Etiologies of outpatient medically attended acute respiratory infections among young Ecuadorian children prior to the start of the 2020 SARS‐CoV‐2 pandemic

Abstract Background Implementation of respiratory virus prevention measures requires detailed understanding of regional epidemiology; however, data from many tropical countries are sparse. We describe etiologies of ambulatory pediatric acute respiratory tract infections (ARTI) in Ecuador immediately preceding the onset of the SARS‐CoV‐2 pandemic. Methods Children < 5 years presenting to a designated study site with an ARTI were eligible. Informed consent was obtained. Demographic and clinical data were recorded. A nasopharyngeal swab was collected, processed, and analyzed using multiplex polymerase chain reaction (PCR) for common respiratory pathogens. Rhinovirus/enterovirus positive samples were further characterized by genomic sequencing. Results A total of 820 subjects were enrolled in the study between July 2018 and March 2020. A total of 655 (80%) samples identified at least one pathogen. Rhinoviruses (44%) were most common, followed by enteroviruses (17%), parainfluenza viruses (17%), respiratory syncytial virus (RSV) (15%), and influenza viruses (13%). Enterovirus D68 was the most common enterovirus detected and was among the leading causes of bronchiolitis. Seasonal RSV and influenza virus activity were different along the coast compared with the highlands. Conclusions Ongoing regional surveillance studies are necessary to optimize available and emerging pathogen‐specific preventative measures.

vaccines are readily accessible in developed countries, yet challenges with availability and distribution often limit widespread use in lower income countries. 7,8 Ideally, influenza vaccine recommendations should be based on local influenza surveillance data, but such data are often limited. 9 Similarly, passive immunization against respiratory syncytial virus (RSV), with palivizumab, is safe and effective in preventing severe RSV disease among high-risk infants, yet its cost precludes its use throughout most of the developing world where RSV-associated morbidity and mortality are highest. 10 Cost aside, the lack of reliable longitudinal seasonal data across much of the tropics further challenges efforts to develop robust national public health programs aimed at preventing RSV. Optimal planning and implementation from season to season requires detailed understanding of regional RSV epidemiology.
Ecuador's ARTI disease burden and economic impact were recently estimated to include 14.8 million cases and 17,757 deaths over 5 years, 11 with associated annual costs during childhood exceeding $50 million. 12 Where resources permit, the emergence and availability of multiplex polymerase chain reaction (PCR)-based diagnostics have substantially improved the overall quality of pathogen-specific surveillance data. [13][14][15][16] Prior reports on the specific causes of ARTI in Ecuadorian children focused on hospitalized children living in the highlands with a paucity, or complete absence of virus seasonality along the coast. 14,15,17 Here, we report interim results from our 5-year surveillance study of the etiologies of ARTI in children enrolled during outpatient sick visits at clinics located in Quito (highlands) and Machala (coastal) during the period immediately preceding the onset of the SARS-CoV-2 pandemic.

| Study population
Eligibility for inclusion and recruitment logistics were previously described. 18 Briefly, children < 5 years of age presenting to a studydesignated ambulatory clinic with a suspected ARTI are eligible. For study purposes, an ARTI is defined as the presence or parental report of two or more of the following for fewer than 8 days: temperature ≥ 38 C, nasal congestion/discharge, cough, tachypnea, wheezing, rales, hypoxia, or apnea. Children in foster care, those with parents unable/unwilling to provide consent, and those hospitalized This assay detects and differentiates between rhinovirus-and enterovirus-specific RNA. Briefly, extracted RNA is subjected to reverse transcription and then amplified by targeting a highly conserved sequences of the 5'UTR of rhinoviruses/enteroviruses and using virus-specific primer pairs. Fluorescence-labeled probes included in the amplification mix allow the user to distinguish whether a detected signal represents sequence specific to rhinovirus or enterovirus. Positive, negative, and internal controls were included with each run. All enterovirus positive samples were then further characterized by genomic sequencing. BLAST algorithm software ( Clinical characteristics of subjects by pathogen group detected are summarized in Table 2. Almost half (156/322, 48%) of the respiratory samples collected from subjects diagnosed with nasopharyngitis were positive for the detection of rhinovirus. Clinically, RSV infection was more likely to be associated with wheezing and a clinical diagnosis of LRTI than any other respiratory virus (p < 0.05). Subjects diagnosed with bronchiolitis were identified year-round ( Figure 2). RSV was the most common pathogen identified among the 238 subjects diagnosed with bronchiolitis (61, 26%) followed by enteroviruses (60, 25%), rhinoviruses (51, 21%), and PIVs (43, 18%) ( Figure 1), whether or not the subjects were stratified by age. Enteroviruses were more commonly detected among children with bronchiolitis in Quito (48/161, 30%) than in Machala (10/77, 13%) (p < 0.05) (Table 3) Rhinovirus-and enterovirus-associated bronchiolitis was observed throughout the study period. PIV-associated bronchiolitis was biphasic, clustering between May and July and again between October and January, whereas RSV bronchiolitis occurred from November to May.

| Pathogen-specific results
The most commonly detected pathogen group, accounting for 285/655 (44%) positive samples, were the rhinoviruses ( where detection peaked between the months of October and December ( Figure 3). In contrast, a clear seasonal peak of PIV type 3 activity was not observed in Machala.
RSV was detected in 99 (15%) samples (  (Figure 3). In contrast, RSV detection was first noted in Quito        [27][28][29] There are currently little publicly available and/or published data regarding IV epidemiology from the existing surveillance systems in Ecuador. Data published a decade ago are consistent with our findings here that show year-round IV activity. 17 Understanding recent and current IV epidemiology is necessary to optimize disease prevention strategies. Although influenza vaccine recommendations across equatorial regions should be determined by regional epidemiology and active surveillance, such data are often unavailable, leaving public health decisions on the timing and formulation of annual vaccine programs to be made based solely on geographic location.
Results from this study, which continues through the calendar year 2023, and future efforts that focus on factors that help to explain differing circulation patterns of respiratory virus activity across different areas of Ecuador and other equatorial communities are increasingly important as new and emerging prevention measures become available.