AETIOLOGICAL STRUCTURE OF ACUTE RESPIRATORY TRACT INFECTIONS AMONG CHILDREN YOUNGER THAN 5 YEARS IN BULGARIA

Acute respiratory tract infections (ARI) are a leading cause of morbidity and hospital admissions among infants and young children. This study aims to determine the viral aetiology of ARI and the clinical significance of the most common respiratory viruses in children aged <5 years in Bulgaria. During the period October 2017-March 2019, nasopharyngeal specimens were collected from children younger than 5 years in different country regions. Real-time PCR analysis was performed for detection of influenza viruses A/B, respiratory-syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza viruses (PIV) types 1, 2 and 3, rhinoviruses (RV), adenoviruses (AdV) and bocaviruses (BoV).


INTRODUCTION
Acute respiratory infections (ARI) are associated with a large number of doctor visits, hospital admissions, significant mortality, serious health and social consequences. With respect to the anatomical localisation, ARIs are classified as upper and lower respiratory tract infections. Upper respiratory tract involvement usually occurs as a mild or moderate illness. The high incidence of these infections is associated with prolonged circulation of pathogens in the community. Lower respiratory tract involvement leads to severe complications as bronchitis, bronchiolitis, pneumonia, often requiring hospitalisation. In 2010, 11.9 million episodes of severe and 3 million episodes of very severe ARI have led to the admission of infants and young children to hospital worldwide (1). Bronchiolitis occurs in children <2 years of age and most often has viral aetiology, whereas pneumonia can be caused by bacteria, viruses or other pathogens. Pneumonia is the leading infectious cause of mortality in children <5 years of age (2). A large spectrum of viruses is associated with ARI and the major causative pathogens are influenza viruses, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza viruses (PIV) 1/2/3, rhinoviruses (RV), adenoviruses (AdV) and bocaviruses (BoV). This study aims to determine the role of influenza viruses and 8 other respiratory viruses in the development of ARI in children younger than 5 years of age during two consecutive seasons in Bulgaria.

MATERIAL AND METHODS
A total of 953 children aged <5 years, ambulatory-

ADDRESS FOR CORRESPONDENCE:
Ivelina Trifonova, Department of Virology, National Centre of Infectious and Parasitic Diseases, 44A Stoletov Blvd. 1233 Sofia, Bulgaria Tel: +359 2 9318 132; e-mail: trifonova.ivelina@abv.bg treated or hospitalised for influenza-like illness (ILI) or acute respiratory illness (ARI) in different regions of the country were examined in the study. Of them, 444 (46%) had complications -laryngotracheitis, bronchiolitis, pneumonia or central nervous system (CNS) infections (febrile seizures, brain oedema, meningitis, encephalopathy, encephalitis). Viral nucleic acids were automatically extracted from respiratory specimens using a commercial ExiPrep Dx Viral DNA/RNA Kit (Bioneer) according to the manufacturer's instructions. Laboratory testing was conducted at the National Laboratory "Influenza and Acute Respiratory Diseases" recognised by WHO as a National Influenza Centre. Detection and typing/subtyping of influenza viruses was carried out by real-time RT-PCR method with SuperScript III Platinum® One-Step Quantitative RT-PCR System (Invitrogen). All samples were initially tested for influenza A and B viruses using primers and probes donated by CDC Atlanta and those positive for influenza A and B were subsequently tested for A(H1N1)pdm09 and A(H3N2), B/Yamagata and B/Victoria, respectively. All samples were also examined by singleplex real-time RT-PCR assays for RSV, HMPV, PIV 1/2/3, RV, AdV and BoV using specific primers/probes and AgPath-

ID One
Step RT-PCR Kit (Applied Biosystems). Primers and probes were identical to those previously described (3).   Single infections were detected in 568 (59.6%) patients; 88 (9.2%) children were co-infected with two and 7 (0.7%) children -with three viruses. Among the children with mono-infections, the most commonly determined pathogen was RSV, followed by influenza A(H1N1)pdm09. AdV, RV,

Seasonal distribution of viral agents
The greatest number of respiratory viruses was detected in specimens obtained in December 2018, February 2017 and May 2018 (Fig. 3). Influenza and RSV infections were more prevalent in winter. RV, BoV and HMPV infections occurred predominantly during fall and spring in Bulgaria.

