Background and objective: During the 2009 influenza A (H1N1) pandemic, 60% of the respiratory specimens collected from patients under surveillance for influenza-like illness (ILI) in São Paulo, Brazil, tested negative for influenza A and B by Real-Time RT-PCR (RT-qPCR) assays. In this retrospective study, we identified the diversity of other respiratory viruses associated with ILI during the pandemic period and added the benefit of RT-qPCR for their detection.

Methods: Five-hundred and forty-two influenza RT-qPCR negative respiratory specimens from ILI patients collected from Jan - Dec 2009, that also tested negative by a commercial Indirect Immuno Fluorescence (IIF) assay for the Respiratory Syncytial Virus (RSV), Parainfluenza (PIV) PIV1, PIV2, PIV 3 and Adenovirus (AdV) were retested by a panel of RT-qPCR assays for the other respiratory viruses, including RSV, Human Metapneumovirus (HMPV), PIV 1-4, AdV, Human Coronaviruses (HCoV) OC43, 229E, NL63 and HKU1, Rhinovirus (RV), Enterovirus (EV) and Human Bocavirus (HBoV).

Results: Of the 542 negative specimens, 428 (78%) were positive by RT-qPCR for another respiratory virus: In descending order: RV (149, 27%); RSV (41, 8%); AdV (32, 6%); HMPV (29, 5%); PIV3 (19, 4%); PIV1 (3, 1%); HCoV-OC43 (9, 2%); HBoV (9, 2%); PIV4 (6, 1%); HCoV-HKU1 (6, 1%); EV (5, 1%); HCoV-229E 4 (4, 1%); HCoV-NL63 (3, 1%). Co-detections of two viruses (98, 18%) and three viruses (12, 2%) were common.

Conclusion: This retrospective study revealed a high prevalence of respiratory viruses other than influenza associated with ILI during the pandemic period and highlights the superiority of RT-qPCR over IIF for ILI surveillance.