Analysis of National Surveillance of Respiratory Pathogen for Children and Adolescents’ Community Acquired Pneumonia

Background: Respiratory infection in particular community acquired pneumonia (CAP) in children is a major disease that ranks high in outpatient and inpatient cases. The causes of CAP vary depending on the individual susceptibility, epidemiological characteristics of the community, and season. We performed this study to establish nationwide surveillance network system and identify the causative agents for CAP and antibiotics resistance in Korean children with CAP.. Methods: The monitoring network was composed of the 28 secondary and tertiary medical institutions. Upper and lower respiratory samples were assayed using culture or Polymerase chaini reaction (PCR) from Aug 2018 to May 2020. Results: A total of 1023 cases were registered in patients with CAP, and 264 cases (25.8%) were isolated by culture, S. aureus 131 cases (12.8%), S. pneumoniae 92 cases (9%), H. inuenzae 20 cases (2%). PCR of atypical pneumonia pathogen revealed 422 cases of M. pneumoniae (41.3%). Respiratory virus showed positive rates in 65.7% by multiplex PCR test and of them, human rhinovirus was most highest with 312 cases (30.5%). The proportion of mixed infection was 49.2%. The rate of antibiotics resistance showed similar results as previous reports. Conclusions: It will identify the pathogens that cause respiratory infections, and analyze the current status of antibiotic resistance to provide scientic evidence for management policies of domestic respiratory infection. Also, in preparation for the new epidemic, including COVID19, monitoring of respiratory infections in children and adolescents, has become more important, and research should be continuously conducted in the future. test culture. Virus (Respiratory syncytial virus (RSV) A, B, Inuenza virus (IFV) A, B, Parainuenza virus (PIV) 1, 2, 3, 4, Adenovirus (ADV), Human Rhinovirus (HRV), Human metapneumovirus (HMPV), Coronovirus (CoV) 229E, NL63, OC43, Bocavirus (BoV), Human Enterovirus (HEV)) were examined using multiplex PCR of antibiotics resistance in this study showed similar results as previous reports. Penicillin sensitivity of pneumococcus in the study of adult pneumonia was 42.9 % in 2001, but 100 percent in the 2010. 19,20 According to treatment guideline of CAP, S. pneumoniae has a low resistance rate for the penicillin and quinolone and relatively high resistance rate for some cephalosporin and macrolide. 34 Our results also showed high resistance rates for second-generation cephalosporin and macrolide, while low resistance rates for the third-generation cephalosporin, penicillin, and quinolone, which is similar to what was previously reported. In addition, the results of the antibiotic resistance of H. inuenzae, P. aeruginosa, K. Pneumoniae, S. aureus were also conrmed to be similar to the 2017 National Antibacterial Resistance Survey Report. 35 These data can be helpful in forming the basis of empiric antibiotics therapy in child with CAP.


Inclusion criteria
Among patients who have had a cough or a severe fever as a major symptom, patients diagnosed with pneumonia in the chest X-ray were included during the study period. However, it was de ned as a child under 18 years of age who had no history of using antibiotics within ve days of the visit period.

Exclusion criteria
The upper respiratory tract infected patients, which is accompanied by rhinitis symptoms such as runny nose and stuffy nose, patients who had chronic underlying diseases or immune suppresive disease, and patients with a history of antibiotic use within ve days were excluded.

Study participants
For each patient, a questionnaire with clinical and epidemiological features were completed. The questionnaire applied includes the following information: patients information (age, birth date, gender, name of hospital, specimen type, history of hospitalization, underlying disease, status of vaccination, siblings, clinical information (fever, cough, sputum, rhinorrhea, vomiting, sore throat, etc, radiologic ndings, vital signs, breathing sound, O 2 saturation), treatment (prescribed antibiotics, hospitalization period, sequelae) (Additional le 2).

