Investigation of occurrence patterns of respiratory syncytial virus A and B in infected-patients from Cheonan, Korea

Background Respiratory infections caused by viruses affect the lower respiratory tract; these infections are severe in patients with underlying diseases and can even lead to death. Respiratory syncytial virus (RSV), one of the causative agents of respiratory viral infections, is the most common cause of pneumonia and bronchiolitis in children and adults. Methods Respiratory specimens (nasopharyngeal aspirate, nasal swab, throat swab, etc.), which were sent to the Department of laboratory medicine from January 2012 to December 2018 for detection of respiratory viruses via real time reverse transcription PCR (Real time RT-PCR) were used in this study. RSV detected by real-time RT-PCR were analyzed on the basis of co-infection, sex and age of the patients, and year and month of sample collection. Results During the study period, we observed that the RSV detection rate was 12.8% (n = 1150/9010); the detection rate of RSV-A (7.1%) was higher than that of RSV-B (5.8%). The detection rate of RSV was the highest at 36.5% in December, and RSV-A and RSV-B were in vogue every year. Co-infection rate of RSVs was the highest in the patients over 80 years of age; RSVs showed the highest Co-infection with Rhinoviruses. Conclusions During the study period, prevalence was different among the two subtypes of RSV, and the average age of RSV-B-positive patients was higher than that of RSV-A. Co-infection rate tended to increase every year. RSVs cause mild as well as severe infections. There are reports of serious clinical progress as RSVs cause overlapping infections with other viruses and increase the risk of secondary bacterial infections. Thus, further research on RSV should be done.


Background
Respiratory infectious diseases are a primary reason behind frequent inpatient and outpatient visits of infants and young children. Respiratory viruses cause lower respiratory tract infections or contribute to acute exacerbation of asthma [1]; the viruses can also cause severe respiratory infections in children with bronchial pulmonary dysplasia, immunodeficiency, or congenital heart disease. Major viruses responsible for respiratory infections are adenovirus, coronavirus, human rhinovirus, influenza virus, human metapneumovirus, parainfluenza virus, and respiratory syncytial virus (RSV) [2].
RSV is an RNA virus, which belongs to the genus Orthopneumovirus, and it is the most common cause of bronchiolitis and pneumonia in children under 1 year of age [3,4]. Nearly all children are infected with RSV at least once before the age of two [5], and RSV bronchiolitis in infants is a major reason for their hospitalization [6]. Most people infected with RSV recover within one to 2 weeks, but it causes pneumonia in in older or immunocompromised individuals, which requires hospitalization. Individuals with congenital heart defects, premature infants, children immediately after open heart surgery, and immunocompromised patients (such as bone marrow transplant or organ transplant patients), also previously healthy children are severely affected by RSV infections [7,8].
To date, there are no RSV vaccines or specific antiviral agents, and symptomatic treatment is under way. The most important treatment for acute bronchiolitis, mainly caused by the respiratory syncytial virus (RSV), is oxygen and hydration. Drug therapy is not recommended, and it improves clinical symptoms in the short term, but it does not affect cure [9]. Most adults have neutralizing antibodies for RSV, but reinfection is also common [10]. The epidemiological and clinical characteristics of RSV-A and B infections differ [11].
The clinical symptoms of most of respiratory viral infections are similar so we cannot easily distinct; thus, for early diagnosis of RSV infection it is important to identify the causative virus. Thus, a real-time PCR based method, which is faster than the conventional PCR method, is required for quick diagnosis and rapid treatment of RSV infections, which can also decrease antibiotic resistance.
In Korea, sample monitoring of patients with respiratory diseases has been conducted since 2011, and RSV infections have been reported to increase gradually [12]. In this study, RSV infections were diagnosed with a single-step real-time reverse transcription (RT)-PCR based method from samples monitored at a single institute between January 2012 and December 2018; these samples were analyzed according to the year and month of sample collection, and sex and age of the patients. The epidemic and co-infection patterns and the differences according to RSV subtypes were also analyzed. In addition, the relationship of RSV infections with temperature was analyzed using the Meteorological Agency's climate data [13].

Respiratory specimens
We analyzed a total of 9010 respiratory specimens from all inpatients with respiratory symptoms (nasopharyngeal aspirate, nasal swab, throat swab), which were sent to the Department of laboratory medicine, Dankook university from January 2012 to December 2018 for identification of respiratory viruses via real-time RT-PCR. The samples were either immediately tested or refrigerated at 4°C and tested within 24 h.

Extraction of RNA
The collected specimens were treated with the QIAamp® MinElute® Virus Spin Kit (Qiagen, Hilden, Germany) for RNA extraction.

Real-time RT-PCR
The synthesized cDNA was then amplified and probed for RVs with the AdvanSure™ RV real-time RT-PCR (LG Life Science, Seoul, Korea) according to the manufacturer's instructions. Briefly, 5 μl of the cDNA was added to a tube containing 5 μl of primer probe mix and 10 μl of one-step RT-PCR premix. For the reverse transcription step, this mixture was incubated at 50°C for 10 min. Denaturation was done at 95°C for 30 s, followed by 10 cycles of PCR (15 s at 95°C, 30 s at 53°C, and 30 s at 60°C). Subsequently, 30 additional cycles of PCR were completed for the detection of fluorescence signals (15 s at 95°C, 30 s at 53°C, 30 s at 60°C). During the study, the same kits were used in the experiment and there was no change.

