儿童肺炎流行特征、病原体诊断方法评价和肺炎链球菌多重PCR分型方法研究
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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。
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 China
Information 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 Children
Acute 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 Pneumoniae
The 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.
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。
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 China
Information 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 Children
Acute 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 Pneumoniae
The 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.
引文
[1]Rudan I, Tomaskovic L, Boschi-Pinto C, et al. Global estimate of the incidence of clinical pneumonia among children under five years of age[J]. Bulletin of the World Health Organization,2004,82:895-903.
[2]Rudan I, Boschi-Pinto C, Biloglav Z, et al. Epidemiology and etiology of childhood pneumonia.[J]. Bull World Health Organ,2008,86(5):408-416.
[3]Leowski J. Mortality from acute respiratory infections in children under 5 years of age:global estimates.[J]. World Health Stat Q,1986,39(2):138-144.
[4]Bryce J, Boschi-Pinto C, Shibuya K, et al. WHO estimates of the causes of death in children.[J]. Lancet,2005,365(9465):1147-1152.
[5]WHO. the Global burden of disease 2004 update[G].2008.
[6]刘玉琳,林良明.1991-1993年中国5岁以下儿童肺炎死亡监测结果[J].中华儿科杂志,1996,34(6)：365-368.
[7]全国5岁以下儿童死亡调查协作组.中国5岁以下儿童死亡抽样调查[J].中华儿科杂志,1994,32(3)：149-152.
[8]王艳萍,缪蕾,钱幼琼,等.1996至2000年全国5岁以下儿童死亡监测主要结果分析[J].中华预防医学杂志,2005,39(4)：260-264.
[9]PACE. The world pneumonia day[Z].2009:2009.
[10]Mulholland K. Magnitude of the problem of childhood pneumonia.[J]. Lancet, 1999,354(9178):590-592.
[11]Vuori E, Peltola H, Kallio M J, et al. Etiology of pneumonia and other common childhood infections requiring hospitalization and parenteral antimicrobial therapy. SE-TU Study Group.[J]. Clin Infect Dis,1998,27(3):566-572.
[12]赵国昌,王晓红,朱启镕.上海地区儿童急性肺炎病原学和临床流行病学研究[J].2003：4.
[13]华春珍,俞惠民,陈志敏,等.小儿下呼吸道感染的细菌病原学分析[J].中国当代儿科杂志,2006,8(5)：365-368.
[14]秦铭,季纯珍,田曼,等.儿童急性下呼吸道感染细菌病原学分析[J].江苏医药,2005,31(5)：330-332.
[15]李浩.儿童下呼吸道感染的病原菌及其耐药性分析[J].中国医师杂志,2006,8(9)：1276-1277.
[16]车大钿,陆权,陆敏,等.2000年上海地区儿童急性下呼吸道感染的病原学 研究[J].中国当代儿科杂志,2004,6(2)：136-138.
[17]车大钿,陆敏,张泓,等.上海地区儿童呼吸道感染致病菌耐药性研究[J].中国当代儿科杂志,2006,8(4)：338-340.
[18]汤丽霞,谭斯艺,黄燕.儿童上呼吸道医院感染病原菌监测及药敏分析[J].广西医学,2006,28(12)：1926-1927.
[19]Zhao G, Black S, Shinefield H, et al. Serotype distribution and antimicrobial resistance patterns in Streptocccus pneumoniae isolates from hospitalized pediatric patients with respiratory infections in Shanghai, China[J]. Pediatr Infect Dis J,2003,22(8):739-742.
[20]Shann F. Etiology of severe pneumonia in children in developing countries. [J]. Pediatr Infect Dis,1986,5(2):247-252.
[21]Hu H, He L, Dmitriev A, et al. The role of Haemophilus influenzae type b in fatal community-acquired pneumonia in Chinese children.[J]. Pediatr Infect Dis J,2008,27(10):942-944.
[22]王亚娟,姚德秀,燕润菊,等.肺炎链球菌在儿童急性下呼吸道感染中的检测[J].中华微生物学和免疫学杂志,1999,19(6)：460.
[23]Pneumococcal conjugate vaccine for childhood immunization--WHO position paper.[J]. Wkly Epidemiol Rec,2007,82(12):93-104.
[24]Scott J A. The preventable burden of pneumococcal disease in the developing world.[J].Vaccine,2007,25(13):2398-2405.
[25]O'Brien K, Wolfson L, Pwatt J, et al. Burdern of disease caused by streptococcus pneumonia in children younger than 5years, global estimates[J]. Lancet,2009, 374:893-902.
[26]Yao K H, Yang Y H. Streptococcus pneumoniae diseases in Chinese children: past, present and future.[J]. Vaccine,2008,26(35):4425-4433.
[27]董柏青；唐振柱；林枚；李翠云；谭冬梅；梁大斌；等.广西南宁地区5岁以下儿童细菌性脑膜炎的流行病学监测[J].中华流行病学杂志,2004,25(5)：391-395.
[28]Siber G R, Klugman K P, Makela P H. Pneumococcal vaccines:the impact of conjugate vaccine [M]. Washington, DC:ASM Press,2008.
[29]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[30]O'Brien K L, Moulton L H, Reid R, et al. Efficacy and safety of seven-valent conjugate pneumococcal vaccine in American Indian children:group randomised trial.[J]. Lancet,2003,362(9381):355-361.
[31]丁绍卿,叶人邦,袁曾麟.肺炎链球菌血清学分型的研究[J].中国生物制品学杂志,1988,l(1)：18-22.
[32]肺炎球菌分型研究协作组.我国18省(市)肺炎球菌血清型及其感染的流行病学研究[J].中华流行病学杂志,1989,10(3)：133-137.
[33]姚开虎,陆权,邓力,等.2000-2002年三家儿童医院分离的肺炎链球菌血清型分布及其对b内酰胺类抗生素敏感性的变化[J].中华儿科杂志,2006,44(12)：928-932.
[34]杨帆,张婴元,Mcgee L,等上海市791名健康儿童222株肺炎链球菌携带株研究[J].中华医学杂志,2001,81(10)：589-592.
[35]Juven T, Mertsola J, Waris M, et al. Etiology of community-acquired pneumonia in 254 hospitalized children.[J]. Pediatr Infect Dis J,2000,19(4):293-298.
[36]王玲,董书平,赵桂珍,等.推广儿童急性呼吸道感染标准病例管理降低0-4岁儿童死亡率[J].中国初级卫生保健,1997,11(3)：28-29.
[37]李香兰,徐能义.包头市学龄前儿童呼吸道疾病调查[J].包头医学院学报,1996,12(3)：20-22.
[38]韩丽春,殷淑珍,景志忠,等.包头市3岁以下儿童四种常见疾病发病情况分析[J].包头医学,2003,27(2)：13-14.
[39]Li Z, Wang L, He G, et al. The incidence of neonatal pnemoniae in urban and rural areas of Beijing [J]. Chinese Medical Journal,1985,98(5):311-315.
[40]艾银凤,孙守兰.宁夏山区5岁以下儿童急性呼吸道感染标准病例管理分析[J].中国妇幼保健,2000,15(4)：246-247.
[41]刘倩,赵珊.青岛市0-4岁儿童急性呼吸道感染监测结果分析[J].青岛医学院学报,1994,30(3)：232-235.
[42]程萍.运用ARI管理适宜技术降低婴幼儿肺炎死亡率[J].江苏预防医学,1996(02).
[43]徐桂玲,李林祥.云南省华宁县5岁以下儿童急性呼吸道感染监测分析[J].中国初级卫生保健,2000,14(6)：36-38.
[44]周燕燕,陈敏.广州地区5岁以下儿童肺炎发病率季节性分析[J].中国儿童保健杂志,2000,8(1)：35-36.
[45]Jiang Z, Wang J, Zhaori G, et al. Pneumonia--one of the major health problems of infants and children in China[J]. Chinese Medical Journal,1992,105(1):81-86.
[46]邵广荣.流行性喘憋性肺炎1167例调查报告[J].南通医学院学报,1998,18(3)：408.
[47]黄文红,陈丽娜.鲤城区0-4岁儿童急性呼吸道感染监测结果分析[J].海峡预防医学杂志,1999,5(2)：21.
[48]张春灵.2001-2005年吉林市1427例5岁以下儿童死亡分析[J].中国妇幼保健,2006,21(23)：3269-3271.
[49]杨东梅,王敏,王静竹.吉林省2005年5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2007,22(6)：781-782.
