1 INTRODUCTION

Each year, nearly 33 million cases of acute lower respiratory tract infection (ALRTI) associated with Respiratory Syncytial virus (RSV) are diagnosed in children under 5 years old.1, 2 RSV is the principal cause of bronchiolitis in young children3 and is globally the leading cause of hospital admissions in infants.4, 5 Although mortality due to RSV is very low among children in high-income countries,6 yet it plays an important role in the hospital resources utilization.7, 8 RSV is responsible for nearly 16 times more infant hospitalizations than influenza.9

In temperate climates, RSV circulation starts generally around November and end in March–April.2, 10

Age, prematurity, birth close to the start of the RSV season, and presence of chronic conditions have been commonly identified as potential risk factors for RSV-associated disease.7, 11, 12 However, by the age of two, almost all children are infected by RSV.13

Despite the high unmet medical need, a solution to protect all infants at risk to develop an RSV-associated disease is not yet available, but several candidates are expected to be licensed in coming years. Therefore, understanding the disease burden by month of age and by month of birth to determine who will benefit the most from these vaccination or monoclonal antibodies strategies is valuable to support future immunization policies and recommendations. Usually, burden-of-disease data are estimated using International Statistical Classification of Diseases and Related Health Problems (ICD) diagnosis codes on syndromic surveillance14 or hospital discharges data.15 Despite strengths of these studies, they usually do not report laboratory confirmed RSV information. To overcome this issue, we conducted a prospective active-surveillance study during 4 years in Valencia Region, Spain. The objective of this study was to better quantify the incidence of RSV-associated hospitalized disease by season, age, and month of birth. In addition, we aimed to describe the clinical presentation of the RSV-associated disease and to determine the risk factors of infants hospitalized with laboratory confirmed RSV.

2 METHODS 2.1 Study population

The study was conducted from 2014 through 2018 in 4 to 10 hospitals (depending on the season) in the Valencia Region: at Hospital General Universitario de Castellón (Castellón, Spain), Hospital Universitario de La Plana (Villarreal, Spain), Hospital Universitario y Politécnico La Fe (Valencia, Spain), Hospital Universitario Doctor Peset (Valencia, Spain), Hospital Universitario de La Ribera (Alzira, Spain), Hospital Universitario San Juan de Alicante (San Juan de Alicante, Spain), Hospital General Universitario de Elda (Elda, Spain), Hospital General Universitario de Alicante (Alicante, Spain), and Hospital Universitario del Vinalopó (Elche, Spain). The catchment area of these hospitals was well defined; they covered 21% (4 hospitals) to 46% (10 hospitals) of total inhabitants of the Valencia Region. From November to March/April every year, except in 2017/2018 season (from September to June), the active surveillance of RSV was set up. RSV circulation period was defined as the weeks between the first of at least two consecutive weeks with two or more RSV cases and the week prior to the first of at least two consecutive weeks without RSV cases. Hospitalized patients from all age groups meeting inclusion criteria were enrolled in the study. For the current publication, we considered only infant population aged <1 year.

2.2 Study design

The methodology of the active-surveillance network has been already described in previous publications.16-18 Full-time dedicated nurses screened consecutive hospitalized patients discharged from the Emergency Department with a diagnosis possibly related to a respiratory infection (Table S1). Patients were included in the study if they were resident in the catchment area of one of the participating hospitals, non-institutionalized, and not discharged from a previous admission in the last 30 days. The onset of symptoms that led to hospitalization was required to be 7 days prior to admission, and patients had to be in hospital between 8 and 48 h before their inclusion in the study. Infants under 1 month of age who left the hospital after delivery with no incidents (who were not admitted after birth in the neonatal unit) and who were subsequently hospitalized after a period of 1 week in the community were susceptible to be included in the study.

The Ethics Research Committee of the Dirección General de Salud Pública-Centro Superior de Investigación en Salud Pública (DGSP-CSISP) approved the protocol of the study. All caregivers signed written informed consent prior to inclusion of their infants in the study.