Collection of samples
Samples were obtained from sputum, bronchoalveolar lavage, nasopharyngeal aspiration, or nasopharyngeal swab. Nasopharyngeal aspirates were obtained within 24 hours after enrollment. A suction catheter was used to passed through the nose into the lower part of the pharynx. A total 2 ml nasopharyngeal aspirates was obtained and sent to laboratory for analysis within 48 hours. Nasal swab was obtained by inserting a swab into both nostrils parallel to the pallate. Throat swab was obtained from the posterial pharyngeal and tonsillar areas. Sputum or bronchoalveolar lavage specimens were also collected if possible. Culture for bacterial pathogens (S. pneumoniae, H. in uenzae, S. aureus, K. pneumoniae, P. aeruginosa) and antimicrobial susceptibility test were performed. And atypical pneumonia pathogen (M. pneumoniae, C. pneumoniae, B. pertussis) were identi ed using polemerase chain reaction (PCR) test and culture. Virus The detection of pathogen A bacterial pathogen A bacterial pathogens were detected by culture, and cultured pathogens were perfomed antimicrobial susceptibility test. If the nal identi cation of the bacteria is ambiguous or requires accurate identi cation, we con rmed the results by using VitekII (Biomerieux, Hazelwood, USA) to verify the infection and 16S rRNA test was performed.

Atypical pneumonia pathogen
Atypical pneumonia pathogen was detected by PCR method. Nucleic acid was extracted from a total volume of 1ml of sample and puri ed. The cyclic Temperature settings were 94 ℃ 20sec, 58 ℃ 20 sec, 72 ℃ 20 sec ampli ed by 35 cycles with the last 72 ℃ 7min. And M. pneumoniae, C. pneumoniae and B. pertussis were con rmed by using Allplex TM PneumoBacter Assay (Seegene, Seoul, South Korea).

A viral pathogen
For the multiplex RT-PCR, viral genomic RNA and DNA were extracted from a total volume of 1ul of sample by the guanidinium thiocyanate extraction method. The lysis buffer included 500 molecules of the cloned ampli ed product used as internal control in each reaction tube and then excluded false negative results. Three independent multiplex reverse transcription nested RT-PCT assays able to detect from 1 to 10 copies of viral genomes were performed. One nested RT-PCR was performed using speci c primer for PIV (1,2,3,4), ADV, HEV, HMPV and another nested RT-PCR was prepared with speci c primers for HRV (A, B, C), CoV (229E, NL63, OC43), BoV (1,2,3,4) and third nested RT-PCR was using speci c primer for RSV (A, B), IFV (A, B, subtype H1, H3, H1pdm09) by using Allplex TM Respiratory Panel (Seegene, Seoul, South Korea).

The susceptibility test of antibiotics
In this study, the antibiotic susceptibility test of isolated bacteria measures minimum inhibitory concentration (MIC) using MicroScanR Microbiology System (Dade Behring, Tokyo, Japan) The criteria for judging antibioitics resistance follow the criteria of the Clinical Laboratory Standard Institute (CLSI).

Results of respiratory pathogens according to age
Respiratory viruses were the most common pathogen of CAP under the age of 3 years. Rate of viruses and M. pneumoniae was similar at the age of 4-6 years. From 7 years of age, bacteria was predominant for CAP; M. pneumoniae was the most common bacterial pathogen, followed by S. aureus. Interestingly, incidence of S. pneumoniae was more common than S. aureus only at the age of 1-3 years.
According to the positive rate of M. pneumoniae by age, the positive rate was highest at at age 7 to 11 (179 cases), followed at age 4 to 6 (121 cases), at age 1 to 3 (56 cases), at age 12 years of age and older (48 cases) and at less than 12 months (14 cases). Five cases of C. pneumoniae were detected at 7-11 years of age. Five cases of B. pertussis were detected at the age of 1-3 in three cases and 7-11 in two cases. When comparing the virus detection rate by age, the positive rate was the highest among those under 12 months, followed by those aged 1-3 and 4-6 and 7-11 and 12 or older. (Table 2)  (Figure 4).