[50]张静,王丽,张焕丽,等.2000-2004年大连市5岁以下儿童死亡监测分析[J].中国妇幼保健,2006,21(6)：813-815.
[51]王辛荑,杨柳.沈阳市和平区2005-2006年5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(8)：1051-1052.
[52]钟新艳.2000-2005年太原市5岁以下儿童死亡情况分析[J].中国健康教育,2007,23(3)：214-215.
[53]刘清华,郝波.北京市1992年5岁以下儿童死亡重点死因——肺炎的分析[J].中华儿童保健杂志,1994,2(4)：277-278.
[54]胡凤贤.5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(1)：51-52.
[55]杜小云,郭小菊.勉县5岁以下儿童肺炎死亡十年对比分析[J].中国儿童保健杂志,1999,7(4)：248-249.
[56]杨珏玲,吴文献.陕西省妇幼卫生项目县婴幼儿生存水平分析[J].中华儿童保健杂志,1994,2(1)：32-34.
[57]王秀霞,马振侠,赵红梅.2000-2003年银川市5岁以下儿童死因分析与干预措施[J].宁夏医学院学报,2005,27(4)：300-301.
[58]刘奇杰,王巧玲,艾银凤.宁夏2003年5岁以下儿童死亡情况的监测[J].宁夏医学杂志,2004,26(8)：524-525.
[59]胡艾莉.西宁市1995年-2004年5岁以下儿童生命监测结果分析[J].青海医药杂志,2006,36(8)：81-83.
[60]乌丽娅,迪丽拜尔.新疆24个县妇女儿童健康状况初步评价[J].中国妇幼保健,2000,15(9)：582-584.
[61]张梅,侯莉.1998-2002年徐州市5岁以下儿童死亡分析[J].江苏卫生保健,2003(04).
[62]王菁,姜梅芳.苏州市1993-1999年5岁以下儿童死亡率及死因变化[J].中国优生优育,2002,13(1)：16-18.
[63]陈雅芬,蒋新液.无锡市1993-1997年5岁以下儿童死因分析[J].江苏卫生 保健,1999：101-102.
[64]汤书晶,王道良,李海龙.萧山市1996年0-4岁儿童死因分析[J].右江民族医学院学报,1998,20(72)：159-160.
[65]赵正言,郑凯航.浙江省O-4岁儿童生存水平的研究[J].中国妇幼保健,1996,11(5)：41-43.
[66]王佩茗.衢州市1995年五岁以下儿童死亡分析[J].浙江预防医学,1998,10(11)：678-681.
[67]陈少华,杨政坚.2004年三明市5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(2)：191-192.
[68]林小远.宁德市20002004年5岁以下儿童死亡监测分析[J].中国妇幼保健,2006,21(10)：1360-1361.
[69]宋杰.开封县1991-2000年5岁以下儿童死亡回顾分析[J].临床医学,2003,23(7)：25-26.
[70]刘智兰.信阳市1991-1995年5岁以下儿童死亡监测结果分析[J].河南医药信息,1997,5(8)：31-32.
[71]赵悦淑,刘安民.1989年河南省五岁以下儿童肺炎死亡调查[J].河南医科大学学报,1994,29(2)：160-162.
[72]熊忠贵,徐育松.湖北省1991-2001年5岁以下儿童死亡监测结果分析[J].中国预防医学杂志,2004,5(3)：179-181.
[73]张桂莲,姚秀英.双峰县O-4岁儿童肺炎监测结果分析[J].实用预防医学,1994,1(4)：235.
[74]欧明才,范明清.四川省50个县5岁以下儿童死亡及其有关因素分析[J].中国卫生事业管理,1995：36-39.
[75]夏凉,李顺年,肖发兴,等.武隆县妇幼卫生基础工作调查分析[J].中国初级卫生保健,1994,8(1)：30-32.
[76]甘慧君.秀山县1998-2001年5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2003,18(11)：702.
[77]董应芬.晋宁县2001-2005年5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(13)：1783-1784.
[78]邝建明,蒋玉欢,邵小飞,等.1999-2003年5岁以下儿童死亡分析[J].中国妇幼保健,2005,20(21)：2855-2856.
[79]苏云,刘楚芹,温丽倩,等.肇庆市1998-2002年5岁以下儿童生命监测结果与干预措施[J].中国妇幼保健,2004,19(5)：69-70.
[80]赵慧贞,张红忠.珠海市1994-2002年5岁以下儿童死亡原因分析[J].中国 初级卫生保健,2003,17(11)：56-57.
[81]龚研宁,黄赏琼.5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2004,19(9)：92-94.
[82]彭修道,韦茂国.海南省5岁以下儿童死亡基础调查研究[J].海南医学,1994,5(3)：182-186.
[83]曹佳莉.连云港市1997-1999年5岁以下儿童死亡原因的调查与分析[J].齐齐哈尔医学院学报,2001,22(11)：1320-1321.
[84]李明敏,许云娥.成都市1997年5岁以下儿童死因分析[J].四川省卫生管理干部学院学报,2000,19(1)：57-58.
[85]陈莹莹.1989年湖北省5岁以下儿童死亡回顾调查[J].湖北医科大学学报,1995,16(4)：373-375.
[86]聂忠华,邹芸芸.万安县5岁以下儿童死亡原因10年动态对比分析[J].江西医学院学报,2003,43(1)：121-123.
[87]魏红光,宋成义,张玉珍,等.甘肃省酒泉市肃州区2000-2004年新生儿死亡原因分析[J].卫生职业教育,2006,24(1)：112-113.
[88]梁颖.佛山市2000-2004年5岁以下儿童死亡分析[J].实用预防医学,2005,12(5)：1122-1123.
[89]王勇.广西儿童1999-2000年新生儿死亡监测情况分析[J].中国儿童保健杂志,2003,11(2)：128-129.
[90]刘龙池.隆回县0-6天新生儿死亡监测分析[J].医学理论与实践,2006,19(12)：1449-1450.
[91]谢艳玲,杨勤.湖北省5岁以下儿童肺炎死亡对策的研究[J].湖北预防医学杂志,2003,14(4)：17-19.
[92]蒋维国,蒋明仁.20年住院新生儿肺炎发病率及死亡率回顾分析[J].卫生职业教育,2005,23(23)：42.
[93]南京市儿童医院.2226新生儿肺炎住院病例分析[J].中华儿科杂志,1979,17(4)：216-219.
[94]杜梅.新生儿细菌性肺炎病原菌及耐药情况分析[J].中国误诊学杂志,2006,6(2)：257-258.
[95]桂林艳.新生儿肺炎病原菌及药敏分析[J].中国误诊学杂志,2007,7(5)：973-974.
[96]裘淑琴,葛传生,范一新,等.264例小儿细菌性肺炎的病原与临床[J].中华儿科杂志,1987,25(1)：10-11.
[97]马晓路,徐迎春,郑季彦,等.新生儿肺炎的病原及临床研究[J].浙江预防 医学,2005,17(1)：6-8.
[98]汪洁,罗新声.新生儿肺炎的病原体诊断[J].新生儿科杂志,1997,12(2)：62-63.
[99]尤灿,李先斌,黄彩芝,等.2164例新生儿肺炎需氧菌的分布及耐药性分析[J].中国感染控制杂志,2004,3(4)：345-347.
[100]李先斌,黄铭华,江训良,等.新生儿感染性肺炎病原菌分布及耐药性分析[J].实用预防医学,2003,10(5)：671-673.
[101]李坚,宋燕燕,李小晶,等.新生儿肺炎病原学及临床研究——附169例分析[J].中国妇幼保健,2004,19(10)：95-97.
[102]陈淑贞,蔡应木,杨启平.新生儿肺炎的病原学分析[J].中国微生态学杂志,2004,16(1)：45-46.
[103]林菁.新生儿肺炎细菌病原学及药敏结果分析[J].国际医药卫生导报,2001：46.
[104]曾令威.新生儿吸入性肺炎102例的病原菌分布及耐药性分析[J].广西医学,2007,29(6)：879-880.
[105]徐广范,于新环,胡锦华.儿童大叶性肺炎151例临床分析[J].牡丹江医学院学报,2007,28(1)：23.
[106]董枫,高鹏.儿童肺炎病原菌检测及药敏分析[J].大连医科大学学报,1998,20(1)：54-55.
[107]王惠萍,庞延凯.婴儿急性细菌性肺炎的临床流行病学分析[J].中华流行病学杂志,1994,15(4)：209-211.
