Resurgence of Respiratory Virus after Relaxation of COVID-19 Containment Measures: A Real-World Data Study from a Regional Hospital of Italy

The SARS-CoV-2 virus spread in the Northern Hemisphere during the 2019/2020 influenza seasons and it persisted in the 2021/2022 season. A cocirculation of SARS-CoV-2 and influenza viruses was expected in Italy during the winter seasons. This study aims to investigate the prevalence of influenza and respiratory syncytial viruses observed in a hospital in Verona Province, Italy hospital during these past three winter seasons and to compare our data with national and global surveillance reports on the transmission of respiratory viruses in the preceding decade. Our findings clearly demonstrated the extremely low prevalence of influenza virus among hospitalized patients and outpatients during the first two COVID-19 winter seasons, with a reemergence of respiratory syncytial virus in the late 2021. Containment measures may have played an important role in temporarily stopping the circulation of respiratory viruses, but after relaxation, in 2021, we experienced an unusual increase of respiratory syncytial viruses at the beginning of the winter season.


Introduction
Infuenza is a contagious disease of the respiratory tract caused by human infuenza virus types A, B (INF-A/B), or C. Infuenza infections typically show seasonality, with epidemic transmission intensity between autumn and spring. Tese annual epidemics constitute an important public health problem with substantial morbidity and mortality, and they have a considerable economic impact [1]. Another common cause of lower respiratory tract infection is respiratory syncytial virus (RSV), that is classifed into two types, RSV-A and -B. RSV is responsible for numerous hospitalizations of children in their frst years of life, which add to the seasonal burden on health-care services [2]. To date, vaccines to prevent RSV disease or curative treatment options are not available.
Te pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Italy and the rest of the Northern Hemisphere during the 2019-2020 winter season and continues to circulate even during the season 2020/21 and 2021/22. On January 31 st 2020, following the recommendations of WHO PHEIC [3], the Council of Ministers declared a state of health emergency due to the SARS-CoV-2 epidemic. To contain the epidemic transmission of SARS-CoV-2, Italy introduced nonpharmaceutical interventions (NPI), including social distancing, face mask, hands hygiene, and announced the frst complete lockdown of social activities between March and May 2020.
Te clinical presentation, transmission routes, and seasonality of SARS-CoV-2 resemble those of INF-A/B, and it can be difcult to distinguish the two infections. Te Department of Infectious Tropical Diseases and Microbiology (DITM) at the IRCCS Sacro Cuore Don Calabria Hospital, therefore, implemented a diagnostic routine to test all persons for whom SARS-CoV-2 testing was requested, using a multiplex molecular assay that includes testing for SARS-CoV-2, INF-A/B, and RSV-A/B.
Here, we describe the diagnostic yield of INF and RSV testing in our catchment area since the start of the SARS-CoV-2 pandemic, and we compare our data with Italian and global virus surveillance data.

Study Design.
We performed a retrospective observational analysis of INF-A/B and RSV-A/B molecular diagnostic test results obtained from patients who were referred to IRCCS Sacro Cuore Don Calabria Hospital (Italy). Nasopharyngeal swab samples processed by the routine diagnostics were collected during the seasons 2019/ 2020 (from week 46 in 2019 to week 12 in 2020), 2020/2021 (from week 47 in 2020 to week 17 in 2021) and 2021/2022 (from week 42 in 2021 to week 4 in 2022).

Study Population.
Te results for all diagnostic and screening INF-A/B and RSV-A/B tests (including all positive and negative results) were extracted from the IRCCS Laboratory Information Management System (LIMS) database. Te retrieved data was cleaned by removing duplicate test results. Patients' age and sex were also collected.

Statistical Analysis.
Descriptive statistics were generated to defne the study population. A comparison was made by an unpaired the two-tailed Student's t-test, p < 0.05 was defned as the level of signifcance. Graphpad (GraphPad Software, USA) was used to perform statistical analyses.

Ethics Statement. In accordance with EU General Data Protection Regulation (GDPR) 2016/679 and Good Clinical
Practice standards, sensitive data were not collected. Each observation in the dataset was identifed solely by an alphanumerical code not related to patient identifcation. Ethical clearance for the study protocol was obtained from the competent Ethics Committees of Verona and Vicenza provinces (Prot. 25099/2021).

