RESEARCH ARTICLE
Prevalence and Correlation of Infectious Agents in Hospitalized Children with Acute Respiratory Tract Infections in Central China Jia Liu1☯, Hongwu Ai2☯, Ying Xiong1☯, Fu Li1, Zhou Wen1, Weiyong Liu1, Tongya Li1, Kai Qin1, Jianguo Wu1*, Yingle Liu1* 1 State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, China, 2 Department of Clinical Laboratory, Wuhan Children’s Hospital, Wuhan, China ☯ These authors contributed equally to this work. * [email protected] (JW); [email protected] (YL)
Abstract OPEN ACCESS Citation: Liu J, Ai H, Xiong Y, Li F, Wen Z, Liu W, et al. (2015) Prevalence and Correlation of Infectious Agents in Hospitalized Children with Acute Respiratory Tract Infections in Central China. PLoS ONE 10(3): e0119170. doi:10.1371/journal. pone.0119170 Academic Editor: Charles J. Russell, St. Jude Children's Research Hospital, UNITED STATES Received: October 11, 2014 Accepted: January 10, 2015 Published: March 9, 2015 Copyright: © 2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by research grants from the Major State Basic Research Development Program (973 Program) (2012CB518900), the National Natural Science Foundation of China (31230005 and 81171525), the National Mega Project on Major Infectious Disease Prevention (2012ZX10002006-003 and 2012ZX10004-207). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acute respiratory tract infections (ARTIs) are associated with significant morbidity and mortality worldwide, especially in children under the age of 5 years. Almost 2 million children die from ARTIs each year, and most of them are from developing countries. The prevalence and correlation of pathogens in ARTIs are poorly understood, but are critical for improving case prevention, treatment, and management. In this study, we investigated the prevalence and correlation of infectious agents in children with ARTIs. A total of 39,756 children with one or more symptoms, including fever, cough, sore throat, tonsillitis, pharyngitis, herpangina, pneumonia, and bronchiolitis, were enrolled in the study. All patients were hospitalized in Wuhan Children’s Hospital between October 1, 2010 and September 30, 2012, and were evaluated for infectious agents. Pathogens, including Mycoplasma pneumoniae, influenza A virus, influenza B virus, adenoviruses, respiratory syncytial virus, parainfluenza virus, Legionella pneumophila, Chlamydophila pneumoniae, and Coxiella burnetii, were screened simultaneously in patient blood samples using anti-pathogen IgM tests. Regression analysis was used to reveal correlations among the pathogens. Our results showed that one or more pathogens were identified in 10,206 patients, and that Mycoplasma pneumoniae, adenoviruses, and influenza B virus were the leading infectious agents. Mixed-infections of pathogens were detected in 2,391 cases, with Mycoplasma pneumoniae as the most frequent pathogen. The most common agents in the co-infections were Mycoplasma pneumoniae and influenza B virus. Regression analysis revealed a linear correlation between the proportion of mixed infections and the incidence of multi-pathogen infections. The prevalence of infectious agents in children with ARTIs was determined. Equations were established to estimate multiple infections by single-pathogen detection. This revealed a linear correlation for pathogens in children with ARTIs. This study provides useful information for improving case prevention and management.
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
1 / 14
Infectious Agents of Respiratory Tract Infections
Competing Interests: The authors have declared that no competing interests exist.
