【作者】 张涛；

【导师】 赵根明；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2010， 博士

【摘要】 急性呼吸道感染是儿童最为常见的疾病。在广大发展中国家,肺炎是5岁以下儿童死亡的首要原因。自上世纪70年代以来,儿童肺炎一直也是我国5岁以下儿童的首要死亡原因。然而,迄今为止,我国5岁以下儿童的肺炎死亡率和发病率等流行病学特征仍不清楚。对儿童肺炎病原体的正确诊断是预防和控制儿童呼吸道感染的关键所在。然而,由于难以获取高质量的下呼吸道标本,肺炎病原体的诊断一直是医学界的难题。深部吸痰法是近年发展起来的损伤性小的下呼吸道标本采集方法。为了正确地使用和推广该方法,本研究结合鼻咽拭子的检测结果,对深部吸痰法的有效性和特异性等进行综合性的评价。此外,为了了解我国肺炎链球菌菌株的血清分布和监测其血清型的变更等,迫切需要寻找一种可替代荚膜肿胀试验,且适用于发展中国家流行病学研究的肺炎链球菌分型方法,以控制肺炎链球菌感染,并为制定肺炎链球菌疫苗免疫策略提供依据。第一部分系统性回顾中国儿童肺炎流行病学特征系统的检索了1950-2007年间公开发表的关于儿童肺炎发病、死亡和病原体等相关的文献。通过对NCBI, Cochrane, CNKI, VP,万方等主要数据库的检索和手工检索,共获得6310篇文献。经过筛选、全文阅读、质量评价和文献信息提取等,最终采纳并进入分析的文献共142篇。20世纪90年代,来自黑龙江、内蒙古、宁夏、山东、北京、江苏、云南、广东等地以人群为基础的儿童呼吸道感染监测结果显示,中国5岁以下儿童肺炎发病率为26.6%。儿童年龄越小,发病率越高。随着年龄的增长,肺炎的发病率迅速下降。不同地域儿童肺炎的发病率也有差异。总体来说,南方高于北方,尤其是西南、华南地区比华北、华东地区高。肺炎一直是我国儿童,尤其是5岁以下儿童的首位死亡原因。1991-2000年间,全国儿童死亡调查结果显示儿童总死亡率随着时间有所下降,肺炎在所有死亡原因中占首位。年龄不同,儿童肺炎死亡率也不相同。低年龄组儿童肺炎死亡率高,随着年龄的增长,肺炎死亡率迅速下降。在各地区之间,儿童肺炎死亡率差异较大,西部地区的儿童肺炎死亡率比中东部地区高,而北方地区儿童肺炎死亡率高于南方地区。病死率是反映疾病严重程度的一个重要指标。从年龄上看,新生儿的肺炎死亡率最高,达21.3%(95%CI：19.8%-22.9%)。5岁以下儿童的肺炎病死率明显下降,仅为0.6%(95%CI：0.5%-0.7%)。北方地区儿童肺炎病死率为18.0%o(95%CI：13.7%o-22.2%o),高于南方地区的5.3%(95%CI：4.7%o-5.9%o)。西部地区儿童肺炎病死率高达113%o(95%CI：8.5‰-14.1‰),显著高于中东部地区。儿童肺炎的细菌检出率为31.2%(95%CI：30.7%-31.8%),其中以新生儿肺炎的细菌检出率较高。在阳性分离细菌中,新生儿以金黄色葡萄球菌为较为常见。较大年龄儿童中,肺炎链球菌,流感嗜血杆菌的阳性率也较高。不同年代、不同地区儿童肺炎的细菌学感染谱有所不同。总而言之,我国属于儿童肺炎高负担国家,尤其是经济相对落后地区5岁以下儿童的发病率和死亡率居高不下。第二部分对深部吸痰法诊断儿童肺炎病原体的评价本研究旨在对深部吸痰法诊断下呼吸道感染儿童病原体的可行性、有效性和特异性等进行综合评价。研究以2006年3月-2007年3月期间于苏州儿童医院住院治疗的下呼吸道感染住院儿童(病例组)和无呼吸道感染的外科患儿(对照组)为研究对象,在征得其父母或监护人的知情同意之后,对研究对象父母或监护人进行面对面的问卷调查、病史摘录及病案回顾等以收集相关的信息,同时采集研究对象的鼻咽拭子和深部吸痰标本,送实验室进行细菌学检测,在标本的采集和实验室操作过程中分别采取了严格质量控制和盲法。研究共入选了839名儿童,其中66人因标本采集不合格等原因而被剔除,最终773人(病例组379人,对照组394人)被纳入数据分析。两组研究对象在性别、年龄和入选时间等方面存在差异,并且86%的病例组儿童在入院前使用过抗生素。根据研究假设,对照组儿童的深部吸痰结果的阳性率应为零或很低,病例组儿童的深部吸痰的阳性结果可认为是下呼吸道感染的病原菌。然而,研究结果与研究假设相比有差异。