By contrast, we prospectively evaluated VL in NPAs from 23 untreated infants, children, and adolescents (median age, 12.7 years; range, 0.7 to 17 years) without underlying comorbidities and with uncomplicated influenza attending to the Anna Meyer Children’s University Hospital (Florence, Italy) during July 2009. With parental consent, clinical assessment and NPAs were repeated at 5 and 10 days from symptom onset in children with positive results. RNA was extracted by the QIAamp Viral RNA minikit (Qiagen; Milan, Italy). Two real-time RT-PCRs,2 using the SuperScript III Platinum One-Step qRT_PCR (Invitrogen; Carlsbad, CA), and primers and probes targeting the influenza A virus M gene and the hemagglutinin gene of the A(H1N1), respectively, were performed. The positive control was the strain A/Italy/05/2009(H1N1)v isolated in our laboratory (accession numbers GQ251032 to GQ251039). For the quantitative assay targeting the hemagglutinin gene, serial dilutions (-2 to -7 containing 1 × 105.5 to 1 × 100.5 tissue culture infectious doses 50 equivalents [TCID50]/mL) of the positive control strain were used to construct the calibration curve. Approximately one TCID50 contained 1,000 viral genome copies. The estimated limit of the quantitative RT-PCR was 3.1 TCID50 equivalents/mL (approximately 3,100 copies/mL). Virus concentrations (expressed in TCID50 equivalents) were log transformed and reported (Table 1) as mean ± SD. One girl still had a positive result 12 days after onset of fever. Duration of fever in all 23 children was 3 days (interquartile range, 3 to 5 days).