Introduction Lower respiratory tract infections (LRTI) (primarily pneumonia) are one of the leading causes of death worldwide in infants and children, especially in developing country. There are approximately almost 2 million children die from ARTIs each year [1–2]. “Typical” bacteria (e.g., Streptococcus pneumonia) as the principal agent of community-acquired pneumonia (CAP) in children have been widely investigated [3]. Recent studies showed that atypical pathogens are also important cause of LRTI resulting in mild to life threatening illness, which should be obtained more attention. The most common atypical pathogens include Mycoplasma pneumonia (M. pneumophila), Legionella pneumophila (L. pneumophila), Chlamydophila pneumoniae (C. pneumonia) and respiratory viruses [2]. These atypical pathogens were listed as common agents of CAP in American Community-acquired Pneumonia Diagnosis and treatment guidelines in Adult (2007) and Chinese Community-acquired Pneumonia Management Guidelines in Children (trial, 2007). M. pneumoniae, C. pneumoniae, and L. pneumophila cause mild, moderate or severe acute respiratory tract infections in children, responsible for 10% to 30% of CAP in children respectively [4]. M. pneumoniae is a frequent cause of hospitalization among children as young as 2 years of age and can even necessitate ventilatory assistance. L. pneumophila can occur across all age groups, but C. pneumoniae has emerged as an important cause of pneumonia in both adults and children as young as 2 years old. Coxiella burnetii (C. burnetii) is the etiologic agent of Q fever, a known pathogen that causes fever, pneumonia, and intravascular infections [5,6]. Studies of CAP have traditionally focused little on viral causes[2]. Currently, viral infections are also involved with 80% of episodes of CAP in children under 2 years old and over 40% of older children [6–11]. The existing studies have showed that respiratory syncytial virus (RSV) and influenza viruses (Flu) are important pathogens among the hospitalized and outpatient children presenting with ARTI [5, 6].Adenovirus virus (ADV) and parainfluenza viruses (PIV) are also associated with a substantial proportion of ALRI in infants and young children [7–9]. The etiology of LRTI can be established in only 30–50% of cases using conventional methods and unidentified etiology causes inappropriate antibiotic usage, antibiotic resistance, unintended adverse reactions and increased cost. Therefore, rapid, sensitive diagnostic methods and pathogen-directed therapy are important. However, Regarding atypical pathogens are difficult to isolate, conventional culture methods require longer test times and a facility able to perform these tests. In addition, the appropriate sample is critical for the aetiologic diagnosis. Sputum, representing lower-airway secretions, can rarely be obtained from children [2,7]. New rapid detection of multiple viruses and bacteria has been developed lately [8,9]. IgM antibody first appeared in the process of infection, but the last time not long. So it was regarded as diagnostic criteria of early infection. Specific IgM appeared in one week, peak within three weeks after infections, thus more valuable for early diagnosis in children based on Chinese Expert Consensus of Diagnosis and Treatment of Mycoplasma Pneumoniae in Children. With advances in methods aimed to detect pathogens, some pediatric patients with ALRT infections are infected simultaneously by multiple pathogens [10,11]. And it is suggested that co-infections are medically relevant, and effective treatment for severe respiratory tract infections ultimately requires diagnosis of all involved pathogens [12]. Interestingly, recent studies have provided statistical evidence that co-infection is not random, and that co-infection occurs more frequently with certain pathogens than with others. However, Studies designed to identify multiple pathogens simultaneously are limited, and information regarding mixed infections is lacking in China. And preferential interactions among specific pathogens remain uncertain [13].
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
2 / 14
Infectious Agents of Respiratory Tract Infections
In this study, we analyzed the data collected a total of 39,756 hospitalized children with ARTIs in Wuhan Children’s Hospital, the largest children’s hospital in Central China. To better understand the epidemiological and etiological characteristics of the infections, all blood specimens were tested simultaneously by immunofluorescence assay for IgMs of nine pathogens. In addition, co-infection with specific pathogens and multiple infections among pathogens were investigated. The prevalence and correlation of pathogens in children with ARTIs were identified and may be useful for the prevention and treatment of ARTIs.
Materials and Methods Study Patients Written informed consent was obtained from the guardians of the children. The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the College of Life Sciences, Wuhan University, in accordance with its guidelines for the protection of human subjects. Between October 1, 2010 and September 30, 2012, a total of 39,756 hospitalized children aged 0–15 years with respiratory tract infections at Wuhan Children’s Hospital, the largest children’s hospital in Central China, were enrolled in this study prospectively. The respiratory tract infections were divided into upper and lower respiratory tract infections. The symptoms of upper respiratory tract infections include fever, cough, sore throat, tonsillitis, pharyngitis, and herpangina. Pneumonia and bronchiolitis were considered lower respiratory tract infections. Infected patients with one or more of the symptoms were included in the study. Detailed demographic information on age and gender was documented, and laboratory data were collected from the patients’ medical files.