Statistical analysis
The data used in this study are laboratory data of Cheonan Hospital of Dankook University. The hospital has an average of 900,000 inpatients and outpatients per year, and samples of suspected respiratory infections from January 1, 2012 to December 31, 2018. The results obtained by collecting and performing RT-PCR tests were used. All statistical analyses were performed using SPSS 26 program for windows. The characteristics of this study were frequency analysis and cross-analysis was used for positive patients and climatic factors were compared using the student t test.
RSV detected by real-time RT-PCR was analyzed based on co-infection, sex and age of the patients, and year and month of sample collection. A p-value of less than 0.05 was considered significant.

Climate data
Climate data for the Cheonan region used in this study was provided by the weather data opening portal of the Korea Meteorological Administration.
We observed that the detection rate of RSV-A and RSV-B together was the highest in December at 36.5% (n = 386/1058) and the lowest in June at 0.2% (n = 1/ 636). The detection rate of RSV-A was the highest in November at 23.2% (n = 192/829) and the lowest in June at 0% (n = 0/636). The detection rate of RSV-B was the highest in December at 15.8% (n = 167/1058), the lowest in June at 0.2% (n = 1/636) and July at 0.2% (n = 1/590) (Fig. 4). In addition, as a result of analyzing RSV-A, B and analysis of climatic factors, it is observed that the detection rate is high when the average temperature is low ( Table 2).
The mean co-infection rate during the study was 27.7% (n = 319/1150). The co-infection rate of RSV-B was higher than that of RSV-A. Both RSV-A and RSV-B showed the most co-infection with Rhinovirus (Table 3)       were 7 co-infections of RSV-A and RSV-B. Of the 43 triple infections, 22 were due to RSV-A and 24 were due to RSV-B. Among the triple infections of RSV-A and RSV-B, 10 and 11 patients, respectively, were coinfected with Adenovirus and Rhinovirus (Table 4).

Discussion
During the study, the total RSV detection rate was 12.8% (n = 1150/9010) and the average age of patients was 4.1 years (median age: 0.4 years, range year). The average age of RSV-A infected patients was 2.9 years and the average age of RSV-B infected patients was 5.5 years.
We also observed an alternating pattern between RSV-A and RSV-B infections during the study period. In a study in Busan, Korea, RSV-A and B types showed an alternating trend similar to our results. Although hostimmunity against RSV-B is active longer than that of RSV-A, RSV-A is considered to be more dominant [14], but more detailed studies, such as the study related to immunity in the population are needed.
In this study, we observed that the detection rate of RSV-B in January, March, April, May, June, and September was higher than that of RSV-A. Similar studies in Korea have reported that RSV-A prevailed between November and December, with RSV-B being dominant in January and February [13]. Studies conducted in temperate climates and RSV, such as in Korea, have reported to be prevalent between October and April [15][16][17]. Compared with similar studies in China over a similar period, the results do not show as clear alternating pattern [18].
The detection rate in the group of under 1-yearold patients was the highest at 24.6% (n = 740/3005). The average age of RSV-B infected patients was 5.5 years (median age: 0.6 years, range year), which was higher than the average age of RSV-A infected patients, 4.1 years (median age: 0.4 years, range year). Some studies have reported that RSV-B infections occur in patients older than the patients infected by RSV-A [19].
The co-infection rate of RSV-B was higher than that of RSV-A. We observed that except for the quadruple infections detected during the study period, the average age of the patients increased as the number of pathogens increased in co-infections. The highest rate of double infections was observed in the 80-year-old patients (80%, n = 8/10), followed by (50%, n = 1/2) the 20-29-year-old patients, and the (40%, n = 4/10) 10-19-year-old patients. The results are similar to other studies that coinfection is most common in RSV, rhinovirus and bacterial pathogens [20].
Co-infection rate tended to increase gradually. The coinfection rate in 2017 was the highest at 35.5% (n = 55/ 155) and the lowest in 2013 was 17.6% (n = 23/131). Follow-up studies of duplicate infections with respiratory bacteria as well as duplicate infections between respiratory viruses are also needed.
Previous studies have reported respiratory infections in up to 11.5-45.4% of the children, with the most common causal organisms being RSV and rhinoviruses [21,22]. This differs depending on several factors, such as age, detection time, detection method, and detection area. Coinfection with RSV and Metapneumovirus has been reported to increase the relative risk of pediatric intensive care unit (ICU) admission of patients by 10-fold [23]. It has been reported that co-infection with RSV and Rhinovirus shows a five-fold increase in the severity of RSV infections [24].
The prevalence of RSV is generally reported to be high in places with low mean temperature and high relative humidity. Some subtypes of RSV are prevalent at equal rates, but one subtype prevailed [13], and some studies reported that RSV-B infections occurred in patients older than the patients that were infected with RSV-A [19]; however, RSV-A has been reported to have higher severity than RSV-B infections [25,26].
In addition to being recognized as a primary cause of mild as well as severe respiratory infections, reports show serious clinical implications of these infections, as RSVs cause overlapping infections with other viruses and increase the risk of secondary bacterial infections in patients with low immunity. Thus, research on RSVs should be continued by broadening the parameters under consideration. This study is a retrospective study that has not been conducted according to the clinical symptoms of the test subjects. We could not analyze the genotypes of the viruses and their distribution. Analyzing the prevalent RSV genotypes in each community is likely to improve the ecological understanding of RSV infections. Further research is required to address these issues.