2.3 Laboratory procedures

Nasopharyngeal and nasal (FLOQSwabs, Copan, Italy) swabs were obtained within the first 48 h of admission from each patient fulfilling the inclusion criteria. Both swabs were combined in a tube of viral transport media (Copan, Italy) and frozen between −50°C and −20°C until shipped refrigerated to a centralized Virology laboratory at FISABIO-Public Health. One third of the viral transport media volume was used to extract total nucleic acids using an automated silica-based method (Nuclisens Easy-Mag, BioMérieux, Lyon, France). Extracted nucleic acids were tested for RSV, influenza, and other respiratory viruses (a total of 19) by multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR), following WHO protocols19 with the qScript XLT One-Step RT-qPCR ToughMix (Quanta BioSciences, MD, USA) in a Lightcycler 480II apparatus (Roche Diagnostics, Spain).

2.4 Statistical analysis 2.4.1 RSV-associated hospitalization incidence rates by season

We calculated the RSV hospitalization incidence rates per 100,000 infants-season overall and by hospital. The catchment area of each participating hospital along the different seasons was considered as the denominator. The RSV circulation period was estimated by epidemiological weeks, using EPIWEEK STATA module.

2.4.2 RSV-associated hospitalization incidence rates by age and birth month

According to age (0 to 11 months) and birth months (January to December), we calculated the RSV hospitalization incidence rates per 100,000 infants-season. The numerator was the number of RSV cases by age or by birth month for each season. The denominator was estimated by dividing the catchment population under 1 year old by 12. Due to the different duration of the seasons and to allow comparisons between them, the RSV hospitalization incidence rates were provided per 100,000 infants-week and per 100,000 infants-month (restricted to the RSV circulation period).

2.4.3 Risk factors of infants hospitalized with laboratory confirmed RSV

Comparison between RSV positive and RSV negative hospitalizations was conducted based on the following parameters: (1) birth month, (2) age (in months), (3) prematurity (<29 weeks, 29 to <37 weeks, ≥37 weeks), (4) associated comorbidities (chronic cardiovascular disease, chronic obstructive pulmonary disease [COPD], bronchitis, or any other chronic respiratory disease except asthma, anemia, and renal impairment), (5) contact with kids, and (6) kindergarten/school attendance and exposure to tobacco. Either Pearson Chi-squared or Fisher's exact tests were performed, as appropriate, to obtain p values.

2.4.4 Impact of age on RSV-associated hospitalization

A negative binomial regression, a generalization of the Poisson regression model that addresses the over-dispersion issue, was performed to assess the impact of age (≤3 months, >3 months) on RSV hospitalization incidence rates, after adjusting by calendar month at hospital admission (restricted from November to March), hospital and season. The population denominator was included as an offset. The adjusted relative risks (aRR) and their 95% confidence intervals (CIs) were provided.

2.4.5 RSV-associated disease—RSV positivity rate according to ICD-10 discharge diagnoses by season and age

Hospital discharge information using the 10th revision of ICD diagnosis codes (ICD-10) was retrieved for each infant included in the study. RSV-associated disease was defined based on the following ICD-10 codes recorded at hospital discharge: Lower Respiratory Infection (LRI): J09-J22, bronchiolitis: J21 and pneumonia: J12-J18. These outcomes were described by season and age group (<3 months, 3 to 5 months, and 6 to 11 months). Laboratory confirmed RSV results were used to determine RSV positivity rates for each disease outcome.

All statistical analyses were carried out in Stata version 14 (StataCorp, College Station, Texas) and R (Viena, Austria). All probabilities were two-tailed, and p values <0.05 were considered significant.