即无呼吸道感染症状的对照组儿童的深部吸痰标本细菌阳性率高,如流感嗜血杆菌阳性率高达27.2%(95%CI：22.8%-31.5%),卡他莫拉菌的阳性率为22.1%(95%CI：18.0%-26.2%),肺炎链球菌的阳性率为8.4%(95%CI：5.6%-11.1%)。病例组儿童由于受到大量使用的抗生素的影响,深部吸痰标本的细菌阳性率相对较低,如流感嗜血杆菌阳性率为15.3%(95%CI：11.7%-18.9%),卡他莫拉菌的阳性率为4.7%(95%CI：2.6%-6.9%),肺炎链球菌的阳性率为10.3%(95%CI：7.2%-13.3%)。两组对象深部吸痰法的流感嗜血杆菌和卡他莫拉菌的阳性率差异有统计学意义。59名对照组儿童鼻咽部携带了肺炎链球菌,其中20名儿童的深部吸痰标本肺炎链球菌阳性,即深部吸痰法的假阳性率为33.9%(95%CI：20.7-47.0%)。鼻咽拭子和深部吸痰法检测结果一致性较高,Kappa值位于0.37至0.64之间。可见深部吸痰法的标本受到上呼吸道正常菌群的干扰,假阳性率高,特异性较差。当然,深部吸痰法也有其可取之处。虽然受到广泛应用的抗生素的影响,但与鼻咽拭子相比,深部吸痰法的细菌分离率高。根据对采样部位的分析,结合鼻咽拭子的检测结果,深部吸痰法的检测结果可以反映细菌向下侵袭的趋势,对下呼吸道感染病原体的诊断有提示作用。结合临床检验的结果,如结合革兰氏染色和瑞氏染色,局部痰液标本中找到G+球菌和脓细胞,有助于肺炎链球菌等常见呼吸道感染细菌的分离；血液中C反应蛋白水平和中性粒白细胞计数等,可以有助于下呼吸道感染病原体的诊断。综上所述,深部吸痰法检测下呼吸道感染病原菌的特异性较差,容易受到上呼吸道携带细菌的干扰,不适合直接用于儿童肺炎病原体的诊断。第三部分肺炎链球菌多重PCR分型方法的研究本研究结合我国儿童肺炎链球菌常见的血清型和基因库序列分析,设计血清型特异的PCR引物,以初步建立肺炎链球菌多重PCR分型方法。通过与传统的肺炎链球菌血清分型方法—荚膜肿胀试验和经典的分子分型方法多位点序列分型方法(MLST)进行比较,研究和评价多重PCR的分子生物学分型方法用于判别肺炎链球菌血清分型的可行性、敏感性和特异性,旨在寻找一种适合于我国流行病学调查和研究的肺炎链球菌血清分型方法。根据肺炎链球菌血清型相关基因cps的结构和组成,以及国内既往研究结果,设计我国较为常见血清型1,3,5,6,7F,9V 14,15A,15B／C,18C,19F,19A,20,22 F,23F等的特异引物,并优化建立了多重PCR反应体系。从临床分离的肺炎链球菌中成功区别血清型19F,19A,23F,14,18C,6A/B,15A,15B／C等常见血清型。对多重PCR的的初步分型结果,进行普通PCR反应验证和PCR反应产物的测序等确认分型结果的准确性。多重PCR分型结果与荚膜肿胀试验分型结果相比,63株肺炎链球菌中,两者分型结果一致的有52株菌,结果一致性达82.5%(95%CI：73.2%-91.9%),灵敏度为89.2%(95%CI：79.2%-99.2%),阳性预测值82.5%(95%CI：70.7%-94.3%)。在分型结果不一致的11株菌中,7株荚膜肿胀试验尚未分出具体血清型别,多重PCR方法可区分其血清型,分别为血清型23F,19A,18C,6A/B等非常常见的血清型。此外,多重PCR方法还识别了两种血清型19F和23F共存的现象。多重PCR分型结果与MLST分型结果相比,45株肺炎链球菌血清分型结果一致的有40株,结果一致性达88.9%(95%CI：79.7%-98.1%),灵敏度为88.4%(95%CI：78.8%-98.0%),阳性预测值100%。可见多重PCR分型方法与荚膜肿胀试验和MLST分型结果一致性高,分型结果准确,可以同时筛查多种血清型,具有快速、便捷、客观等优点,其分型成本为各种分型方法中最低。此外,多重PCR方法可以对荚膜阴性肺炎链球菌菌株进行分型,也可识别多种血清型共存的现象,还可直接用于临床标本的肺炎链球菌感染的检测和分型。今后可将其分型范围扩展至更多血清型,并在现场应用和进一步评价该方法。总之,肺炎链球菌多重PCR分型方法是一种经济、快速、便捷、客观的分型方法,适用于发展中国家和大规模的流行病学调查。更多还原

【Abstract】 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.更多还原