[108]刘秀云,江载芳.小儿重症社区获得性肺炎31例病原分析[J].中国实用儿科杂志,2005,20(12)：749-750.
[109]毕晶,王美英.保定儿童医院2726例肺炎患儿多种病原检测与临床研究[J].中国医药导报,China medical herald,2006,3(32):60-61.
[110]张中馥,高国林,张建明,等.小儿肺炎318例痰培养细菌学分析[J].临床荟萃,2005,20(16)：938-939.
[111]上官芹,孙志良.儿童细菌性肺炎致病菌的耐药性分析[J].中国妇幼保健,2006,21(11)：1528-1529.
[112]张秀莲.不同月龄阶段婴儿肺炎病原体检测的临床意义[J].中原医刊,2006,33(18)：7-8.
[113]罗海燕,祝益民,李贵南,等.重症监护病房婴幼儿重症肺炎细菌学监测及临床分析[J].实用儿科临床杂志,2007,22(16)：1238-1240.
[114]龙元珠,刘建梅,吴爱民.小儿肺炎235例病原学临床分析[J].实用临床医 学(江西),2004,5(5)：84.
[115]李启先.儿童肺炎病原学分析[J].蚌埠医学院学报,2002,27(3)：245.
[116]杨英.660例小儿肺炎病原学和耐药性分析[J].现代医药卫生,2007,23(20)：3007-3009.
[117]罗蓉,黄英.重庆地区小儿肺炎下呼吸道分泌物病原耐药性分析[J].实用儿科临床杂志,2006,21(4)：224-225.
[118]张晓芬,陈全景,王春晖,等.十堰地区893例小儿细菌性肺炎病原学及药敏结果分析[J].郧阳医学院学报,2005,24(1)：37-38.
[119]考验,姚静婵,等.小儿肺炎细菌学检测及药敏试验[J].浙江预防医学,2001,13(11)：5-6.
[120]王峰,张竹馨,王玉屏.178例小儿肺炎分离菌分布和耐药性分析[J].中国微生态学杂志,2005,17(6)：458-459.
[121]刘纯义,张侃,陆必森,等.细菌性重症肺炎患儿的病原菌分析及治疗对策[J].小儿急救医学,2005,12(2)：115-117.
[122]宋玫,蔡桂丰.116例小儿肺炎分离菌的耐药性分析[J].中国微生态学杂志,2000,12(6)：351-352.
[123]李玉凤,祝俭平.516例小儿细菌性肺炎细菌学监测及分析[J].右江民族医学院学报,2007,29(4)：607-609.
[124]谢永武,宋玫,宝杰,等.小儿肺炎细菌学分类及抗生素耐药性分析[J].宁夏医学杂志,2006,28(6)：437-439.
[125]姜德昭,黄明春.武鸣县儿童肺炎病原学及正常咽部带菌的研究[J].广西医学院学报,1991,8(1)：1-4.
[126]Garenne M, Ronsmans C, Campbell H. The magnitude of mortality from acute respiratory infections in children under 5 years in developing countries. [J]. World Health Stat Q,1992,45(2-3):180-191.
[127]Williams B G, Gouws E, Boschi-Pinto C, et al. Estimates of world-wide distribution of child deaths from acute respiratory infections.[J]. Lancet Infect Dis,2002,2(1):25-32.
[128]Dowell S F, Kupronis B A, Zell E R, et al. Mortality from pneumonia in children in the United States,1939 through 1996.[J]. N Engl J Med,2000,342(19):1399-1407.
[129]Sazawal S, Black R E. Effect of pneumonia case management on mortality in neonates, infants, and preschool children:a meta-analysis of community-based trials.[J]. Lancet Infect Dis,2003,3(9):547-556.
[130]Wubbel L, Muniz L, Ahmed A, et al. Etiology and treatment of community-acquired pneumonia in ambulatory children. [J]. Pediatr Infect Dis J,1999,18(2):98-104.
[131]Rapkin R H. Bacteriologic and clinical findings in acute pneumonia of childhood.[J]. Clin Pediatr (Phila),1975,14(2):130-133.
[132]Ikeogu M O. Acute pneumonia in Zimbabwe:bacterial isolates by lung aspiration. [J]. Arch Dis Child,1988,63(10):1266-1267.
[133]Wang Y, Vuori-Holopainen E, Yang Y, et al. Relative frequency of Haemophilus influenzae type b pneumonia in Chinese children as evidenced by serology[Z].2002:21,271-277.
[134]Michelow I C, Lozano J, Olsen K, et al. Diagnosis of Streptococcus pneumoniae lower respiratory infection in hospitalized children by culture, polymerase chain reaction, serological testing, and urinary antigen detection.[J]. Clin Infect Dis,2002,34(1):E1-E11.
[135]Michelow I C, Olsen K, Lozano J, et al. Epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children.[J]. Pediatrics,2004,113(4):701-707.
[136]Nohynek H, Eskola J, Laine E, et al. The causes of hospital-treated acute lower respiratory tract infection in children.[J]. Am J Dis Child,1991,145(6):618-622.
[137]Ruuskanen 0, Nohynek H, Ziegler T, et al. Pneumonia in childhood:etiology and response to antimicrobial therapy.[J]. Eur J Clin Microbiol Infect Dis,1992, 11(3):217-223.
[138]Falade A G, Mulholland E K, Adegbola R A, et al. Bacterial isolates from blood and lung aspirate cultures in Gambian children with lobar pneumonia.[J]. Ann Trop Paediatr,1997,17(4):315-319.
[139]Wubbel L, Muniz L, Ahmed A, et al. Etiology and treatment of community-acquired pneumonia in ambulatory children.[J]. Pediatr Infect Dis J,1999,18(2): 98-104.
[140]Korppi M, Katila M L, Kalliokoski R, et al. Pneumococcal finding in a sample from upper airways does not indicate pneumococcal infection of lower airways. [J]. Scand J Infect Dis,1992,24(4):445-451.
[141]Nohynek H, Eskola J, Kleemola M, et al. Bacterial antibody assays in the diagnosis of acute lower respiratory tract infection in children.[J]. Pediatr Infect Dis J,1995,14(6):478-484.
[142]Falade A G, Mulholland E K, Adegbola R A, et al. Bacterial isolates from blood and lung aspirate cultures in Gambian children with lobar pneumonia. [J]. Ann Trop Paediatr,1997,17(4):315-319.
[143]Lankinen K S, Salo P, Rapola S, et al. Pneumococcal capsular antigen detection after enrichment culture:an alternative to culture methods in epidemiologic research.[J]. Am J Trop Med Hyg,1997,56(2):211-215.
[144]Lankinen K S, Ruutu P, Nohynek H, et al. Pneumococcal pneumonia diagnosis by demonstration of pneumolysin antibodies in precipitated immune complexes: a study in 350 Philippine children with acute lower respiratory infection.[J]. Scand J Infect Dis,1999,31(2):155-161.
[145]Faden H, Heimerl M, Goodman G, et al. New technique (the NOW test) for rapid detection of Streptococcus pneumoniae in the nasopharynx.[J]. J Clin Microbiol,2002,40(12):4748-4749.
[146]Dominguez J, Blanco S, Rodrigo C, et al. Usefulness of urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal pneumonia in children.[J]. J Clin Microbiol,2003,41(5):2161-2163.
[147]Lankinen K S, Rintamaki S, Syrjanen R, et al. Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens.[J]. J Microbiol Methods,2004,56(2):193-199.
[148]Neuman M I, Harper M B. Evaluation of a rapid urine antigen assay for the detection of invasive pneumococcal disease in children. [J]. Pediatrics,2003,112(6 Pt 1):1279-1282.
[149]von Graevenitz A, Rathbone R R. Branhamella catarrhalis in respiratory secretions:clinical correlation in 16 cases. [J]. South Med J,1981,74(9):1095-1096.
[150]Perera A, Wickramasinghe S, Perera B J, et al. Reliable and easy identification of bacterial acute respiratory infections of childhood.[J]. Ceylon Med J,1998, 43(4):200-205.
[151]Lehtomaki K, Leinonen M, Takala A, et al. Etiological diagnosis of pneumonia in military conscripts by combined use of bacterial culture and serological methods.[J]. Eur J Clin Microbiol Infect Dis,1988,7(3):348-354.
[152]Weber M W, Mulholland E K, Greenwood B M. Respiratory syncytial virus infection in tropical and developing countries.[J]. Trop Med Int Health,1998, 3(4):268-280.
