Received: 19 December 2021 Revised: 21 January 2022 Accepted: 25 January 2022 DOI: 10.1111/irv.12972 ORIGINAL ARTICLE Prevalence and seasonality of viral respiratory infections in a temperate climate region: A 24-year study (1997–2020) Laura García-Arroyo1 | Núria Prim1 | Marga Del Cuerpo1 | Pilar Marín1 | Maria Carme Roig1 | Mnontserrat Esteban1 | Rosa Labeaga1 | Neus Martí1 | Carla Berengua1 | Ignasi Gich2 | Ferran Navarro1,3 | Núria Rabella1,3 1 Microbiology Department. Hospital de la Santa Creu i Sant Pau, Universitat Autònoma Abstract de Barcelona (UAB), Sant Pau Biomedical Background: Few long-term reports have been published on the epidemiology of Research Institute (IIB Sant Pau), Barcelona, Spain respiratory viruses despite their frequent involvement in extremely common infec- 2 CIBER Epidemiología y Salud Pública tions. The aim here was to determine the frequency and distribution of respiratory (CIBERESP), Clinical Epidemiology and Public Health Department, Hospital de la Santa Creu viruses in a temperate climate area (Barcelona, Spain) throughout a 24-year period. i Sant Pau, Sant Pau Biomedical Research Methods: We collected data on all respiratory viruses detected from 1997 to 2020 Institute (IIB Sant Pau), Barcelona, Spain 3 in our institution. Clinical specimens were analyzed mainly by conventional tech- Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona (UAB), niques, and molecular techniques were also used. Barcelona, Spain Results: Of the 59,579 specimens analyzed, 21,382 (35.9%) were positive for at least Correspondence one virus. The number of positive samples during cold months was significantly Núria Rabella, Microbiology Department, higher than in warm months. Respiratory virus infections were detected in patients Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona (UAB), of all ages, above all in children under 3 years of age, who were most frequently Sant Pau Biomedical Research Institute (IIB infected with the respiratory syncytial virus, whereas Influenza A virus predominated Sant Pau), Barcelona, Spain. Email: nrabella@santpau.cat in the other groups, especially in adults. A clear demographic and seasonal pattern was established for some viruses. Circulation of other respiratory viruses during the FLUAV H1N1pdm09 and SARS-CoV-2 pandemics was observed. Conclusions: This long-term study provides new knowledge about the prevalence of respiratory viruses in a Mediterranean region. Throughout the study period, the fre- quency of some viruses remained constant, whereas others varied with the year. A clear demographic and seasonal pattern was established for some viruses. Patients suffering from severe respiratory infections should be examined for a range of respi- ratory viruses regardless of gender, age, or season. KEYWORDS climate, epidemiology, influenza, prevalence, respiratory viruses, seasonality 1 | I N T RO DU CT I O N They have a wide range of clinical presentations, ranging from com- mon colds to severe lower respiratory tract infections. As the same Respiratory viral infections are extremely common and among the clinical presentation may be caused by different viruses and the same main causes of medical consultations, especially in pediatric patients. virus may manifest with different symptoms, microbiological diagnosis This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd. 756 wileyonlinelibrary.com/journal/irv Influenza Other Respi Viruses. 2022;16:756–766. 