Discussion
Most of the studies about the causative agents of CAP in children are limited by the di culty of obtaining adequate specimens. This study is meaningful in that it was the rst prospective extensive study investigating respiratory pathogen of children with CAP at Korea. In this study, the rate of pathogen detection in children with CAP was 70.5%. There are studies about the causative agent of CAP in many countries. In Taiwan study, at least one pathogen was identi ed in 68.3% of children with CAP, 10 in China study, causative pathogen was identi ed in 70.1% of the hospitalized children with CAP at one hospital. 11 In a US study of 3 years, causative pathogen was identi ed in 81% of patients under the age of 18 hospitalized with CAP. 2 In study of Finland, 85% was detected in hospitalized children with pneumonia. 12 The difference in detection rate in each country seems to be due to the difference in sample types and methods. The sample types collected in this study were nasopharyngeal swab/aspirate and sputum, and the causative pathogens were identi ed through PCR and culture.
To detect viruses, the same method (PCR test) is used through nasopharyngeal swab/aspirate in other countries. However, unlike this study, most tests for bacterial detection were conducted through blood and pleural uid culture, or blood PCR tests. As a result, it is thought that detection rate of the pathogen in each study is different. There are few studies about the causative agent of children with CAP in Korea. In 2009, respiratory viruses were identi ed in 49.6% of severe lower respiratory tract infection in child. 13 But unlike our study,there was no data for bacteria. Recently, there was also a study on the seasonal pattern in etiologic virus and M. pneumoniae in children hospitalized with CAP in Korea, which showed M. pneumoniae was the most common identi ed with pathogen. 14 It was noticeable that bacterial pathogens as well as viruses were also tested. In general, common bacterial pathogens of CAP in children are S. pneumoniae, H. in uenzae, S. aureus. To differentiate true pathogen from colonization in CAP, culture from blood/pleural uid sample or urinary S. pneumoniae antigens test using immunochromatography is better than nasopharyngeal sample. 1,12 In current study, we did not use this method and used bacterial culture or PCR from nasopharyngeal swab/aspirates. However, detection rate of bacteria from blood culture in CAP is very low and pleural culture is limited to only children with parapneumonic effusion. It is di cult to determine these bacterias as the causative pathogen in sample of nasopharyngeal swab/aspirate. S. pneumoniae and H. in uenzae are common ora in upper respiratory tract and up to two-thirds of children younger than 5 years are colonized with common bacterial pathogens in the upper respiratory tract. Identi cation of bacteria from the upper respiratory tract does not always mean pathogen of CAP, therefore physicians should consider clinical relevance for ascertaining bacterial etiologies of pneumonia. 15 Over the past 20 years, more than 20 cases of B. pertussis have been reported every year in Korea. During 2001-2008, an average of 11.5 cases of B.
pertussis per year, 16 and it was reported a gradual increase from 2009 to 2012. 17 In particular, 24.5% of Korean adolescents and adults with chronic cough were positive against B. pertussis in 2015. 18 Due to the lack of data on B. pertussis in pediatric age in Korea, it was important to obtain epidemiological data on B. pertussis through the results of the surveillance network study. In this study, B. pertussis was isolated from ve children, which was less than we expected. All were found between January and April and with two of them were younger than three months old, while the other three were aged one, six and nine years old. Due to the National Immunization Program by Korean government, incidence of B. pertussis is low in children, but gradual increase in adolescents and adults requires the need for booster injection. Based on this data, it will be necessary to accumulate various epidemiological data on pertussis through the construction of a continuous monitoring network in children and adolescents in Korea. The common pathogens of CAP in adults were S. pneumoniae, K. pneumoniae, and P. aeruginosa, in a prospective multicenter study in Korea, while S. pneumoniae, M. pneumoniae, K pneumoniae in other study. 19,20 Unlike adults, the most common cause of CAP in children vary according to age. Several studies have previously reported that respiratory viruses are the leading cause of CAP, which can be detected in more than 50% of the cases. 21,22 However, this results may vary by studies and age groups. Similar to our research, in Peru study of patients who were hospitalized for pneumonia under the age of 18, M. pneumoniae was more frequently detected than respiratory viruses. 23 Meanwhile in Taiwan study, S. pneumoniae was the most common pathogen, than any other pathogens, and the detection rate of pneumococcus was much higher than in our study. 10 This may attribute to relatively lower vaccination rates in Taiwan and Korean government has started pneumococcal vaccination to every infants and children as National Immunization Program since 2014, which may rapidly decrease the frequency of S.