[153]赵国昌,王晓红,朱启镕.上海地区儿童急性肺炎病原学和临床流行病学研究[J].2003：4.
[154]盛朝凯,刘岚,刘昌,等.儿童呼吸道感染时3种病原菌检出率及耐药性观察[J].临床儿科杂志,2004,22(2)：94-96.
[155]Mulholland K. Global burden of acute respiratory infections in children: implications for interventions.[J]. Pediatr Pulmonol,2003,36(6):469-474.
[156]Bratcher P E, Kim K H, Kang J H, et al. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization.[J]. Microbiology,2010,156(Pt 2):555-560.
[157]Park I H, Pritchard D G, Cartee R, et al. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae.[J]. J Clin Microbiol,2007,45(4):1225-1233.
[158]Llull D, Munoz R, Lopez R, et al. A single gene (tts) located outside the cap locus directs the formation of Streptococcus pneumoniae type 37 capsular polysaccharide. Type 37 pneumococci are natural, genetically binary strains.[J]. J Exp Med,1999,190(2):241-251.
[159]Cartee R T, Forsee W T, Jensen J W, et al. Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.[J]. J Biol Chem,2001,276(52):48831-48839.
[160]Dillard J P, Vandersea M W, Yother J. Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.[J]. J Exp Med,1995,181(3):973-983.
[161]Kolkman M A, van der Zeijst B A, Nuijten P J. Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae.[J]. J Biochem,1998,123(5):937-945.
[162]Garcia E, Llull D, Munoz R, et al. Current trends in capsular polysaccharide biosynthesis of streptococcus pneumoniae [J]. Res. Microbiol.,2000,151:429-435.
[163]Bentley S D, Aanensen D M, Mavroidi A, et al. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.[J]. PLoS Genet,2006, 2(3):e31.
[164]Jiang S, Wang L, Reeves P R. Molecular Characterization of Streptococcus pneumoniae Type 4,6B,8, and 18C Capsular Polysaccharide Gene Clusters[J]. Infection and Immunity,2001,69(3):1244-1255.
[165]Lalitha M K, Thomas K, Kumar R S, et al. Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera.[J]. J Clin Microbiol, 1999,37(1):263-265.
[166]Sorensen U B. Typing of pneumococci by using 12 pooled antisera.[J]. J Clin Microbiol,1993,31 (8):2097-2100.
[167]Brito D A, Ramirez M, de Lencastre H. Serotyping Streptococcus pneumoniae by Multiplex PCR[J]. J Clin Microbiol,2003,41(6):2378-2384.
[168]Gratten M, Montgomery J, Gerega G, et al. Multiple colonization of the upper respiratory tract of Papua New Guinea children with Haemophilus influenzae and Streptococcus pneumoniae.[J]. Southeast Asian J Trop Med Public Health, 1989,20(4):501-509.
[169]Lloyd-Evans N, O'Dempsey T J, Baldeh I, et al. Nasopharyngeal carriage of pneumococci in Gambian children and in their families.[J]. Pediatr Infect Dis J, 1996,15(10):866-871.
[170]Obaro S K, Adegbola R A, Banya W A, et al. Carriage of pneumococci after pneumococcal vaccination.[J]. Lancet,1996,348(9022):271-272.
[171]O'Brien K L, Millar E V, Zell E R, et al. Effect of pneumococcal conjugate vaccine on nasopharyngeal colonization among immunized and unimmunized children in a community-randomized trial.[J]. J Infect Dis, 2007,196(8):1211-1220.
[172]Maiden M C, Bygraves J A, Feil E, et al. Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms. [J]. Proc Natl Acad Sci U S A,1998,95(6):3140-3145.
[173]Enright M C, Spratt B G. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease.[J]. Microbiology,1998,144 (Pt 11):3049-3060.
[174]Pai R, Gertz R E, Beall B. Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates[J]. J Clin Microbiol, 2005,44(1):124-131.
[175]Lawrence E R, Griffiths D B, Martin S A, et al. Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups[JJ. J Clin Microbiol,2003,41(2):601-660.
[176]Morais L, Carvalho M G, Roca A, et al. Sequential multiplex PCR for identifying pneumococcal capsular serotypes from South-Saharan African clinical isolates.[J]. J Med Microbiol,2007,56(Pt 9):1181-1184.
[177]O'halloran D M, Cafferkey M T. Multiplex PCR for Identification of Seven Streptococcus pneumoniae Serotypes Targeted by a 7-Valent Conjugate Vaccine[J]. J Clin Microbiol,2005,43(7):3487-3490.
[178]Dias C A, Teixeira L C M, Carvalho M D G R, et al. Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children[J]. Journal of Medical Microbiology,2007,56:1185-1188.
[179]Rivera-Olivero I A, Blommaart M, Bogaert D, et al. Multiplex PCR reveals a high rate of nasopharyngeal pneumococcal 7-valent conjugate vaccine serotypes co-colonizing indigenous Warao children in Venezuela.[J]. J Med Microbiol, 2009,58(Pt 5):584-587.
[180]Turner P, Hinds J, Gould K, et al. Conventional techniques for detecting nasopharyngeal pneumococcal carriage significantly underestimate the serotype carriage[C]. Tel Aviv, Isreal:2010.
[181]Billal D S, Hotomi M, Suzumoto M, et al. Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.[J]. Eur J Pediatr,2008,167(4):401-407.
[182]Saha S K, Darmstadt G L, Baqui A H, et al. Identification of Serotype in Culture Negative Pneumococcal Meningitis Using Sequential Multiplex PCR: Implication for Surveillance and Vaccine Design[J]. PLOS one,2008,3(10): e3576.
[183]Butler J C, Breiman R F, Campbell J F, et al. Pneumococcal polysaccharide vaccine efficacy. An evaluation of current recommendations.[J]. JAMA,1993, 270(15):1826-1831.
[184]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[185]Whitney C G, Pilishvili T, Farley M M, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease:a matched case-control study.[J]. Lancet,2006,368(9546):1495-1502.
[186]Pelton SI, Huot H, Finkelstein JA, et al., Emergence of 19A as Virulent and Multidrug Resistant Pneumococcus in Massachusetts Following Universal Immunization of Infants With Pneumococcal Conjugate Vaccine[J]. Pediatr Infect Dis J.2007;26(6):468-472.
[187]Mahjoub-Messai F, Doit C, Koeck JL, et al., Population Snapshot of Streptococcus pneumoniae Serotype 19A Isolates before and after Introduction of Seven-Valent Pneumococcal Vaccination for French Children[J]. J Clin Microbiol,2009,47(3):837-840.
[188]Konradsen H B. Validation of serotyping of Streptococcus pneumoniae in Europe.[J]. Vaccine,2005,23(11):1368-1373.
[189]Finch R G, Phillips I. Serological grouping of streptococci by a slide coagglutination method.[J]. J Clin Pathol,1977,30(2):168-170.
[190]Rajalakshmi B, Kanungo R. Cost-effective method of serotyping streptococcus pneumoniae using staphylococcal co-agglutination. [J]. Indian J Med Microbiol,2001,19(4):197-200.
[191]MK Lalitha, R Pai, TJ John, et al. Serotyping of Streptococcus pneumoniae by agglutination assays:a cost-effective technique for developing countries[J]. Bull World Health Organ.,1996,74(4):387-390.
[192]Arai S, Konda T, Wada A. Use of antiserum-coated latex particles for serotyping streptococcus pneumoniae [J]. Microbiol Immunol.,2001,45(2):159-162.
[193]A S, K L M, J J T, et al. Modified latex agglutination test for rapid detection of Streptococcus pneumoniae and haemophilus influenzae in cerebrospinal fluid and direct serotyping of Streptococcus pneumoniae [J]. European journal of clinical microbiology & infectious diseases,1996,15(6):472-477.
[194]Slotved H C, Kaltoft M, Skovsted I C, et al. Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).[J]. J Clin Microbiol, 2004,42(6):2518-2522.
[195]Yu J, Lin J, Jr Benjamin W H, et al. Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies. [J]. J Clin Microbiol,2005,43(1):156-162.
[196]Lin J, Kaltoft M S, Brandao A P, et al. Validation of a multiplex pneumococcal serotyping assay with clinical samples.[J]. J Clin Microbiol,2006,44(2):383-388.
[197]Yu J, Carvalho M G, Beall B, et al. A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.[J]. J Med Microbiol,2008,57(Pt 2): 171-178.