3 RESULTS 3.1 Description of the study population from 2014/2015 to 2017/2018

The total infant catchment population varied from 8,726 to 18,414 depending on the number of hospitals included in the study. Throughout the four seasons, 2,184 infants with respiratory symptoms were identified at hospitals. A total of 1,494 (68.41%) were included in the study. Among those, 631 (42.24%) were RSV positive (Table 1; Figure 1). Re-infection was not detected in infants included in the study. Most of RSV-associated hospitalizations (81%) occurred in otherwise healthy infants. Out of the 631 RSV positive patients, we subtyped 574 samples of which 64% were found to be RSV A (by season: 67%, 78%, 47%, and 65%, data not shown). Eighty three (13% of total RSV positive infants) out of the 631 RSV positive patients had co-infections, mainly rhinovirus (46%), coronavirus (25%), and bocavirus (14%) (data not shown).

TABLE 1. Study population, screened patients, included patients, PCR-positive patients, and RSV-positive patients by season and hospital Surveillance period Hospital Population Screened Included PCR-positive RSV-positive RSV surveillance period RSV circulation period N N N N N Rate x100,000 infants-seasona N Weeks Persons-time (in weeks) Rate ×100,000 infants-week Rate ×100,000 infants-monthb 2014/2015 (21 weeks, 4.8 months) General Castellón 2,469 174 124 91 54 2187.12 53 16 39,504 134.16 580.93 La Plana 1,707 83 67 51 39 2284.71 39 27,312 142.79 618.30 La Fe 1,672 64 53 28 19 1136.36 19 26,752 71.02 307.53 Dr Peset 3,017 77 57 31 13 430.89 13 48,272 26.93 116.61 La Ribera 2,280 121 83 59 39 1710.53 39 36,480 106.91 462.91 San Juan 1,629 27 21 12 5 306.94 5 26,064 19.18 83.06 Elda 1,690 59 50 30 15 887.57 15 27,040 55.47 240.20 General Alicante 2,391 115 44 29 13 543.71 11 38,256 28.75 124.50 Vinalopó 1,559 27 21 12 5 320.72 5 24,944 20.04 86.79 Overall 18,414 747 520 343 202 1,096.99 199 294,624 67.54 292.46 2015/2016 (24 weeks, 5.5 months) General Castellón 2,384 166 130 101 59 2474.83 59 20 47,680 123.74 535.80 La Fe 2,597 75 48 32 24 924.14 24 51,940 46.21 200.08 Dr Peset 2,147 77 68 55 33 1537.03 32 42,940 74.52 322.68 General Alicante 2,428 113 61 47 30 1235.58 30 48,560 61.78 267.50 Overall 9,556 431 307 235 146 1,527.84 145 191,120 75.87 328.51 2016/2017 (23 weeks, 5.3 months) General Castellón 2,288 136 109 65 36 1,573.43 36 17 38,896 92.55 400.76 La Fe 2,434 73 52 36 25 1,027.12 24 41,378 58.00 251.15 Dr Peset 2,098 118 84 55 33 1,572.93 33 35,666 92.53 400.63 General Alicante 2,327 177 93 66 50 2,148.69 50 39,559 126.39 547.28 Overall 9,147 504 338 222 144 1,574.29 143 155,499 91.96 398.20 2017/2018 (41 weeks, 9.5 months) General Castellón 2,279 163 127 100 52 2,281.70 50 19 43,301 115.47 499.99 La Fe 2,428 76 56 42 18 741.35 17 46,132 36.85 159.56 Dr Peset 1,685 86 39 29 19 1,127.60 19 32,015 59.35 256.97 General Alicante 2,334 177 107 85 50 2,142.25 49 44,346 110.49 478.44 Overall 8,726 502 329 256 139 1,592.94 135 165,794 81.43 352.58 Note: Infants hospitalized in the VAHNSI network, Valencia Region, Spain. Abbreviations: PCR, Polymerase Chain Reaction; RSV: Respiratory Syncytial Virus. a Season as time unit. Rates not comparable among seasons due to the different durations. b Approximating 1 month = 4.33 weeks.