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GARCÍA-ARROYO ET AL. 757 is essential to determine the etiological agent causing the infection.1 been performed by GeneXpert® Dx (Cepheid), and since January The most common respiratory viruses are respiratory syncytial virus 2015, this kit has also targeted RSV. A panel for the molecular detec- (RSV), adenoviruses (AdV), influenza virus types A and B (FLUAV and tion of respiratory viruses (FilmArray™) was introduced in the routine FLUBV), human metapneumovirus (hMPV), parainfluenza virus types workflow of the laboratory in May 2014, allowing the detection of 1–3 (PIV-1, PIV-2, and PIV-3), rhinoviruses (RV), enteroviruses (EV), PIV4 and different coronaviruses (HuCoV-229E, HuCoV-HKU1, and coronaviruses (CoV). Classical virological diagnosis of respiratory HuCoV-NL63, and HuCoV-OC43). At the beginning of 2020, several infections is based on cell culture and/or antigen detection tech- molecular methods were set up for the detection of SARS-CoV-2 niques. Molecular techniques have improved the capacity to detect (GeneXpert®, Simplexa®, VIASURE® Real Time PCR Detection Kit, these and other viruses in respiratory tract specimens, especially SARS-CoV-2 ELITe MGB® Kit, cobas® SARS-CoV-2 Test, and Alinity 2,3 those that cannot be identified by conventional methods. m SARS-CoV-2 assay). The few long-term studies on respiratory virus epidemiology pub- Specimens received from March 2020 were only studied for the 4–9 lished to date show seasonal patterns of circulation in defined geo- presence of SARS-CoV-2 due to the exceptionality of that year, and graphical areas. However, most reports cover shorter periods.10–14 they were analyzed separately. The epidemiological studies carried out in Spain are restricted to a few seasons,15 certain viruses,16 or particular groups of patients,17 and to our knowledge, none have focused on the prevalence of sev- 2.2 | Statistical analyses eral respiratory viruses within a broad population over a long period of time. Statistical analysis was performed by means of SPSS v26 (IBM Corp, The aim of the present study was to determine the frequency and Armonk, New York, USA). Continuous data and categorical data were distribution of respiratory viruses in a Mediterranean area throughout analyzed using the t test and Chi-square test, respectively. a 24-year period, including the first months of the SARS-CoV-2 pandemic. 3 | RE SU LT S 2 | MATERIALS AND METHODS 3.1 | General results 2.1 | Study design and data collection In the 1997–2019 period, 59,579 respiratory specimens were received by the laboratory, with a median of 2,590 per year (range We collected all the available data on respiratory viruses detected 952–4,883). Of these, 49,712 samples were processed by conven- from January 1997 to March 2020 in our institution, a tertiary referral tional methods and the other 9,867 only by molecular methods. A teaching hospital covering an area of 407,550 inhabitants in Barcelona total of 21,382 samples (35.9%) were positive for at least one virus. (Spain). We analyzed the circulation of respiratory viruses, including Overall, 21,939 virus detections were made, with more than one iden- frequency and seasonal distribution, the relationship between the dif- tified in 551 specimens (2.6%). The rate of positivity was significantly ferent viruses detected, and two baseline parameters of patients (age higher in samples acquired during the cold versus warm months, and sex). The Ethics Committee of Hospital de la Santa Creu i Sant Pau matching the seasonal distribution of samples received (p < 0.001). approved the research (approval number: IIBSP-VIR-2014-41) and In 2020, a total of 66,616 respiratory specimens were collected, waived the need for consent. 2,903 during the three first months of the year, and 832 (28.6%) Patients were grouped according to their age: groups A (under respiratory virus detections were made. All specimens received after 6 months), B (6–12 months), C (1–2 years), D (3–5 years), E (6– the WHO declaration of the SARS-CoV-2 pandemic were analyzed 17 years), F (18–29 years), G (30–39 years), H (40–59 years), and I only for SARS-CoV-2. A total of 6,566 samples were positive for (over 60 years). All patients under 18 years of age were assigned to SARS-CoV-2 in 2020. the pediatric population. Patients were classified as of unknown age when this information was unavailable. The