[198]Batt S L, Charalambous B M, Mchugh T D, et al. Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.[J]. J Clin Microbiol,2005,43(6):2656-2661.
[199]Lawrence E R, Arias C A, Duke B, et al. Evaluation of Serotype Prediction by cpsA-cpsB Gene Polymorphism in Streptococcus pneumoniae [J]. J Clin Microbiol,2000,38(4):1319-1323.
[200]Moreno J, Hernandez E, Sanabria O, et al. Detection and Serotyping of Streptococcus pneumoniae from Nasopharyngeal Samples by PCR-Based Multiplex Assay[J]. J Clin Microbiol,2005,43(12):6152-6154.
[201]Billal D S, Hotomi M, Tasnim S, et al. Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction.[J]. ORL J Otorhinolaryngol Relat Spec,2006,68(3):135-138.
[202]Kong F, Wang W, Tao J, et al. A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy-or wzx-specific PCR[J]. Journal of Medical Microbiology,2005,54: 351-356.
[203]Kong F, Gilbert G L. Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes[J]. Journal of Medical Microbiology,2003,52:1047-1058.
[204]Kong F, Brown M, Sabananthan A, et al. Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes[J]. J Clin Microbiol,2006,44(5):1887-1891.
[205]Wang H, Kong F, Jelfs P, et al. Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.[J]. Appl Environ Microbiol,2004,70(3):1483-1486.
[206]Zhao Z, Kong F, Gilbert G L. Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.[J]. Clin Vaccine Immunol,2006,13(1):145-149.
[207]Wang Q, Wang M, Kong F, et al. Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.[J]. J Microbiol Methods,2007,68(1):128-136.
[1]Siber G R, Klugman K P, Makela P H. Pneumococcal vaccines:the impact of conjugate vaccine [M]. Washington, DC:ASM Press,2008.
[2]Park I H, Pritchard D G, Cartee R, et al. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae.[J]. J Clin Microbiol, 2007,45(4):1225-1233.
[3]Bratcher P E, Kim K H, Kang J H, et al. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization.[J]. Microbiology,2010,156(Pt 2):555-560.
[4]Butler J C, Breiman R F, Campbell J F, et al. Pneumococcal polysaccharide vaccine efficacy. An evaluation of current recommendations.[J]. JAMA,1993, 270(15):1826-1831.
[5]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[6]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[7]Whitney C G, Pilishvili T, Farley M M, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease:a matched case-control study.[J]. Lancet,2006,368(9546):1495-1502.
[8]Emergence of 19A as Virulent and Multidrug Resistant Pneumococcus in Massachusetts Following Universal Immunization of Infants With Pneumococcal Conjugate Vaccine[J].
[9]Population Snapshot of Streptococcus pneumoniae Serotype 19A Isolates before and after Introduction of Seven-Valent Pneumococcal Vaccination for French Children[J].
[10]Konradsen H B. Validation of serotyping of Streptococcus pneumoniae in Europe.[J].Vaccine,2005,23(11):1368-1373.
[11]Finch R G, Phillips I. Serological grouping of streptococci by a slide coagglutination method.[J]. J Clin Pathol,1977,30(2):168-170.
[12]Rajalakshmi B, Kanungo R. Cost-effective method of serotyping streptococcus pneumoniae using staphylococcal co-agglutination.[J]. Indian J Med Microbiol, 2001,19(4):197-200.
[13]Mk L, R P, Tj J, et al. Serotyping of Streptococcus pneumoniae by agglutination assays:a cost-effective technique for developing countries[J]. Bull World Health Organ.,1996,74(4):387-390.
[14]Lalitha M K, Thomas K, Kumar R S, et al. Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera.[J]. J Clin Microbiol, 1999,37(1):263-265.
[15]Arai S, Konda T, Wada A. Use of antiserum-coated latex particles for serotyping streptococcus pneumoniae[J]. Microbiol Immunol.,2001,45(2):159-162.
[16]A S, K L M, J J T, et al. Modified latex agglutination test for rapid detection of Streptococcus pneumoniae and haemophilus influenzae in cerebrospinal fluid and direct serotyping of Streptococcus pneumoniae[J]. European journal of clinical microbiology & infectious diseases,1996,15(6) 472-477.
[17]Slotved H C, Kaltoft M, Skovsted I C, et al. Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).[J]. J Clin Microbiol,2004, 42(6):2518-2522.
[18]Yu J, Lin J, Jr Benjamin W H, et al. Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies.[J]. J Clin Microbiol, 2005,43(1):156-162.
[19]Lin J, Kaltoft M S, Brandao A P, et al. Validation of a multiplex pneumococcal serotyping assay with clinical samples.[J]. J Clin Microbiol,2006,44(2):383-388.
[20]Yu J, Carvalho M G, Beall B, et al. A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.[J]. J Med Microbiol,2008,57(Pt 2): 171-178.
[21]Maiden M C, Bygraves J A, Feil E, et al. Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms.[J]. Proc Natl Acad Sci U S A,1998,95(6):3140-3145.
[22]Enright M C, Spratt B G. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease.[J]. Microbiology,1998,144 (Pt 11):3049-3060.
[23]Batt S L, Charalambous B M, Mchugh T D, et al. Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.[J]. J Clin Microbiol,2005,43(6):2656-.2661.
[24]Lawrence E R, Arias C A, Duke B, et al. Evaluation of Serotype Prediction by cpsA-cpsB Gene Polymorphism in Streptococcus pneumoniae [J]. JOURNAL OF CLINICAL MICROBIOLOGY,2000,38(4):1319-1323.
[25]Lawrence E R, Griffiths D B, Martin S A, et al. Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups[J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2003,41(2):601-660.
[26]Brito D A, Ramirez M, de Lencastre H. Serotyping Streptococcus pneumoniae by Multiplex PCR[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2003,41(6): 2378-2384.
[27]Pai R, Gertz R E, Beall B. Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,44(1):124-131.
[28]Moreno J, Herna'ndez E, Sanabria O, et al. Detection and Serotyping of Streptococcus pneumoniae from Nasopharyngeal Samples by PCR-Based Multiplex Assay[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,43 (12):6152-6154.
[29]O'halloran D M, Cafferkey M T. Multiplex PCR for Identification of Seven Streptococcus pneumoniae Serotypes Targeted by a 7-Valent Conjugate Vaccine[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,43(7):3487-3490.
[30]Billal D S, Hotomi M, Tasnim S, et al. Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction.[J]. ORL J Otorhinolaryngol Relat Spec,2006,68(3):135-138.
[31]Billal D S, Hotomi M, Suzumoto M, et al. Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.[J]. Eur J Pediatr,2008,167(4):401-407.
[32]Dias C A, Teixeira L C M, Carvalho M D G R, et al. Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children[J]. Journal of Medical Microbiology,2007,56:1185-1188.
[33]Kong F, Wang W, Tao J, et al. A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy-or wzx-specific PCR[J]. Journal of Medical Microbiology,2005,54: 351-356.
[34]Kong F, Gilbert G L. Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes[J]. Journal of Medical Microbiology,2003,52:1047-1058.
[35]Kong F, Brown M, Sabananthan A, et al. Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2006,44(5):1887-1891.
[36]Wang H, Kong F, Jelfs P, et al. Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.[J]. Appl Environ Microbiol,2004,70(3):1483-1486.
[37]Zhao Z, Kong F, Gilbert G L. Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.[J]. Clin Vaccine Immunol,2006,13(1):145-149.
[38]Wang Q, Wang M, Kong F, et al. Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.[J]. J Microbiol Methods,2007,68(1):128-136.
[2]Rudan I, Boschi-Pinto C, Biloglav Z, et al. Epidemiology and etiology of childhood pneumonia.[J]. Bull World Health Organ,2008,86(5):408-416.
[3]Leowski J. Mortality from acute respiratory infections in children under 5 years of age:global estimates.[J]. World Health Stat Q,1986,39(2):138-144.
[4]Bryce J, Boschi-Pinto C, Shibuya K, et al. WHO estimates of the causes of death in children.[J]. Lancet,2005,365(9465):1147-1152.
[5]WHO. the Global burden of disease 2004 update[G].2008.
[6]刘玉琳,林良明.1991-1993年中国5岁以下儿童肺炎死亡监测结果[J].中华儿科杂志,1996,34(6)：365-368.
[7]全国5岁以下儿童死亡调查协作组.中国5岁以下儿童死亡抽样调查[J].中华儿科杂志,1994,32(3)：149-152.