Specimens Blood samples were collected from each child who was given a case definition. Specimens were obtained from children before clinical treatment (within 24 h after hospital admission to avoid inclusion of hospital-acquired infections and antibiotic interference). Blood samples were collected in vacuum blood tubes without the addition of anticoagulants and were further clarified after clotting. Serum was stored at −20°C on ice until analysis. Specimens were stored at −70°C if analysis was not possible within 24 h after collection.
Pathogen Detection Specimens were tested simultaneously for M. pneumoniae, influenza A virus (IAV), influenza B virus (IBV), AdV, RSV, PIV, L. pneumophila, C. pneumonia and C. burnetii using indirect immunofluorescent assay through PNEUMOSLIDE IgM (VIRCELL, Spain): Each slide has 10 wells, each containing one of the above agent antigens and cell control. Serum samples were diluted 1:1 with Phosphate Buffered Saline (PBS) then treated with anti-human IgG sorbent. The sorbent treated diluted serum was incubated 90 min at 37°C with the 10 slide wells. The slide washed twice with PBS. A fluorescent secondary IgM antibody (Anti-human IgM/FITC) was added to the wells and incubated at 37°C for 30 min, then washed twice with PBS. If positive an IgM response (greenish yellow fluorescence) is obtained [9]. And patients in whom any one of the targeted pathogens was detected using the above methods were regarded as positive. Cases in which a single pathogen was detected are referred to as mono-infections; cases of two or more pathogens are referred to as co-infections or multiple infections, respectively.
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
3 / 14
Infectious Agents of Respiratory Tract Infections
Statistical Analysis General data are presented as a percentage (P), logarithm of the percentage [LN(P)], or mean ± SD. Statistical analyses was performed using Statistics Analysis System (SAS) version 9.0, Statistical Product and Service Solutions (SPSS) version 13.0, and Microsoft Excel 2007. Differences in categorical variables between groups were compared by the χ2 test. A single-tailed Pvalue of
Prevalence and Correlation of Infectious Agents in Hospitalized Children with Acute Respiratory Tract Infections in Central China Jia Liu1☯, Hongwu Ai2☯, Ying Xiong1☯, Fu Li1, Zhou Wen1, Weiyong Liu1, Tongya Li1, Kai Qin1, Jianguo Wu1*, Yingle Liu1* 1 State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, China, 2 Department of Clinical Laboratory, Wuhan Children’s Hospital, Wuhan, China ☯ These authors contributed equally to this work. * [email protected] (JW); [email protected] (YL)
Abstract OPEN ACCESS Citation: Liu J, Ai H, Xiong Y, Li F, Wen Z, Liu W, et al. (2015) Prevalence and Correlation of Infectious Agents in Hospitalized Children with Acute Respiratory Tract Infections in Central China. PLoS ONE 10(3): e0119170. doi:10.1371/journal. pone.0119170 Academic Editor: Charles J. Russell, St. Jude Children's Research Hospital, UNITED STATES Received: October 11, 2014 Accepted: January 10, 2015 Published: March 9, 2015 Copyright: © 2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by research grants from the Major State Basic Research Development Program (973 Program) (2012CB518900), the National Natural Science Foundation of China (31230005 and 81171525), the National Mega Project on Major Infectious Disease Prevention (2012ZX10002006-003 and 2012ZX10004-207). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acute respiratory tract infections (ARTIs) are associated with significant morbidity and mortality worldwide, especially in children under the age of 5 years. Almost 2 million children die from ARTIs each year, and most of them are from developing countries. The prevalence and correlation of pathogens in ARTIs are poorly understood, but are critical for improving case prevention, treatment, and management. In this study, we investigated the prevalence and correlation of infectious agents in children with ARTIs. A total of 39,756 children with one or more symptoms, including fever, cough, sore throat, tonsillitis, pharyngitis, herpangina, pneumonia, and bronchiolitis, were enrolled in the study. All patients were hospitalized in Wuhan Children’s Hospital between October 1, 2010 and September 30, 2012, and were evaluated for infectious agents. Pathogens, including Mycoplasma pneumoniae, influenza A virus, influenza B virus, adenoviruses, respiratory syncytial virus, parainfluenza virus, Legionella pneumophila, Chlamydophila pneumoniae, and Coxiella burnetii, were screened simultaneously in patient blood samples using anti-pathogen IgM tests. Regression analysis was used to reveal correlations among the pathogens. Our results showed that one or more pathogens were identified in 10,206 patients, and that Mycoplasma pneumoniae, adenoviruses, and influenza B virus were the leading infectious agents. Mixed-infections of pathogens were detected in 2,391 cases, with Mycoplasma pneumoniae as the most frequent pathogen. The most common agents in the co-infections were Mycoplasma pneumoniae and influenza B virus. Regression analysis revealed a linear correlation between the proportion of mixed infections and the incidence of multi-pathogen infections. The prevalence of infectious agents in children with ARTIs was determined. Equations were established to estimate multiple infections by single-pathogen detection. This revealed a linear correlation for pathogens in children with ARTIs. This study provides useful information for improving case prevention and management.