Prevalence of INF-A/B
. We retrieved respiratory virus PCR data collected on our site during the 2019/2020 season, from week 46/2019 to week 12/2020. Overall, 19 out of 167 nasopharyngeal swabs (11.3%) were found positive for INF-A and/or INF-B viruses. Te percentage of samples that tested positive for INF-A/B was highest in week 8/2020 (4.2%) and lowest in week 10/2020 (1.2%) (Figure 1(a)). Te highest prevalence of INF-A/B infection was observed in the age group 0-12 years (73.6%) and the lowest in patients aged 13-18 years (0%) (Figure 1(b)). It should be noted that in week 10/2020 Italy started nationwide lockdown measures that included the closing of schools (March 9 th , 2020). After  [4]. Furthermore, our data were in line with data reported by FluNet, the global web-based tool for infuenza virological surveillance. FluNet showed an unusual absence of seasonal infuenza activity in the temperate zone of the Northern Hemisphere compared to previous seasons, although sporadic infuenza detections were reported in Southern Asia and tropical Africa where the transmission intensity remained at interseasonal levels across all countries during 2020/2021 [5][6][7]. During the season 2020/2021, the total seasonal prevalence of non-SARS-CoV-2 respiratory viruses for all specimens tested at IRCCS-SC was exceptionally low. In the whole of Italy, none of the respiratory samples tested positive for INF-A/B. However, during the 2021/2022 season (from week 45/ 2021 to 4/2022), 10 infuenza cases were detected. Eight out of 10 of the 2021/2022 samples were genotyped and identifed as infuenza type A; the subtype of seven of these was H3N2, and the other was H1N1. Two samples were not subtyped due to low viral loads. Interestingly, we observed one case of coinfection, detected during week 2 in a 37year-old woman patient with a Ct for SARS-CoV-2 of 32 and 36 for INF-A. Te genotype resulted to be A/H3N2. InfuNet data recorded low levels of infuenza virus transmission in Italy for the 2021/2022 season (up until the 4 th week of 2022). Overall, 55 INF-A and 5 INF-B strains have been identifed since the start of the season, with the infuenza A/H3N2 subtype as the most prevalent. Globally, very sporadic and localized infuenza outbreaks were reported in the same period, with regional outbreaks of infuenza A(H3N2) occurring in South and Southeast Asia, infuenza B/Victoria in China, and infuenza A(H1N1) in West Africa.

Prevalence of RSV-A/B.
During the 2019/2020 season, we detected 17.3% RSV-A/B positive samples. RSV-A/B detection was most prevalent in the 0-12 years age group (77.7%). We also observed a decreasing trend in RSV-A/B cases after the implementation of the national lockdown in week 10 of 2020 (3.14% vs 0.89%, p < 0.05) (Figure 1(a)). However, as observed for infuenza, the overall prevalence of RSV-A/B in the 2020/2021 season was atypically low. We did not detect any cases of RSV-A/B in our 0-12 years age group. Surprisingly, in the 2021/2022 season, after previous seasons of exceptionally low RSV circulation, a massive increase of RSV cases was observed from week 42 until week 49 of 2021, with a peak in week 47 (17.1%, Figure 2(a)), anticipating its circulation by about 1 month. RSV detection was higher in children aged 0-12 years (53.6%) and the lowest rate of infection was observed in age 13-18 (0.5%) years ( Figure 2(b)). Te number of RSV infections detected in the 2021/2022 season was signifcantly higher than that recorded during the 2019/2020 season (2.30% vs 14.29%, p < 0.01). Data from the USA and Australia showed an inter-seasonal increase in RSV infections that coincided with lifting lockdown restrictions [8,9]. Te resurgence of RSV detections probably resulted from circulation of the virus among a large susceptible pediatric population, and this led to a substantial increase in RSV-related hospitalizations [10]. Tese events illustrate the need for an efective prevention strategy for RSV in infants.

Study Limitations.
Tis study has some limitations. First, because of the retrospective design of the study, important clinical characteristics were not recorded. Second, for the season 2019/2020, we performed less infuenza testing compared to the 2020/2021 and 2021/2022 seasons, and this may lead to an underestimate of the prevalence of infection.

Conclusions
In summary, we reported the trend of infuenza and RSV, during the last three fu seasons, in the course of the COVID-19 pandemic, in our reality. An exceptional low prevalence of INF-A/B was observed during season 2020/ 2021 and, curiously, an unprecedented surge in RSV cases was detected during the last fu season. Tis fnding is related to mitigation strategies that were adopted to contain the COVID-19 pandemic. Our data strongly support the notion that the public health measures adopted by Italy and other countries to contain the SARS-CoV-2 outbreak modifed the epidemiology and transmission of other respiratory viruses.
To date, it is difcult to ascertain which NPI was most effective in limiting the spread of infections. Terefore, further investigations are needed to clarify the strength of associations between public health interventions and their impact on the basic reproductive number of respiratory viruses. Systematic and continuous surveillance for non-SARS-CoV-2 respiratory viruses must be maintained in order to promptly prevent future epidemic surges of old or new respiratory viruses. In addition, eforts are needed to improve the RSV surveillance network.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.