[8]王艳萍,缪蕾,钱幼琼,等.1996至2000年全国5岁以下儿童死亡监测主要结果分析[J].中华预防医学杂志,2005,39(4)：260-264.
[9]PACE. The world pneumonia day[Z].2009:2009.
[10]Mulholland K. Magnitude of the problem of childhood pneumonia.[J]. Lancet, 1999,354(9178):590-592.
[11]Vuori E, Peltola H, Kallio M J, et al. Etiology of pneumonia and other common childhood infections requiring hospitalization and parenteral antimicrobial therapy. SE-TU Study Group.[J]. Clin Infect Dis,1998,27(3):566-572.
[12]赵国昌,王晓红,朱启镕.上海地区儿童急性肺炎病原学和临床流行病学研究[J].2003：4.
[13]华春珍,俞惠民,陈志敏,等.小儿下呼吸道感染的细菌病原学分析[J].中国当代儿科杂志,2006,8(5)：365-368.
[14]秦铭,季纯珍,田曼,等.儿童急性下呼吸道感染细菌病原学分析[J].江苏医药,2005,31(5)：330-332.
[15]李浩.儿童下呼吸道感染的病原菌及其耐药性分析[J].中国医师杂志,2006,8(9)：1276-1277.
[16]车大钿,陆权,陆敏,等.2000年上海地区儿童急性下呼吸道感染的病原学 研究[J].中国当代儿科杂志,2004,6(2)：136-138.
[17]车大钿,陆敏,张泓,等.上海地区儿童呼吸道感染致病菌耐药性研究[J].中国当代儿科杂志,2006,8(4)：338-340.
[18]汤丽霞,谭斯艺,黄燕.儿童上呼吸道医院感染病原菌监测及药敏分析[J].广西医学,2006,28(12)：1926-1927.
[19]Zhao G, Black S, Shinefield H, et al. Serotype distribution and antimicrobial resistance patterns in Streptocccus pneumoniae isolates from hospitalized pediatric patients with respiratory infections in Shanghai, China[J]. Pediatr Infect Dis J,2003,22(8):739-742.
[20]Shann F. Etiology of severe pneumonia in children in developing countries. [J]. Pediatr Infect Dis,1986,5(2):247-252.
[21]Hu H, He L, Dmitriev A, et al. The role of Haemophilus influenzae type b in fatal community-acquired pneumonia in Chinese children.[J]. Pediatr Infect Dis J,2008,27(10):942-944.
[22]王亚娟,姚德秀,燕润菊,等.肺炎链球菌在儿童急性下呼吸道感染中的检测[J].中华微生物学和免疫学杂志,1999,19(6)：460.
[23]Pneumococcal conjugate vaccine for childhood immunization--WHO position paper.[J]. Wkly Epidemiol Rec,2007,82(12):93-104.
[24]Scott J A. The preventable burden of pneumococcal disease in the developing world.[J].Vaccine,2007,25(13):2398-2405.
[25]O'Brien K, Wolfson L, Pwatt J, et al. Burdern of disease caused by streptococcus pneumonia in children younger than 5years, global estimates[J]. Lancet,2009, 374:893-902.
[26]Yao K H, Yang Y H. Streptococcus pneumoniae diseases in Chinese children: past, present and future.[J]. Vaccine,2008,26(35):4425-4433.
[27]董柏青；唐振柱；林枚；李翠云；谭冬梅；梁大斌；等.广西南宁地区5岁以下儿童细菌性脑膜炎的流行病学监测[J].中华流行病学杂志,2004,25(5)：391-395.
[28]Siber G R, Klugman K P, Makela P H. Pneumococcal vaccines:the impact of conjugate vaccine [M]. Washington, DC:ASM Press,2008.
[29]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[30]O'Brien K L, Moulton L H, Reid R, et al. Efficacy and safety of seven-valent conjugate pneumococcal vaccine in American Indian children:group randomised trial.[J]. Lancet,2003,362(9381):355-361.
[31]丁绍卿,叶人邦,袁曾麟.肺炎链球菌血清学分型的研究[J].中国生物制品学杂志,1988,l(1)：18-22.
[32]肺炎球菌分型研究协作组.我国18省(市)肺炎球菌血清型及其感染的流行病学研究[J].中华流行病学杂志,1989,10(3)：133-137.
[33]姚开虎,陆权,邓力,等.2000-2002年三家儿童医院分离的肺炎链球菌血清型分布及其对b内酰胺类抗生素敏感性的变化[J].中华儿科杂志,2006,44(12)：928-932.
[34]杨帆,张婴元,Mcgee L,等上海市791名健康儿童222株肺炎链球菌携带株研究[J].中华医学杂志,2001,81(10)：589-592.
[35]Juven T, Mertsola J, Waris M, et al. Etiology of community-acquired pneumonia in 254 hospitalized children.[J]. Pediatr Infect Dis J,2000,19(4):293-298.
[36]王玲,董书平,赵桂珍,等.推广儿童急性呼吸道感染标准病例管理降低0-4岁儿童死亡率[J].中国初级卫生保健,1997,11(3)：28-29.
[37]李香兰,徐能义.包头市学龄前儿童呼吸道疾病调查[J].包头医学院学报,1996,12(3)：20-22.
[38]韩丽春,殷淑珍,景志忠,等.包头市3岁以下儿童四种常见疾病发病情况分析[J].包头医学,2003,27(2)：13-14.
[39]Li Z, Wang L, He G, et al. The incidence of neonatal pnemoniae in urban and rural areas of Beijing [J]. Chinese Medical Journal,1985,98(5):311-315.
[40]艾银凤,孙守兰.宁夏山区5岁以下儿童急性呼吸道感染标准病例管理分析[J].中国妇幼保健,2000,15(4)：246-247.
[41]刘倩,赵珊.青岛市0-4岁儿童急性呼吸道感染监测结果分析[J].青岛医学院学报,1994,30(3)：232-235.
[42]程萍.运用ARI管理适宜技术降低婴幼儿肺炎死亡率[J].江苏预防医学,1996(02).
[43]徐桂玲,李林祥.云南省华宁县5岁以下儿童急性呼吸道感染监测分析[J].中国初级卫生保健,2000,14(6)：36-38.
[44]周燕燕,陈敏.广州地区5岁以下儿童肺炎发病率季节性分析[J].中国儿童保健杂志,2000,8(1)：35-36.
[45]Jiang Z, Wang J, Zhaori G, et al. Pneumonia--one of the major health problems of infants and children in China[J]. Chinese Medical Journal,1992,105(1):81-86.
[46]邵广荣.流行性喘憋性肺炎1167例调查报告[J].南通医学院学报,1998,18(3)：408.
[47]黄文红,陈丽娜.鲤城区0-4岁儿童急性呼吸道感染监测结果分析[J].海峡预防医学杂志,1999,5(2)：21.
[48]张春灵.2001-2005年吉林市1427例5岁以下儿童死亡分析[J].中国妇幼保健,2006,21(23)：3269-3271.
[49]杨东梅,王敏,王静竹.吉林省2005年5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2007,22(6)：781-782.
[50]张静,王丽,张焕丽,等.2000-2004年大连市5岁以下儿童死亡监测分析[J].中国妇幼保健,2006,21(6)：813-815.
[51]王辛荑,杨柳.沈阳市和平区2005-2006年5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(8)：1051-1052.
[52]钟新艳.2000-2005年太原市5岁以下儿童死亡情况分析[J].中国健康教育,2007,23(3)：214-215.
[53]刘清华,郝波.北京市1992年5岁以下儿童死亡重点死因——肺炎的分析[J].中华儿童保健杂志,1994,2(4)：277-278.
[54]胡凤贤.5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(1)：51-52.
[55]杜小云,郭小菊.勉县5岁以下儿童肺炎死亡十年对比分析[J].中国儿童保健杂志,1999,7(4)：248-249.
[56]杨珏玲,吴文献.陕西省妇幼卫生项目县婴幼儿生存水平分析[J].中华儿童保健杂志,1994,2(1)：32-34.
[57]王秀霞,马振侠,赵红梅.2000-2003年银川市5岁以下儿童死因分析与干预措施[J].宁夏医学院学报,2005,27(4)：300-301.
[58]刘奇杰,王巧玲,艾银凤.宁夏2003年5岁以下儿童死亡情况的监测[J].宁夏医学杂志,2004,26(8)：524-525.