clinical specimens analyzed were nasopharyngeal aspirates, 3.2 | Seasonal distribution of respiratory viruses nasal and pharyngeal exudates, bronchoalveolar lavages, and lung biopsies. The conventional techniques routinely used to identify respi- The different respiratory viruses identified throughout the study ratory viruses were immunochromatography, immunofluorescence period are shown in Table 1. The most frequently detected were 18 (IF), and cell culture (CC), as described in the literature. The respira- FLUAV (31.4%) and RSV (28.7%), which were co-detected in 25.5% tory viruses detected using these techniques were RSV, AdV, FLUAV, (118/462) of all the samples that tested positive for more than one FLUBV, PIV1, PIV2, PIV3, RV, and EV; screening for hMPV was virus. included from December 2008. At the start of 2009, an in-house PCR RSV, FLUAV, AdV, EV, and PIV3 were detected every year was introduced to detect FLUAV, FLUAV H1N1pdm09, and FLUBV. throughout the period prior to the SARS-CoV-2 pandemic. RSV con- Since February 2010, the molecular detection of these viruses has stituted 15.5% to 47.1% of all detected respiratory viruses per year, 758 TABLE 1 Yearly distribution of the respiratory viruses detected throughout the period under study (n = 21 939) a Viruses 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 RSV, n (%) 165 362 178 313 201 265 230 231 315 266 294 (47.1) (36.5) (17.1) (29) (38.7) (41.6) (32.6) (46.8) (38.2) (35.9) (30.6) FLUAV, n (%) 22 381 538 610 42 89 216 20 182 112 226 (6.3) (38.4) (51.7) (56.4) (8.1) (14) (30.6) (4) (22.1) (15.1) (23.5) FLUBV, n (%) 25 11 23 1 9 71 3 0 41 44 58 (7.1) (1.1) (2.2) (0.1) (1.7) (11.1) (0.4) (5) (5.9) (6) AdV, n (%) 58 123 152 68 101 79 122 67 99 133 199 (16.6) (12.4) (14.6) (6.3) (19.5) (12.4) (17.3) (13.6) (12) (17.9) (20.7) EV, n (%) 55 78 102 66 104 81 71 51 88 84 66 (15.7) (7.9) (9.8) (6.1) (20) (12.7) (10.1) (10.3) (10.7) (11.3) (6.9) RV, n (%) 0 1 4 0 4 37 33 62 56 47 71 (0.1) (0.4) (0.8) (5.8) (4.7) (12.6) (6.8) (6.3) (7.4) PIV1, n (%) 1 6 13 7 34 6 13 28 13 2 6 (0.3) (0.6) (1.2) (0.6) (6.6) (0.9) (1.8) (5.7) (1.6) (0.3) (0.6) PIV2, n (%) 16 7 17 1 5 1 4 0 9 0 15 (4.6) (0.7) (1.6) (0.1) (1) (0.2) (0.6) (1.1) (1.6) PIV3, n (%) 8 22 14 15 19 8 14 35 21 53 26 (2.3) (2.2) (1.3) (1.4) (3.7) (1.3) (2) (7.1) (2.5) (7.2) (2.7) hMPV, n (%) - - - - - - - - - - - Total of viruses 350 991 1,041 1,081 519 637 706 494 824 741 961 Samples, n 952 1,943 1,825 2,045 1,930 1,936 2,265 1,745 2,315 2,077 2,438 a The percentage of each virus is calculated considering the number of total viruses each year. b The total number does not include five PIV4 and 19 non-typed PIV. GARCÍA-ARROYO ET AL. 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GARCÍA-ARROYO ET AL. TABLE 1 (Continued) a Viruses 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Total RSV, n (%) 284 239 208 233 115 109 196 276 413 413 389 465 6,160 (39) (15.5) (28.3) (25.3) (16.3) (17.3) (25.2) (24.8) (26.9) (30.2) (21.2) (27.7) (28.1) FLUAV, n (%) 81 728 130 138 308 89 406 441 601 497 640 870 7,367 (11.1) (47.2) (17.7) (15) (43.6) (14.1) (52.3) (39.7) (39.1) (36.3) (34.9) (51.8) (33.6) FLUBV, n (%) 86 101 2 176 52 208 4 110 270 156 518 37 2006 (11.8) (6.5) (0.3) (19.1) (7.4) (33.1) (0.5) (9.9) (17.6) (11.4) (28.3) (2.2) (9.2) AdV, n (%) 126 218 132 146 83 91 44 84 66 112 101 120 2,524 (17.3) (14.1) (18) (15.8) (11.8) (14.5) (5.7) (7.6) (4.3) (8.2) (5.5) (7.1) (11.5) EV, n (%) 81 81 120 72 46 27 29 36 38 55 29 27 1487 (11.1) (5.2) (16.3) (7.8) (6.5) (4.3) (3.7) (3.2) (2.5) (4) (1.6) (1.6) (6.8) RV, n (%) 30 65 55 46 54 37 57 73 78 57 65 75 1006 (4.1) (4.2) (7.5) (5) (7.6) (5.9) (7.3) (6.6) (5.1) (4.2) (3.5) (4.5) (4.6) PIV1, n (%) 1 8 1 3 0 13 0 7 0 2 1 9 174 (0.1) (0.5) (0.1) (0.3) (2.1) (0.6) (0.1) (0.1) (0.5) (0.8) PIV2, n (%) 3 15 1 2 2 2 1 6 5 3 8 4 127 (0.4) (1) (0.1) (0.2) (0.3) (0.3) (0.1) (0.5) (0.3) (0.2) (0.4) (0.2) (0.6) PIV3, n (%) 32 28 33 33 