Screened patients, included patients, PCR-positive patients, and RSV-positive patients by season and overall. Infants hospitalized in the VAHNSI network, Valencia Region, Spain

3.2 Description of the seasons

The study was carried out during four consecutive seasons. The RSV circulation period, comprised of weeks 2,014–2,050 to 2,015–2,012, 2,015–2,048 to 2,016–2,016, 2,016–2046 to 2,017–2,010, and 2,017–2,045 to 2,018–2,011 therefore by season, the duration was 16, 20, 17, and 19 weeks, respectively. The peaks were reached in weeks 2,015–2,001, 2,016–2,001, 2,016–2,050 and 2,017–2,052, corresponding to January in 2014/2015 and 2015/2016 and December in 2016/2017 and 2017/2018 (Figure 2).

Time distribution of hospitalizations by RSV laboratory result. Infants hospitalized in the VAHNSI network, Valencia Region, Spain

3.3 RSV-associated hospitalization incidence rates by season, age, and birth month

The overall RSV-associated hospitalization incidence rates ranged between 1,096.99 (2014/2015) and 1,592.94 (2017/2018) per 100,000 infants-season (Table 1). When calculating rates per month (to allow comparisons between seasons) during the RSV circulating period, RSV hospitalization incidence rates ranged between 292.46 (2014/2015) to 398.20 (2016/2017) per 100,000 infants-month (Table 1).

Substantial variability was detected among hospitals, irrespective of the seasons, and among seasons, irrespective of the hospital. For instance, in 2014/2015, rates varied between 83.06 (San Juan Hospital) and 580.93 (General de Castellón Hospital) per 100,000 infants-month. By season, rates for Doctor Peset Hospital were 116.61, 322.68, 400.63, and 256.97 per 100,000 infants-month, respectively (Table 1).

Every season, highest RSV-associated hospitalization incidence rates were detected in 1 month old infants (ranging between 231.59 and 275.10 per 100,000 infants-week, in 2016/2017 and 2017/2018, respectively), followed by 2 months old infants (between 142.60 and 216.15 per 100,000 infants-week, in 2014/2015 and 2016/2017, respectively). RSV-associated hospitalization incidence rates started to decrease for infants above 2 months old, although it was still high for 3 months old infants (200.71 per 100,000 infants-week) in 2016/2017 (Figure 3).

RSV hospitalization incidence rates per 100,000 infants-week (of RSV circulation) by season and months of age. Infants hospitalized in the VAHNSI network, Valencia Region, Spain

In terms of birth month, highest incidences were found in infants born from August to December, especially from September to November, irrespective of the season (Figure 4). In 2014/2015, 2015/2016, and 2016/2017, highest RSV hospitalization incidence rates were detected in infants born in November (215.94, 188.44, and 247.03 per 100,000 infants-week, respectively). In 2017/2018, highest rate was in infants born in October (253.38 per 100,000 infants-week) (Figure 4). Over the four seasons, higher risk of RSV hospitalization was detected in infants born before or at the beginning of the RSV season, and we observed that 54% of infants hospitalized due to RSV in their first RSV season were born outside of the season (April to October) (Table 2).

RSV hospitalization incidence rates per 100 000 infants-week (of RSV circulation) by season and birth month. Infants hospitalized in the VAHNSI network, Valencia Region, Spain

TABLE 2. Characteristics of patients by season and RSV status 2014/2015 2015/2016 2016/2017 2017/2018 RSV+ % RSV− % p RSV+ % RSV− % p RSV+ % RSV− % p RSV+ % RSV− % p Month of birth 202 100 318 100 0.001 146 100 161 100 0.041 144 100 194 100 0.017 139 100 190 100 <0.001 September 14 6.93 29 9.12 14 9.59 12 7.45 31 21.53 24 12.37 18 12.95 28 14.74 October 34 16.83 40 12.58 28 19.18 13 8.07 25 17.36 30 15.46 35 25.18 18 9.47 November 53 26.24 51 16.04 30 20.55 30 18.63 32 22.22 27 13.92 22