[59]胡艾莉.西宁市1995年-2004年5岁以下儿童生命监测结果分析[J].青海医药杂志,2006,36(8)：81-83.
[60]乌丽娅,迪丽拜尔.新疆24个县妇女儿童健康状况初步评价[J].中国妇幼保健,2000,15(9)：582-584.
[61]张梅,侯莉.1998-2002年徐州市5岁以下儿童死亡分析[J].江苏卫生保健,2003(04).
[62]王菁,姜梅芳.苏州市1993-1999年5岁以下儿童死亡率及死因变化[J].中国优生优育,2002,13(1)：16-18.
[63]陈雅芬,蒋新液.无锡市1993-1997年5岁以下儿童死因分析[J].江苏卫生 保健,1999：101-102.
[64]汤书晶,王道良,李海龙.萧山市1996年0-4岁儿童死因分析[J].右江民族医学院学报,1998,20(72)：159-160.
[65]赵正言,郑凯航.浙江省O-4岁儿童生存水平的研究[J].中国妇幼保健,1996,11(5)：41-43.
[66]王佩茗.衢州市1995年五岁以下儿童死亡分析[J].浙江预防医学,1998,10(11)：678-681.
[67]陈少华,杨政坚.2004年三明市5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(2)：191-192.
[68]林小远.宁德市20002004年5岁以下儿童死亡监测分析[J].中国妇幼保健,2006,21(10)：1360-1361.
[69]宋杰.开封县1991-2000年5岁以下儿童死亡回顾分析[J].临床医学,2003,23(7)：25-26.
[70]刘智兰.信阳市1991-1995年5岁以下儿童死亡监测结果分析[J].河南医药信息,1997,5(8)：31-32.
[71]赵悦淑,刘安民.1989年河南省五岁以下儿童肺炎死亡调查[J].河南医科大学学报,1994,29(2)：160-162.
[72]熊忠贵,徐育松.湖北省1991-2001年5岁以下儿童死亡监测结果分析[J].中国预防医学杂志,2004,5(3)：179-181.
[73]张桂莲,姚秀英.双峰县O-4岁儿童肺炎监测结果分析[J].实用预防医学,1994,1(4)：235.
[74]欧明才,范明清.四川省50个县5岁以下儿童死亡及其有关因素分析[J].中国卫生事业管理,1995：36-39.
[75]夏凉,李顺年,肖发兴,等.武隆县妇幼卫生基础工作调查分析[J].中国初级卫生保健,1994,8(1)：30-32.
[76]甘慧君.秀山县1998-2001年5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2003,18(11)：702.
[77]董应芬.晋宁县2001-2005年5岁以下儿童死亡分析[J].中国妇幼保健,2007,22(13)：1783-1784.
[78]邝建明,蒋玉欢,邵小飞,等.1999-2003年5岁以下儿童死亡分析[J].中国妇幼保健,2005,20(21)：2855-2856.
[79]苏云,刘楚芹,温丽倩,等.肇庆市1998-2002年5岁以下儿童生命监测结果与干预措施[J].中国妇幼保健,2004,19(5)：69-70.
[80]赵慧贞,张红忠.珠海市1994-2002年5岁以下儿童死亡原因分析[J].中国 初级卫生保健,2003,17(11)：56-57.
[81]龚研宁,黄赏琼.5岁以下儿童死亡监测结果分析[J].中国妇幼保健,2004,19(9)：92-94.
[82]彭修道,韦茂国.海南省5岁以下儿童死亡基础调查研究[J].海南医学,1994,5(3)：182-186.
[83]曹佳莉.连云港市1997-1999年5岁以下儿童死亡原因的调查与分析[J].齐齐哈尔医学院学报,2001,22(11)：1320-1321.
[84]李明敏,许云娥.成都市1997年5岁以下儿童死因分析[J].四川省卫生管理干部学院学报,2000,19(1)：57-58.
[85]陈莹莹.1989年湖北省5岁以下儿童死亡回顾调查[J].湖北医科大学学报,1995,16(4)：373-375.
[86]聂忠华,邹芸芸.万安县5岁以下儿童死亡原因10年动态对比分析[J].江西医学院学报,2003,43(1)：121-123.
[87]魏红光,宋成义,张玉珍,等.甘肃省酒泉市肃州区2000-2004年新生儿死亡原因分析[J].卫生职业教育,2006,24(1)：112-113.
[88]梁颖.佛山市2000-2004年5岁以下儿童死亡分析[J].实用预防医学,2005,12(5)：1122-1123.
[89]王勇.广西儿童1999-2000年新生儿死亡监测情况分析[J].中国儿童保健杂志,2003,11(2)：128-129.
[90]刘龙池.隆回县0-6天新生儿死亡监测分析[J].医学理论与实践,2006,19(12)：1449-1450.
[91]谢艳玲,杨勤.湖北省5岁以下儿童肺炎死亡对策的研究[J].湖北预防医学杂志,2003,14(4)：17-19.
[92]蒋维国,蒋明仁.20年住院新生儿肺炎发病率及死亡率回顾分析[J].卫生职业教育,2005,23(23)：42.
[93]南京市儿童医院.2226新生儿肺炎住院病例分析[J].中华儿科杂志,1979,17(4)：216-219.
[94]杜梅.新生儿细菌性肺炎病原菌及耐药情况分析[J].中国误诊学杂志,2006,6(2)：257-258.
[95]桂林艳.新生儿肺炎病原菌及药敏分析[J].中国误诊学杂志,2007,7(5)：973-974.
[96]裘淑琴,葛传生,范一新,等.264例小儿细菌性肺炎的病原与临床[J].中华儿科杂志,1987,25(1)：10-11.
[97]马晓路,徐迎春,郑季彦,等.新生儿肺炎的病原及临床研究[J].浙江预防 医学,2005,17(1)：6-8.
[98]汪洁,罗新声.新生儿肺炎的病原体诊断[J].新生儿科杂志,1997,12(2)：62-63.
[99]尤灿,李先斌,黄彩芝,等.2164例新生儿肺炎需氧菌的分布及耐药性分析[J].中国感染控制杂志,2004,3(4)：345-347.
[100]李先斌,黄铭华,江训良,等.新生儿感染性肺炎病原菌分布及耐药性分析[J].实用预防医学,2003,10(5)：671-673.
[101]李坚,宋燕燕,李小晶,等.新生儿肺炎病原学及临床研究——附169例分析[J].中国妇幼保健,2004,19(10)：95-97.
[102]陈淑贞,蔡应木,杨启平.新生儿肺炎的病原学分析[J].中国微生态学杂志,2004,16(1)：45-46.
[103]林菁.新生儿肺炎细菌病原学及药敏结果分析[J].国际医药卫生导报,2001：46.
[104]曾令威.新生儿吸入性肺炎102例的病原菌分布及耐药性分析[J].广西医学,2007,29(6)：879-880.
[105]徐广范,于新环,胡锦华.儿童大叶性肺炎151例临床分析[J].牡丹江医学院学报,2007,28(1)：23.
[106]董枫,高鹏.儿童肺炎病原菌检测及药敏分析[J].大连医科大学学报,1998,20(1)：54-55.
[107]王惠萍,庞延凯.婴儿急性细菌性肺炎的临床流行病学分析[J].中华流行病学杂志,1994,15(4)：209-211.
[108]刘秀云,江载芳.小儿重症社区获得性肺炎31例病原分析[J].中国实用儿科杂志,2005,20(12)：749-750.
[109]毕晶,王美英.保定儿童医院2726例肺炎患儿多种病原检测与临床研究[J].中国医药导报,China medical herald,2006,3(32):60-61.
[110]张中馥,高国林,张建明,等.小儿肺炎318例痰培养细菌学分析[J].临床荟萃,2005,20(16)：938-939.
[111]上官芹,孙志良.儿童细菌性肺炎致病菌的耐药性分析[J].中国妇幼保健,2006,21(11)：1528-1529.
[112]张秀莲.不同月龄阶段婴儿肺炎病原体检测的临床意义[J].中原医刊,2006,33(18)：7-8.
[113]罗海燕,祝益民,李贵南,等.重症监护病房婴幼儿重症肺炎细菌学监测及临床分析[J].实用儿科临床杂志,2007,22(16)：1238-1240.
[114]龙元珠,刘建梅,吴爱民.小儿肺炎235例病原学临床分析[J].实用临床医 学(江西),2004,5(5)：84.
[115]李启先.儿童肺炎病原学分析[J].蚌埠医学院学报,2002,27(3)：245.