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
1 / 14
Infectious Agents of Respiratory Tract Infections
Competing Interests: The authors have declared that no competing interests exist.
Introduction Lower respiratory tract infections (LRTI) (primarily pneumonia) are one of the leading causes of death worldwide in infants and children, especially in developing country. There are approximately almost 2 million children die from ARTIs each year [1–2]. “Typical” bacteria (e.g., Streptococcus pneumonia) as the principal agent of community-acquired pneumonia (CAP) in children have been widely investigated [3]. Recent studies showed that atypical pathogens are also important cause of LRTI resulting in mild to life threatening illness, which should be obtained more attention. The most common atypical pathogens include Mycoplasma pneumonia (M. pneumophila), Legionella pneumophila (L. pneumophila), Chlamydophila pneumoniae (C. pneumonia) and respiratory viruses [2]. These atypical pathogens were listed as common agents of CAP in American Community-acquired Pneumonia Diagnosis and treatment guidelines in Adult (2007) and Chinese Community-acquired Pneumonia Management Guidelines in Children (trial, 2007). M. pneumoniae, C. pneumoniae, and L. pneumophila cause mild, moderate or severe acute respiratory tract infections in children, responsible for 10% to 30% of CAP in children respectively [4]. M. pneumoniae is a frequent cause of hospitalization among children as young as 2 years of age and can even necessitate ventilatory assistance. L. pneumophila can occur across all age groups, but C. pneumoniae has emerged as an important cause of pneumonia in both adults and children as young as 2 years old. Coxiella burnetii (C. burnetii) is the etiologic agent of Q fever, a known pathogen that causes fever, pneumonia, and intravascular infections [5,6]. Studies of CAP have traditionally focused little on viral causes[2]. Currently, viral infections are also involved with 80% of episodes of CAP in children under 2 years old and over 40% of older children [6–11]. The existing studies have showed that respiratory syncytial virus (RSV) and influenza viruses (Flu) are important pathogens among the hospitalized and outpatient children presenting with ARTI [5, 6].Adenovirus virus (ADV) and parainfluenza viruses (PIV) are also associated with a substantial proportion of ALRI in infants and young children [7–9]. The etiology of LRTI can be established in only 30–50% of cases using conventional methods and unidentified etiology causes inappropriate antibiotic usage, antibiotic resistance, unintended adverse reactions and increased cost. Therefore, rapid, sensitive diagnostic methods and pathogen-directed therapy are important. However, Regarding atypical pathogens are difficult to isolate, conventional culture methods require longer test times and a facility able to perform these tests. In addition, the appropriate sample is critical for the aetiologic diagnosis. Sputum, representing lower-airway secretions, can rarely be obtained from children [2,7]. New rapid detection of multiple viruses and bacteria has been developed lately [8,9]. IgM antibody first appeared in the process of infection, but the last time not long. So it was regarded as diagnostic criteria of early infection. Specific IgM appeared in one week, peak within three weeks after infections, thus more valuable for early diagnosis in children based on Chinese Expert Consensus of Diagnosis and Treatment of Mycoplasma Pneumoniae in Children. With advances in methods aimed to detect pathogens, some pediatric patients with ALRT infections are infected simultaneously by multiple pathogens [10,11]. And it is suggested that co-infections are medically relevant, and effective treatment for severe respiratory tract infections ultimately requires diagnosis of all involved pathogens [12]. Interestingly, recent studies have provided statistical evidence that co-infection is not random, and that co-infection occurs more frequently with certain pathogens than with others. However, Studies designed to identify multiple pathogens simultaneously are limited, and information regarding mixed infections is lacking in China. And preferential interactions among specific pathogens remain uncertain [13].