21 31 13 35 23 27 26 16 553 (4.4) (1.8) (4.5) (3.6) (3) (4.9) (1.7) (3.1) (1.5) (2.0) (1.4) (1) (2.5) hMPV, n (%) 5 60 53 73 25 22 27 44 42 46 56 58 511 (0.7) (3.9) (7.2) (7.9) (3.5) (3.5) (3.5) (4) (2.7) (3.4) (3.1) (3.5) (2.3) Total of viruses 729 1543 735 922 706 629 777 ,112 1,536 1,368 1,833 1,681 21,915b Samples, n 2,131 4,152 2,246 2,609 2,047 2,088 2,345 3,069 4,224 3,788 4,883 4,526 59,579 a The percentage of each virus is calculated considering the number of total viruses each year. b The total number does not include five PIV4 and 19 non-typed PIV. 759 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 760 GARCÍA-ARROYO ET AL. the detection rate being significantly higher in 1997 and 2004 (p < 0.001). Among the viruses detected in the cold months, RSV (p < 0.001); FLUAV ranged from 4% to 56.4%, with rates highest in detection was highest from November to January, reaching a maxi- 1999, 2000, 2014, and 2019 (p < 0.001); AdV, 4.3% to 20.7%, with a mum in December. The prevalence of FLUAV was highest from peak in 2007 (p < 0.001); EV, 1.6 to 20%, being highest in 2001 and December to March, normally reaching a peak in January, whereas for 2010 (p < 0.001); and PIV3, 1% to 7.2%, with the highest rate in 2004 FLUBV, it was from January to March, with a peak usually 1 month and 2006 (p < 0.001). After the introduction of routine testing for after FLUAV. hMPV was most prevalent from February to May, and hMPV, this virus was detected every year (2008–2019) at rates rang- PIV-2, from September to December, with a peak usually in ing from 2.7% to 7.9%, with the highest in 2010 and 2011 (p < 0001) November. (Table 1). The other respiratory viruses in this study were detected The other respiratory viruses were not detected every year. throughout the year: AdV was most prevalent from November to FLUBV was not observed in 2004 but otherwise constituted 0.1% to May; EV and RV usually peaked in autumn and spring; PIV-1, from 33.1% of all viruses detected each year, the highest detection rates September to April with a peak in October; and PIV-3, from April to being in 2013, 2016, and 2018 (p < 0.001); RV ranged from 0.1% to June and October to December. 12.6%, with the highest rate observed in 2004 (p < 0.001); PIV-1, 0.1% to 6.6%, with peaks in 2001 and 2004; and PIV-2, 0.1% to 4.6%, peaking in 1997 (p < 0.001) (Table 1). 3.3 | Respiratory viruses during the FLUAV H1N1 The monthly distribution of each respiratory virus and their sea- pdm09 pandemic sonal patterns during the 23 years prior to the SARS-CoV-2 pandemic are shown in Figures 1 and 2, respectively. The relation between sea- The circulation of other respiratory viruses during the FLUAV H1N1 son and prevalence was statistically significant for all viruses pdm09 pandemic (April 2009 to March 2010) was analyzed. Overall, F I G U R E 1 Respiratory viruses during the 23-year period study (1997/2019). Monthly distribution of each respiratory virus detected (part 1 of 2). The depicted area for each virus is adapted to the total number of viruses detected. The large line on each year marks every January 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GARCÍA-ARROYO ET AL. 761 RV, hMPV, and PIV constituted 57% of the detections (Table 2), with the expected seasonal distribution. 3.4 | Correlation between respiratory viruses and the age/sex of patients The highest number of samples was collected from patients over 60 years (group I) and the lowest from those between 18–29 years (group F). The detection frequency for each respiratory virus varied between the different age groups (Table 3), the differences being sta- tistically significant for all studied viruses (p < 0.001). Maximum virus detection occurred in patients of groups B and C (i.e., infants from 6 months to 2 years), with 51.7% and 51.3% of samples testing posi- tive, respectively. At least one virus was detected in 38.9%, 44.8%, and 32.1% of the samples from groups