pneumoniae pneumonia. 7 Rate of M. pneumoniae had surged from the fall of 2019, which abruptly ended after COVID19 outbreak in 2020. M. pneumoniae is a major pathogen of CAP in children and adolescents. 24 Recent study showed that mycoplasma was responsible for about 20-30 % of CAP at the age of three to four years, and during the epidemic, M. pneumoniae infected even children of 2 years old. 6,24,25 M. pneumoniae pneumonia epidemics occur every 3-5 years. 24,26,27 It was reported that epidemics of M. pneumoniae pneumonia occurred in 2007, 2011, and 2015. 28,29 According to the trend cycle, the results of this study con rmed that the mycoplasma epidemic has been re-oating in 2019. Interestingly, rate of M. pneumoniae decreased abruptly after COVID 19. This may attribute to social distancing, mask wearing, hand washing, and online schooling.
The seasonal difference in detection rates for viruses is consistent with the existing data. We found that the respiratory viruses composed of major pathogen for CAP in young children under the age of 3 years. It is known that viruses may cause pneumonia, either directly or by rendering the host more susceptible to bacterial infection. 11 In studies of CAP in childen, mixed infections were identi ed in 34-41% of child with CAP, and mixed viral-bacterial infection showed highest rate. 11,30−32 On the other hand, two US studies have reported lower rate (23-26%) of mixed infection. 2,31 In this study, the proportion of mixed infection was 49.2%, which was higher than in previous studies. The mixed infection of virus, bacteria and atypical bacterial pathogen were 3.6%. It is assumed that the difference of coinfection rate and pattern of coinfection may be related to seasonal, geographical and racial factors. And it may also vary depending on the type of sample or laboratory testing method.
Although viruses area a major cause of childhood CAP, a majority of children with pneumonia receive antibiotics. Empiric use of antibiotics remains as cornerstone of treatment in the absence of results in causative agent of pneumonia. In the U.S. guidelines, amoxillin is used in previously healthy outpatients who have been properly vaccinated if they suspect mild and moderate bacterial pneumonia. And it is recommended to use macrolides if the CAP caused by atypical pneumonia pathogen is suspicious. 33 In our study, empiric antibiotics prescription rate was high, and of them, macrolide prescription rate was the highest. The high macrolide prescription rate may be attributable to the epidemics of M. pneumoniae in 2019.
The rate of antibiotics resistance in this study showed similar results as previous reports. Penicillin sensitivity of pneumococcus in the study of adult pneumonia was 42.9 % in 2001, but 100 percent in the 2010. 19,20 According to treatment guideline of CAP, S. pneumoniae has a low resistance rate for the penicillin and quinolone and relatively high resistance rate for some cephalosporin and macrolide. 34 Our results also showed high resistance rates for secondgeneration cephalosporin and macrolide, while low resistance rates for the third-generation cephalosporin, penicillin, and quinolone, which is similar to what was previously reported. In addition, the results of the antibiotic resistance of H. in uenzae, P. aeruginosa, K. Pneumoniae, S. aureus were also con rmed to be similar to the 2017 National Antibacterial Resistance Survey Report. 35 These data can be helpful in forming the basis of empiric antibiotics therapy in child with CAP.
Study Limitations respiratory tract. Due to the di culty in collecting sputum from lower respiratory tract in young children, this may remain as limitation in children. Secondly, even though this was nationwide multi-center study, number of samples was relatively lower than expected, especially in outpatient clinic. The number of samples decreased from February 2000. This is the beginning of COVID19 epidemic, and is affected by the decrease in respiratory infections, including children and adolescents, as well as adults, and the disappearance of respiratory viruses. In particular, since February 2020, the number of pneumonia patients has decreased sharply due to the restriction of group activities such as highintensity social distance, the use of masks and hand washing. Also the inde nite postponement of opening at kindergarten and schools, and home classes has also been an important factor in the decrease in infection. Third, laboratory results such as white blood cell count or C-reactive protein were not investigated. In the future, it is required to match pathogens with clinical symptoms.