Study On The Epidemiology Of Children Pneumonia, Evaluation Of The Etiologic Diagnosis Method, And Exploring Multiplex PCR Method For Determining Capsular Serotypes Of Pneumococci

Acute respiratory infection (ARI) is the most common diseases and the cause of hospitalization in children. Pneumonia is the leading cause of less than 5 years children mortality in the developing country. Despite comprehensive attempts to delineate the causative pathogens among children with pneumonia, accurate determination of the pathogen of pneumonia is limited because the currently available diagnostic tests are inadequate. Nasotracheal aspiration (NTA) provides samples for testing the microbiological etiology of pneumonia in children; it is used both in routine diagnosis of children with pneumonia and as a research tool in some pediatric hospitals in China. Although NTA is a possible new method for pneumonia etiologic diagnosis, and had been used in China, its sensitivity and specificity have not been formally evaluated. The intent of this study was to evaluate the utility of NTA in the etiological diagnosis of children pneumonia. In addition, the capsular polysaccharides are essential for virulence and are the target for all current pneumococcal vaccines. Although as a golden serotyping method for the Streptococcus pneumoniae(S. pneumoniae), Quellung test is complicated and costly, thus it can't be used as screening method in the developing countries to investigate the serotypes of S. pneumoniae, which was very important for the immunization strategy. So the other objective of the study is to looking for a simple, highly efficient and cheaper method to determine the serotype of S. pneumoniae.Chapter 1 Systematic Review on the Epidemiology of Children Pneumonia in ChinaInformation on pneumonia burden and epidemiology from Chinese children are limited. The purpose of this study is to review the data on the burden of pneumonia in Chinese children from 1950 to 2007, based on the English and Chinese publications.Databases:Pubmed (Medline 1950-2007), Cochrane library, Chinese journal full-text database (CNKI,1990-2007), China Science and Technology Periodical Database (VP,1990-2007), Wanfang Data Resource System (1990-2007),3) and the related magazines were searched for published studies pre 1990 that reported the pneumococcal related disease in Chinese population. The key words'pneumonia', 'Streptococcus Pneumoniae','pneumococcus', and'Chinese'or'China'etc. were used. In total, there were 6310 publications had been searched and after title screening, full text reading, score evaluation and information abstract, finally 142 publications had been included to data analysis.In 1990s, data of the population base monitoring study on ARI from Heilongjiang, Neimenggu, Ningxia, Shandong, Beijing, Jiangsu, Yunnan, Guangdong et al. showed the pneumonia incidence of children less than 5 years old was about 0.266 episodes per children-year. The incidence decreased quickly with age, and varied in the different part of China. For example, incidence from south area was higher than that of north area, western was higher than eastern and middle China.Pneumonia is the leading death cause of<5 years children in the past decades in China. From 1991 to 2000, the national surveillance study showed the pneumonia related mortality of<5 years children was 1513 cases per 100,000 children-year in 1991, decrease to 129 cases per 100,000 children-year in 2005. The pneumonia mortality decreased quickly with age, and varied in the different part of China. For instance, penumonia mortality from western China was higher than that of eastern and middle China, while the mortality from northern China was higher than southern China, which was different from the pneumonia incidence.Case fatality rate (CFR) was an important index for the severity of diseases. The results from the publications shown, the CFR of infants'pneumonia was highest in China, which was account for 21.3%(95%CI:19.8%-22.9%). While the CFR of the children less than 5 years old decreased quickly, which was 0.6%(95%CI: 0.5%-0.7%). The CFR varied in different part of China as well. For example, it was 18.0‰(95%CI:13.7‰-22.2‰) in children from northern China, which was higher than that of children southern China 5.3%(95%CI:4.7‰-5.9‰); CFR was 11.3‰(95%CI:8.5‰-14.1‰) in children form western China, which was higher than that of middle and eastern China.In total the bacterial positive rate of the sputum, blood et al samples from children pneumonia was 31.2%(95%CI:30.7%-31.8%). Positive rate from neonatal pneumonia was the higher than that from other children. S. aureus was the common bacterial isolated from the neonatal pneumonia. While, in the older children's pneumonia, S. pneumoniae and H. influenzae were the common pathogens. The infection spectrum varied in different times and areas of China.In a word, pneumonia was still one of the greatest burdens in China. More efforts are needed to improve the prevention and treatment of the children pneumonia.Chapter 2 Evaluation of the Nasotracheal Aspiration Method for the Pathogen Detection of Pneumonia in ChildrenAcute lower respiratory infection (ALRI) is a leading cause of children mortality and a major cause of hospitalization for children in China. However, the diagnosis of pathogen in ALRI is problematic. The purpose of this study was to evaluate the utility of nasotracheal aspiration (NTA) in the diagnosis of pneumococcal ALRI.A hospital-based study was carried out from March 2006 through March 2007 in Soochow University Affiliated Children's Hospital. Children≤3 years with ALRI were selected from the respiratory wards, and children receiving elective surgery and without respiratory infection symptoms were recruited as controls. With the informed consent of the children's parents, the children were enrolled. A face to face interview, chart review and abstract were carried out by the study coordinator with the structure questionnaire to collect the demographic and clinical information. Nasopharyngeal swabs (NPS) and NTA samples were obtained in the next morning after their admission. Specimen