A, D, and E, respectively. RSV was the most frequently detected virus in patients under 3 years of age. FLUAV was predominant in the other groups, especially in patients over 18 years of age. The distribution of each virus was analyzed within the different age groups (Figure 3). RSV was the most frequent virus in infants up to 6 months of age (group A) (p < 0.001), with 43.1% (2,625/6,086) of all RSV cases identified in this group. The frequency of hMPV was highest in infants of group C (31.5%; 161/511) and group A (30.7%; 157/511) (p < 0.001). Of all FLUAV cases, 30.4% (2,225/7,313) and 15.3% (1,119/7,313) corresponded to groups I and C, respectively, and 34.7% of FLUBV detections (695/2,003) to group I. AdV was detected most frequently in group C (p < 0.001), with 40.1% (998/2,491) of all positive samples corresponding to this group of infants. Detection of EV and RV was higher in pediatric patients than in adults (p < 0.001). Both EV (n = 1461) and RV (n = 1003) were most frequently detected in group A (detection rates of 41.4% and 40.1%, respectively). Detection of parainfluenza viruses was also higher in pediatric patients than in adults (p < 0.001), with the highest rates obtained in group A: 41.8% PIV-1 (69/165), 34.6% PIV-2 (44/127), and 37.2% PIV-3 (204/548). Regarding the gender of patients, 54.6% (n = 32,587) and 44.8% (n = 26,688) of specimens were from males and females, respectively. Respiratory viruses were detected in 11,394 samples from males and 9,873 from females. Although more specimens were received from male patients, the percentage of positivity was higher for females F I G U R E 2 Seasonal pattern of each respiratory virus (shown (36.9%) than for males (34.9%) (p < 0.001). Differences in the positiv- from July to June). The depicted area for each virus is adapted to the ity rates between the genders stemmed from the groups of patients total number of viruses detected more than 18 years old. 3,512 samples were screened for respiratory viruses during this 3.5 | Circulation of other respiratory viruses during period, 1,068 of which tested positive (30.4%) with a total of 1,080 the SARS-CoV-2 pandemic virus detections (Table 2). Four-hundred seven specimens tested posi- tive for FLUAV by PCR. IF/CC resulted in 673 virus detections, RSV The circulation of other respiratory viruses besides SARS-CoV-2 was being the most frequently detected, followed by AdV and EV. During recorded in 2020. This year was analyzed separately and divided in the pandemic, FLUAV was the predominant respiratory virus detected two periods, before and after the WHO declaration of the SARS- (43%), reaching a peak in November 2009, whereas RSV, AdV, EV, CoV-2 pandemic on March 15, 2020. In the first period, 2,903 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 762 GARCÍA-ARROYO ET AL. TABLE 2 Respiratory viruses detected during the FLUAV H1N1 pandemics (April 2009–march 2010) Samples/detection method Conventional, n Molecular, n Total Total 1,961 1,551 3,512 Positive 661 407 1,068 Viruses, n (%)b RSV 260 ND 260 FLUAV 57 407 464a FLUBV 2 0 2 AdV 128 ND 128 EV 79 ND 79 RV 54 ND 54 PIV-1 8 ND 8 PIV-2 13 ND 13 PIV-3 26 ND 26 hMPV 46 ND 46 Total of viruses, n 673 407 1,080b Abbreviation: ND, not detected by the molecular technique used. a FLUAV corresponded to 43% of the total of viruses detected in this period. b Two viruses were detected in 12 samples. specimens were analyzed for the presence of respiratory viruses other infections appeared early, RSV outbreaks tended to be delayed. In than SARS-CoV-2, and 832 (28.6%) of them were positive: FLUAV contrast, in our experience, the seasonal pattern of RSV was similar (n = 401), FLUBV (n = 223), RSV (n = 104), AdV (n = 46), hMPV every year, regardless of FLUAV. (n = 28), RV (n = 16), EV (n = 13), and PIV-1 (n = 1). In the second The detection frequency of the viruses in our study is similar to period, 905 specimens were analyzed for