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
2 / 14
Infectious Agents of Respiratory Tract Infections
In this study, we analyzed the data collected a total of 39,756 hospitalized children with ARTIs in Wuhan Children’s Hospital, the largest children’s hospital in Central China. To better understand the epidemiological and etiological characteristics of the infections, all blood specimens were tested simultaneously by immunofluorescence assay for IgMs of nine pathogens. In addition, co-infection with specific pathogens and multiple infections among pathogens were investigated. The prevalence and correlation of pathogens in children with ARTIs were identified and may be useful for the prevention and treatment of ARTIs.
Materials and Methods Study Patients Written informed consent was obtained from the guardians of the children. The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the College of Life Sciences, Wuhan University, in accordance with its guidelines for the protection of human subjects. Between October 1, 2010 and September 30, 2012, a total of 39,756 hospitalized children aged 0–15 years with respiratory tract infections at Wuhan Children’s Hospital, the largest children’s hospital in Central China, were enrolled in this study prospectively. The respiratory tract infections were divided into upper and lower respiratory tract infections. The symptoms of upper respiratory tract infections include fever, cough, sore throat, tonsillitis, pharyngitis, and herpangina. Pneumonia and bronchiolitis were considered lower respiratory tract infections. Infected patients with one or more of the symptoms were included in the study. Detailed demographic information on age and gender was documented, and laboratory data were collected from the patients’ medical files.
Specimens Blood samples were collected from each child who was given a case definition. Specimens were obtained from children before clinical treatment (within 24 h after hospital admission to avoid inclusion of hospital-acquired infections and antibiotic interference). Blood samples were collected in vacuum blood tubes without the addition of anticoagulants and were further clarified after clotting. Serum was stored at −20°C on ice until analysis. Specimens were stored at −70°C if analysis was not possible within 24 h after collection.
Pathogen Detection Specimens were tested simultaneously for M. pneumoniae, influenza A virus (IAV), influenza B virus (IBV), AdV, RSV, PIV, L. pneumophila, C. pneumonia and C. burnetii using indirect immunofluorescent assay through PNEUMOSLIDE IgM (VIRCELL, Spain): Each slide has 10 wells, each containing one of the above agent antigens and cell control. Serum samples were diluted 1:1 with Phosphate Buffered Saline (PBS) then treated with anti-human IgG sorbent. The sorbent treated diluted serum was incubated 90 min at 37°C with the 10 slide wells. The slide washed twice with PBS. A fluorescent secondary IgM antibody (Anti-human IgM/FITC) was added to the wells and incubated at 37°C for 30 min, then washed twice with PBS. If positive an IgM response (greenish yellow fluorescence) is obtained [9]. And patients in whom any one of the targeted pathogens was detected using the above methods were regarded as positive. Cases in which a single pathogen was detected are referred to as mono-infections; cases of two or more pathogens are referred to as co-infections or multiple infections, respectively.
PLOS ONE | DOI:10.1371/journal.pone.0119170 March 9, 2015
3 / 14
Infectious Agents of Respiratory Tract Infections
Statistical Analysis General data are presented as a percentage (P), logarithm of the percentage [LN(P)], or mean ± SD. Statistical analyses was performed using Statistics Analysis System (SAS) version 9.0, Statistical Product and Service Solutions (SPSS) version 13.0, and Microsoft Excel 2007. Differences in categorical variables between groups were compared by the χ2 test. A single-tailed Pvalue of
