invitrogen). All DNA and cDNA were used immediately for nested multiplex PCR for 20 respiratory pathogens as described previously.20 Tables 1a and 1b in the online supplement summarize sequences and amplicon sizes of the outer and inner sets of PCR primers. Briefly, each nested multiplex PCR assay detected four pathogens. Group 1 comprised influenza A and B group-specific and subtypes H1N1, H3N2, H5N1-specific primers; group 2 comprised parainfluenza viruses (PIV-1, PIV-2, PIV-3, PIV-4a, and PIV-4b); group 3 comprised RSV A and B, HRV, and enterovirus; group 4 comprised HCoV-OC43, HCoV-229E, SARS-CoV, and HMPV; and group 5 comprised M pneumoniae, C pneumoniae, HBoV, and adenovirus. Both the first and second rounds of PCR were conducted in 20-μl reaction mixtures using fast thermal cycler (Applied Biosystems; Foster City, CA). Two microliters of cDNA was used as the template for the first round of PCR for groups 1 to 4, whereas 8 μL of the extracted preparation was used for group 5. In the second round of PCR, a 0.2-μL aliquot of the first-round PCR product was used as a template. Table 1c in the online supplement summarizes the PCR conditions of five multiplex nested PCR assays. The PCR products were stained by SYBR Safe (Invitrogen) and visualized by electrophoresis in 1.5% agarose gels. Four corresponding positive controls and one negative control (sterile water) sample were included for each group simultaneously. In order to prevent PCR contamination, reagent preparation, sample processing, and nested PCR assays were performed in separate rooms away from where amplified products were analyzed. Aerosol-resistant pipette tips were used throughout the experiments.