the presence of respiratory that of previous reports.5,9,24 The differences observed may be viruses other than SARS-CoV-2, and 30 (3.3%) of them were positive: explained by the type of population under study, the season in which FLUAV (n = 9), AdV (n = 8), RV (n = 7), FLUBV (n = 3), hMPV (n = 1), the study was performed, its length, and/or the methodologies EV (n = 1), and PIV-1 (n = 1). The results from 2020 were not used.4,10,12,19 Thus, we observed a lower RSV frequency compared to included in the statistical analysis. studies focusing only on a pediatric population.4,9,10,12 The detection frequency of FLUAV and FLUBV also differed from the literature, as although infections caused by these viruses follow a yearly epidemical 4 | DISCUSSION pattern, their intensity fluctuates.4,6,7,9,12 Furthermore, some viruses were found in lower frequencies (especially hMPV, RV, EV, and some The aim of the present study was to describe the frequency and distri- PIV) than in other studies using molecular methods.13,20–22 bution of respiratory viruses in a specific geographical area with a In temperate climates, respiratory viruses are usually expected temperate climate over a 24-year period up to 2020. In the 23 years during the cold months, whereas in tropical climates, they may be prior to the SARS-CoV-2 pandemic, the overall positivity rate was found throughout the year or are associated with the rainy sea- 36.8% of all the samples analyzed, which is in accordance with other son.4,7,9,12,19,25,26 In the present study, RSV, influenza viruses, and studies also using conventional methods.4,5,10–12,19 The main differ- hMPV were mainly detected in winter and early spring and hardly ences with other reports are due to the different viruses evaluated, as detected in summer, in agreement with other studies performed in not all include RV, EV, or herpes viruses.4,5,11 As expected, our positiv- temperate climates.6,12,22,27–29 RSV was the first virus detected at the ity rates are lower than in studies using molecular techniques, which start of the cold season, displaying a marked seasonal distribution are more sensitive and can identify a wider range of viruses than con- between November and January. FLUAV was found mainly between ventional methods.6,13,14,20–22 December and March. Although not detected every year, FLUBV cir- The phenomenon of respiratory viral interference, in which infec- culated mainly between January and March, usually after the FLUAV tion by a particular virus may interfere with the timing or rate of other epidemics,30 and was followed by hMPV, usually between February viral outbreaks, is well documented.22 The long period of our study and May. allowed us to observe variations in frequency and distribution of Infections caused by other respiratory viruses such as AdV and respiratory viruses over time. The highest positivity rates for some PIV-3 were observed throughout the year, in accordance with other viruses were observed in years with a low circulation of FLUAV; this studies in temperate climates.3,6,28 EV and RV showed a bimodal dis- was the case for PIV-1 in 2001 and 2004, AdV and EV in 2001, and tribution, with seasonal peaks in autumn and spring, as previously RV and PIV-3 in 2004. Van Asten et al23 reported that when FLUAV reported.3,6 Other PIVs do not regularly circulate throughout the year 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GARCÍA-ARROYO ET AL. 763 TABLE 3 Distribution of viruses considering the range of age. Samples from patients of unknown age (n = 492) were not included Group Group B Group C Group D Group E Group F Group G Group H Group Virus/age group A<6m 6–12 m 1–2 y 3–5 y 6–17 y 18–29 y 30–39 y 40–59 y I > 60 y Total Specimen, n 13,874 5,219 8,162 2,872 3,918 1,675 2,514 6,216 14,637 59,087 Positive specimen, n (%)a 5,398 2,699 4,185 1,288 1,259 442 648 1,445 3,821 21,185 38.9% 51.7% 51.3% 44.8% 32.1% 26.4% 25.8% 23.2% 26.1% Viruses, n (%)b RSV 2,625 1,001 1,137 183 77 39 71 191 762 6,086 47.3% 35.6% 26.4% 13.9% 6.0% 8.7% 10.9% 13.0% 19.7% FLUAV 921 543 1119 471 466 296 401 871 2225 7,313 16.6% 19.3% 25.9% 35.9% 36.3% 66.2% 61.4% 59.5% 57.4% FLUBV 117 72 182 202 351 54 105 225 695 2,003 2.1% 2.6% 4.2% 15.4% 27.3% 12.1% 16.1% 15.4% 17.9% AdV 405 617 998 211 158 14 21 34 33 2,491 7.3% 22.0% 23.1% 16.1% 12.3% 3.1% 3.2% 2.3% 0.9% EV 605 217 359 106 95 6 9 28 36 1,461 10.9% 7.7% 8.3% 8.1% 7.4% 1.3% 1.4% 1.9% 0.9% RV 402 122 182 57 72 16 24 53 75 1,003 7.2% 4.3% 4.2% 4.3% 5.6% 3.6% 3.7% 3.6% 1.9% PIV-1 69 21 32 14 9 2 3 10 5 165 1.2% 0.7% 0.7% 1.1% 0.7% 0.4% 0.5% 0.7% 0.1% PIV-2 44 17 26 18 9 0 1 6 6 127 0.8% 0.6% 0.6% 1.4% 0.7% - 0.2% 0,4% 0.2% PIV-3 204 98 117 14 28 18 15 31 23 548 3.7% 3.5% 2.7% 1.1% 2.2% 4.0% 2.3% 2.1% 0.6% hMPV 157 101 161 37 20 2 3 15 15 511 2.8% 3.6% 3.7% 2.8% 1.6% 0.4% 0.5% 1.0% 0.4% Mixed detection 154 112 135 28 30 5 5 19 54 542 Total of viruses, n 5,549 2,809 4,313 1,313 1,285 447 653 1,464 3,875 21,708c Abbreviations: m, months; y, years. a Percentage of positive samples considering each group of age. b Percentage of each virus out of the total of viruses in each group of age. c The total number does not include five PIV4 and 19 non-typed PIV. or every year and vary between different geographic areas.10,28 In our consultation, and therefore, the majority remains undetected. RSV study, PIV-1 was detected essentially in autumn and nearly every was mainly diagnosed in children under 3 years of age, which may be year, whereas PIV-2 was mainly found between September and partially explained by the high percentage of pediatric specimens December. (71.5%) included in our study, but also reflects that RSV infections are In April 2009, the WHO announced the outbreak of the FLUAV the main cause of respiratory infection (bronchiolitis) in infants.10 An H1N1 pdm09 pandemic, which lasted until March 2010. During this increase in active screening of patients of all ages for RSV has brought period, when FLUAV detection was a priority, it was the most fre- to light its role in respiratory infections in adults.5,8 quently detected virus (43% of all respiratory viruses). However, over We observed an increasing tendency in FLUAV detection with half of the respiratory viruses detected during the 2009/2010 pan- patient age, as described in the literature.5,22,29 This may be explained demic corresponded to viruses other than FLUAV, in agreement with by the fact that new variants of influenza viruses appear each year other regional laboratories.31 Given that the clinical manifestations of and previously developed immunization may not be effective against 21 influenza viruses are not specific and the possibility of simultaneous the new variants. circulation, screening for other respiratory viruses should also be car- Our results showed that children aged 5 to 18 years were the ried out during epidemics/pandemics. population with most FLUBV infections, which is in agreement with Overall, the highest positivity rates were observed in the pediatric other studies.10,32 Infections caused by AdV or hMPV were diag- population. All respiratory viruses may cause mild and self-limited nosed mainly in children under 3 years. RV and EV infections were reinfections throughout a lifespan without requiring medical also predominantly detected in pediatric populations, especially in 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 764 GARCÍA-ARROYO ET AL. FIGURE 3 Occurrence of each respiratory virus in relation to the group of age. Respiratory viruses are distributed in percentage according to the age those under 6 months and between 1 and 3 years, as previously changed the study of respiratory viral infections. Accordingly, the reported.11 highest number of specimens for the detection of respiratory viruses Infections caused by PIV-1 and PIV-3 were mainly observed in received in our laboratory in 1 year during the previous 