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Viruses are frequent causal agents of acute respiratory infections and the most common are influenza virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), human metapneumovirus (HMPV), rhinovirus (RV), adenovirus (AdV) and the four endemic human coronaviruses (HCoV) -229E, -NL63, -OC43, -HKU1. Multiplex real-time PCR platforms are becoming increasingly common in laboratories mostly in relation to the increased diagnostic sensitivity and reduced turnaround time. The aim of our study was to determine the prevalence of respiratory viruses in a population of patients within the S.S. Antonio e Biagio e Cesare Arrigo General Hospital catchment area of Alessandria, Italy, from January 2016 to June 2020. Therefore, we retrospectively analyzed the results of multiplex real-time PCR performed on nasopharyngeal swabs collected from consecutive patients with symptoms of respiratory infection. A total of 572 patients were included in the study subdivided as follows: pediatric 197/572 (34.4%), adults 200/572 (35%) and elderly 175/572 (30.6%). Among all samples, 235/572 (41.1%) were positive for a respiratory virus, of whom 189/235 (80.4%) were monomicrobial. The prevalence was: 15.5% (89/572) of rhinovirus/enterovirus (RV/EV); 9.4% (54/572) of RSV; 8.9% (51/572) of influenza virus; 5.4% (31/572) of AdV; 3.1% (18/572) of HCoV; 2.8% (16/572) of HPIV; and 2.3% (13/572) of HMPV. RV/EV were the pathogens most frequently involved in coinfections (34.7%, 16/46), followed by AdV (19.6%, 9/46) and influenza virus (19.6%, 9/46). Samples collected from the pediatric group were more frequently positive. The prevalence of positive pediatric samples compared to adults and elderly, respectively was: 28.4% (56/197) for RV/EV vs 10.5% (21/200) vs 6.9% (12/175), p<0.0001; 18.8% (37/197) for RSV vs 2% (4/200) vs 7.4% (13/175), p<0.0001; 13.7% (27/197) for AdV vs 1% (2/200) vs 1.1% (2/175), p<0.0001; and 6.6% (13/197) for HPIV vs 0.5% (1/200) vs 1.1% (2/175), (p<0.0001). With regard to seasonality, a significantly higher prevalence of influenza virus (p<0.0001) and RSV (p=0.029) was found during winter, with peaks in January-February. AdV peaked during winter 2018-2019 (p=0.004), while HCoV were detected with a significantly higher prevalence during winter 2019-2020 (p=0.037). With regard to HPIV, a significant peak from summer to fall 2018 was observed (p=0.016). Most viral respiratory infections have seasonal patterns and the prevalence of respiratory viruses varies according to the method, geographic area and population considered. Knowledge of local epidemiology is therefore crucial for implementation of prevention and treatment strategies.