Clinical characteristics
During early childhood respiratory viruses may cause serious complications affecting the respiratory tract (laryngotracheitis, bronchiolitis, pneumonia) or CNS (febrile seizures, brain oedema, meningitis, encephalopathy, encephalitis). This study also analysed the involvement of influenza viruses, RSV, HMPV, PIV1/2/3, RV, AdV and BoV in development of the complications mentioned above. A total of 85, 198, 121 and 32 cases of laryngotracheitis, bronchiolitis, pneumonia and CNS complications were investigated. At least one virus was detected in 80%, 79.3%, 61.2% and 53.1% of cases with these syndromes, respectively. Fig.  4 represents the number and proportion (%) of patients infected with respiratory viruses in the groups with different clinical diagnosis. Among patients with laryngotracheitis, RVs were the most frequently identified viruses (21.2%), followed by RSV (20%) and BoV (20%). Among patients with bronchiolitis, RSV, RV and BoV were the most common pathogens -37.9%, 15.7% and 14.1%, respectively. RSV was the most commonly identified virus in patients with pneumonia (23.1%) followed by influenza A(H1N1)pdm09 (9.9%) and RV (8.3%). In total, influenza viruses were responsible for 19% of pneumonia cases. Most (34.4%) neurological complications were associated with influenza infections.

DISCUSSION
Respiratory tract infections are a leading cause of morbidity and mortality in children <5 years around the world. This study aimed to describe the circulation of 12 respiratory viruses among infants and young children in Bulgaria during the 2017/2018 and 2018/2019 seasons. The involvement of these pathogens in the development of some serious diseases of the respiratory tract and CNS was also analysed. The high level (69.6%) of detection of respiratory viruses found in this study is comparable to other surveys (4,5). Mixed infections were identified at a lower percentage (14.3%) compared to other studies (6,7). In a systematic review, Goka et al. reported incidence of mixed viral infections ranging from 5% to 62% (8). A high percentage of mixed infections is likely to be observed in settings with high population density and a large number of children attending childcare facilities. Some authors suggest that there is a link between co-infection and disease severity, especially co-infections involving RSV (9,10,11). The relationship between clinical severity and infection status with single vs. multiple respiratory pathogens remains inconclusive. Influenza epidemics are characterised by variations in the types/subtypes of influenza viruses involved, their virulence and clinical manifestation.
In Bulgaria, during the 2017/2018 winter season, B/Yamagata was the predominantly circulating influenza virus, while during the next 2018/2019 season A(H1N1) pdm09 was prevalent. Similar predominance of B/Yamagata and A(H1N1)pdm09 viruses was observed in most European countries (12). RSV is the most important pathogen of ARI among infants and young children and the major causative agent of bronchiolitis and pneumonia (13,14). In our study, RSV was the most frequently detected virus in all examined patients and a leading cause of bronchiolitis (37.9% of cases) and pneumonia (23.1% of cases). RV infections occur early and periodically in life, mainly as a common cold. However, they are also associated with severe illnesses in infants and young children (15,16). In this study, RVs were the 3rd most frequently identified viruses; they were also the most common pathogen in cases of laryngotracheitis (21.2% of cases) and one of the leading causative agents of bronchiolitis and pneumonia. According to literature data, HBoVs were found as relatively frequent respiratory pathogens in children younger than 5 years of age (17,18,19). In this study, BoVs ranked fourth in frequency among other respiratory viruses. In agreement with other studies, they were involved in co-infections at a significant rate -41.6% (17). Spread of BoVs was observed throughout the year, reaching peaks in the autumn and spring months. The proportion of AdV infections found among Bulgarian children with ARI was 6.3% which was similar to the findings of other researchers (20). AdV were characterised by a significant incidence of co-infections. HMPV is a relatively new pathogen with prevalence in the paediatric population ranging from 5-25% (21). The results of this study showed 4% frequency of HMPV infections and 6.6% incidence in cases of bronchiolitis. PIV type 1, 2, 3 and 4 show different clinical and epidemiological characteristics. PIV types 1 and 2 are leading causes of laryngotracheobronchitis (croup), while PIV-3 is frequently associated with bronchiolitis and pneumonia in infants and young children (22). PIVs were identified at low frequency -3.9% of the studied children, mainly in cases of laryngotracheitis. A limitation of this study is that human coronaviruses, which also cause ARI, were not included in the examinations. The study highlights the role of 11 respiratory viruses in the aetiology of paediatric respiratory infections. RSV followed by influenza viruses, RVs and BoVs were found to be the most common causative agents of ARI in children younger than 5 years during two consecutive seasons (2017/2018 and 2018/2019) in Bulgaria. These pathogens were the main cause of complications such as laryngotracheitis, bronchiolitis and pneumonia that require hospital treatment. Timely and accurate diagnosis of viral respiratory infections is important in order to reduce the need for unnecessary lab tests and antibiotic use and to improve infection prevention and control measures.