23-year infants under 6 months, whereas those caused by PIV-2 were predom- period was 4,883, whereas in 2020, it was 66,616. That year, the inant not only in these patients but also in children aged 1 to 3 years. number of specimens processed for the detection of respiratory These results are comparable to those of Henrickson,33 who reported viruses other than SARS-CoV-2 was 3,808, and 862 such detections that children suffered more severe PIV-1 and PIV-2 infections in their were made, 96.5% of them before the pandemic declaration (data not first year, and PIV-3 caused symptoms similar to those of RSV. published). Little information is available about the circulation of respi- The overall rate of virus detection in the present study was higher ratory viruses other than SARS-CoV-2 during the rest of 2020 in females than in males, in contrast with previously reported because all efforts were directed to SARS-CoV-2. data.4,11,19,34 This difference was observed only in adult female A strength of this study is the use of gold standard methods, patients, which might be partly due to their having closer contact with namely, viral culture and antigen detection immunofluorescence, young children compared to men.27 together with the high number of samples and the long study period, In our study, using antigen detection and viral isolation methods, as they provide a global vision of the prevalence of respiratory viruses co-detections were obtained in 2.6% of positive samples. These data in the area. A limitation of our study is that it was not based on sys- are similar to those obtained by other studies based on conventional tematic surveillance. Additionally, conventional methods are not the 4,10 methods and lower than those only using molecular methods, most suitable to detect certain viruses (e.g., rhinoviruses, cor- where the frequency of co-detections ranges from 10% to onaviruses, and some parainfluenza viruses), although they are more 50%.14,21,22 The clinical impact of co-detection remains unknown, reliable than molecular methods for the detection of an infection in although a more severe clinical course has been associated with the progress. The changes in methodology (mainly for FLUAV, FLUBV, or 35 detection of more than one respiratory virus. RSV) during the study period could have affected the number of In this work, the most frequent co-detection was of RSV and detections, but the detection rates of the viruses in question were FLUAV, as reported in the literature,5 whereas other authors have very similar in years when only conventional methods were used. found RV and AdV to be the most common,21,22 attributing it to their This long-term study therefore broadens our knowledge of the year-round circulation. In our study, cases of co-detection were prevalence of respiratory viruses in a Mediterranean region. The fre- observed mainly during the winter months, the season associated with quency of some viruses was constant throughout the study period, RSV and FLUAV circulation, and coinciding with the maximum number whereas others varied with the year. Clear demographic and seasonal of virus detections. patterns were evident for some viruses. During the influenza A pan- The impact of the COVID-19 pandemic, declared during the prep- demic, other respiratory viruses were detected in more than half of aration of this manuscript, on society and the medical network patients suffering respiratory infections. Other respiratory viruses 17502659, 2022, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/irv.12972 by Readcube (Labtiva Inc.), Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GARCÍA-ARROYO ET AL. 765 were also detected during the first year of the SARS-CoV-2 pan- India: 1970–2020. Front Microbiol 2021;12:723850. doi: demic.36 We conclude that patients suffering from severe respiratory 10.3389/fmicb.2021.723850 10. 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