handling in the lab was blinded as to group.In total, we screened 839 hospitalized children in Children's Hospital of Soochow University for inclusion in the study; 66 who did not meet entry criteria for their group were excluded. Data from the remaining 773 children (379 with ARLI and 394 controls) were entered into the analysis. The mean age and sex proportion was different in the two groups. Antibiotics had been administered to 86%of ALRI children within one week before admission; 80% had received one or moreβ-lactams, and 22% had received macrolides. The rate of antibiotic use in the control children was<1%.Contrary to the very low numbers expected, S. pneumoniae was isolated from 8.4%(95%CI:5.6%-11.1%) of NTA samples from control children, H. influenzae from 27.2%(95%CI:22.8%-31.5%), and M. catarrhalis from 22.1%(95%CI: 18.0%-26.2%). Possible suppress by the abuse antibiotics, the isolation ratio from the ALRI children was lower than control children. For example, S. pneumoniae was isolated from 10.3%(95%CI:7.2%-13.3%) of NTA samples from the ALRI children, H. influenzae from 15.3%(95%CI:11.7%-18.9%), and M. catarrhalis from 4.7% (95%CI:2.6%-6.9%). The difference of isolation rate was significant in the two children groups. Forty-four children with ALRI (11.6%) had NPS samples positive for S. pneumoniae, of whom 29 (65.9%) also had positive NTA samples (Pearsonχ2, P<0.001). There were 59 (15.0%) control children who had NPS samples positive for S. pneumoniae; of these,20 (33.9%; 95% CI:20.7%-47.0%) also had positive NTA samples (Pearsonχ2, P<0.001), which was called as false positive rate in the study. In addition, the agreement analysis was carried out, and the kappa value between NPS and NTA varied from 0.37 to 0.64. So, we can conclude that the specificity of the NTA test is poor. Its results are highly correlated with those of NPS, and thus in most cases it measures only carriage.However, although was suppress by the antibiotics, the bacterial positive rate from the NTA sample was higher than that of NPS samples. According to the site of sampling and the results from the NTA and NPS, NTA could indicate the trend of bacterial invading down. Analysis of NTA samples'cytology, including the presence of polymorphonuclear cells, and others clinical test, including C reaction Protein, and White Blood Cells in the blood, improved the specificity of the test.Therefore, the specificity of the NTA test is poor. Its results are highly correlated with those of NPS, and thus NTA should not be used for patient diagnosis or for epidemiologic studies of ALRI, directly.Chapter 3 Study on the Multiplex PCR Method for Determining Serotype of Streptococcus PneumoniaeThe S. pneumoniae Capsules form a diverse group of polymers that are the most important and most recognized virulence factor of the organism. The capsular polysaccharides are essential for virulence and are the target for all current pneumococcal vaccines. To date,92 distinct capsular serotypes have been recognized, and these differ with respect to the sugars and linkages that make up the repeating units. Quellung test is the golden test for serotyping of S. pneumoniae. It is a biochemical reaction in which anticapsular antibodies bind to the capsule of S. pneumoniae, resulting in the capsule to swell and become more visible, especially under the microscope. But Quellung test is complicated and costly, and it can't be used as a common screening method in the developing country and large scale epidemiology study. The objective of this study is to establish a multiplex PCR method for determining the serotype of S. pneumoniae, which is suitable to be used in the developing countries and epidemiological survey. Basing on the type specific cps gene in the S. pneumoniae chromosome, and the previous study results from China, we designed the type-specific primers for the common serotypes of S. pneumoniae in China, such as serotype/serogroup 1,3,5,6,7F,9V,14,15A,15B/C,18C,19F,19A, 20,22F and 23 F etc. Using the multiplex PCR technique, we successfully identified serotype 19F,19A,23F,14,18C,6A/B,15A and 15B/C etc. The typing results had been confirmed by the general specific PCR and sequence analysis.Compare the results of multiplex PCR and Quellung test, fifty-two of the 63 S. pneumoniae isolates have the same serotype results by the two typing methods, which account for 82.5%(95%CI:73.2%-91.9%), the specificity was 9.2%(95%CI:79.2%-99.2%), and the positive prediction value was 82.5%(95%CI:70.7%-94.3%). For the other 11S. pneumoniae strains, there were 7 strains which untypable by Quellung test, while the multiplex PCR identified as the most common serotypes 23F,19A,18C and 6A/B. More importantly, multiplex PCR recognized a serotype 19F and serotype 23F co-colonized strains.At the same time, we compared the typing result of multiplex PCR with multi loci sequence typing (MLST), and MLST had been assumed as the golden method for molecular typing of S. pneumoniae. In total,40 of the 45 strains have the same serotypes by these two methods, which was 88.9%(95%CI,79.7%-98.1%), the specificity was 88.4%(95%CI:78.8%-98.0%), and the positive prediction value was 100%。In addition, the cost of Quellung test was much higher than multiplex PCR. As we estimated, the cost of Quellung test was about 200 yuan RMB per strain, and 500 yuan RMB per strain by MLST method, but the multiplex PCR only need 15 yuan RMB per strain. Being a molecular typing method, Multiplex PCR could even determine the serotype of the capsular negative strains. And multiplex PCR can be used directly to determine the S. pneumoniae infection and serotypes of S.pneumoniae from the clinical specimen, even not to extract the chromosome DNA. Multiplex PCR could test multi-serotype at one reaction tube by pooling the several serotype primers. In the future, we could expend the typing serotypes of S. pneumoniae, and further evaluate the multiplex PCR method in the field study.In a word, multiplex PCR is a simple, highly efficient and cost method for determining the serotypes of S. pneumoniae, which can be used in epidemiology survey in the developing countries.