[116]杨英.660例小儿肺炎病原学和耐药性分析[J].现代医药卫生,2007,23(20)：3007-3009.
[117]罗蓉,黄英.重庆地区小儿肺炎下呼吸道分泌物病原耐药性分析[J].实用儿科临床杂志,2006,21(4)：224-225.
[118]张晓芬,陈全景,王春晖,等.十堰地区893例小儿细菌性肺炎病原学及药敏结果分析[J].郧阳医学院学报,2005,24(1)：37-38.
[119]考验,姚静婵,等.小儿肺炎细菌学检测及药敏试验[J].浙江预防医学,2001,13(11)：5-6.
[120]王峰,张竹馨,王玉屏.178例小儿肺炎分离菌分布和耐药性分析[J].中国微生态学杂志,2005,17(6)：458-459.
[121]刘纯义,张侃,陆必森,等.细菌性重症肺炎患儿的病原菌分析及治疗对策[J].小儿急救医学,2005,12(2)：115-117.
[122]宋玫,蔡桂丰.116例小儿肺炎分离菌的耐药性分析[J].中国微生态学杂志,2000,12(6)：351-352.
[123]李玉凤,祝俭平.516例小儿细菌性肺炎细菌学监测及分析[J].右江民族医学院学报,2007,29(4)：607-609.
[124]谢永武,宋玫,宝杰,等.小儿肺炎细菌学分类及抗生素耐药性分析[J].宁夏医学杂志,2006,28(6)：437-439.
[125]姜德昭,黄明春.武鸣县儿童肺炎病原学及正常咽部带菌的研究[J].广西医学院学报,1991,8(1)：1-4.
[126]Garenne M, Ronsmans C, Campbell H. The magnitude of mortality from acute respiratory infections in children under 5 years in developing countries. [J]. World Health Stat Q,1992,45(2-3):180-191.
[127]Williams B G, Gouws E, Boschi-Pinto C, et al. Estimates of world-wide distribution of child deaths from acute respiratory infections.[J]. Lancet Infect Dis,2002,2(1):25-32.
[128]Dowell S F, Kupronis B A, Zell E R, et al. Mortality from pneumonia in children in the United States,1939 through 1996.[J]. N Engl J Med,2000,342(19):1399-1407.
[129]Sazawal S, Black R E. Effect of pneumonia case management on mortality in neonates, infants, and preschool children:a meta-analysis of community-based trials.[J]. Lancet Infect Dis,2003,3(9):547-556.
[130]Wubbel L, Muniz L, Ahmed A, et al. Etiology and treatment of community-acquired pneumonia in ambulatory children. [J]. Pediatr Infect Dis J,1999,18(2):98-104.
[131]Rapkin R H. Bacteriologic and clinical findings in acute pneumonia of childhood.[J]. Clin Pediatr (Phila),1975,14(2):130-133.
[132]Ikeogu M O. Acute pneumonia in Zimbabwe:bacterial isolates by lung aspiration. [J]. Arch Dis Child,1988,63(10):1266-1267.
[133]Wang Y, Vuori-Holopainen E, Yang Y, et al. Relative frequency of Haemophilus influenzae type b pneumonia in Chinese children as evidenced by serology[Z].2002:21,271-277.
[134]Michelow I C, Lozano J, Olsen K, et al. Diagnosis of Streptococcus pneumoniae lower respiratory infection in hospitalized children by culture, polymerase chain reaction, serological testing, and urinary antigen detection.[J]. Clin Infect Dis,2002,34(1):E1-E11.
[135]Michelow I C, Olsen K, Lozano J, et al. Epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children.[J]. Pediatrics,2004,113(4):701-707.
[136]Nohynek H, Eskola J, Laine E, et al. The causes of hospital-treated acute lower respiratory tract infection in children.[J]. Am J Dis Child,1991,145(6):618-622.
[137]Ruuskanen 0, Nohynek H, Ziegler T, et al. Pneumonia in childhood:etiology and response to antimicrobial therapy.[J]. Eur J Clin Microbiol Infect Dis,1992, 11(3):217-223.
[138]Falade A G, Mulholland E K, Adegbola R A, et al. Bacterial isolates from blood and lung aspirate cultures in Gambian children with lobar pneumonia.[J]. Ann Trop Paediatr,1997,17(4):315-319.
[139]Wubbel L, Muniz L, Ahmed A, et al. Etiology and treatment of community-acquired pneumonia in ambulatory children.[J]. Pediatr Infect Dis J,1999,18(2): 98-104.
[140]Korppi M, Katila M L, Kalliokoski R, et al. Pneumococcal finding in a sample from upper airways does not indicate pneumococcal infection of lower airways. [J]. Scand J Infect Dis,1992,24(4):445-451.
[141]Nohynek H, Eskola J, Kleemola M, et al. Bacterial antibody assays in the diagnosis of acute lower respiratory tract infection in children.[J]. Pediatr Infect Dis J,1995,14(6):478-484.
[142]Falade A G, Mulholland E K, Adegbola R A, et al. Bacterial isolates from blood and lung aspirate cultures in Gambian children with lobar pneumonia. [J]. Ann Trop Paediatr,1997,17(4):315-319.
[143]Lankinen K S, Salo P, Rapola S, et al. Pneumococcal capsular antigen detection after enrichment culture:an alternative to culture methods in epidemiologic research.[J]. Am J Trop Med Hyg,1997,56(2):211-215.
[144]Lankinen K S, Ruutu P, Nohynek H, et al. Pneumococcal pneumonia diagnosis by demonstration of pneumolysin antibodies in precipitated immune complexes: a study in 350 Philippine children with acute lower respiratory infection.[J]. Scand J Infect Dis,1999,31(2):155-161.
[145]Faden H, Heimerl M, Goodman G, et al. New technique (the NOW test) for rapid detection of Streptococcus pneumoniae in the nasopharynx.[J]. J Clin Microbiol,2002,40(12):4748-4749.
[146]Dominguez J, Blanco S, Rodrigo C, et al. Usefulness of urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal pneumonia in children.[J]. J Clin Microbiol,2003,41(5):2161-2163.
[147]Lankinen K S, Rintamaki S, Syrjanen R, et al. Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens.[J]. J Microbiol Methods,2004,56(2):193-199.
[148]Neuman M I, Harper M B. Evaluation of a rapid urine antigen assay for the detection of invasive pneumococcal disease in children. [J]. Pediatrics,2003,112(6 Pt 1):1279-1282.
[149]von Graevenitz A, Rathbone R R. Branhamella catarrhalis in respiratory secretions:clinical correlation in 16 cases. [J]. South Med J,1981,74(9):1095-1096.
[150]Perera A, Wickramasinghe S, Perera B J, et al. Reliable and easy identification of bacterial acute respiratory infections of childhood.[J]. Ceylon Med J,1998, 43(4):200-205.
[151]Lehtomaki K, Leinonen M, Takala A, et al. Etiological diagnosis of pneumonia in military conscripts by combined use of bacterial culture and serological methods.[J]. Eur J Clin Microbiol Infect Dis,1988,7(3):348-354.
[152]Weber M W, Mulholland E K, Greenwood B M. Respiratory syncytial virus infection in tropical and developing countries.[J]. Trop Med Int Health,1998, 3(4):268-280.
[153]赵国昌,王晓红,朱启镕.上海地区儿童急性肺炎病原学和临床流行病学研究[J].2003：4.
[154]盛朝凯,刘岚,刘昌,等.儿童呼吸道感染时3种病原菌检出率及耐药性观察[J].临床儿科杂志,2004,22(2)：94-96.
[155]Mulholland K. Global burden of acute respiratory infections in children: implications for interventions.[J]. Pediatr Pulmonol,2003,36(6):469-474.
[156]Bratcher P E, Kim K H, Kang J H, et al. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization.[J]. Microbiology,2010,156(Pt 2):555-560.
[157]Park I H, Pritchard D G, Cartee R, et al. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae.[J]. J Clin Microbiol,2007,45(4):1225-1233.
[158]Llull D, Munoz R, Lopez R, et al. A single gene (tts) located outside the cap locus directs the formation of Streptococcus pneumoniae type 37 capsular polysaccharide. Type 37 pneumococci are natural, genetically binary strains.[J]. J Exp Med,1999,190(2):241-251.
[159]Cartee R T, Forsee W T, Jensen J W, et al. Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.[J]. J Biol Chem,2001,276(52):48831-48839.
[160]Dillard J P, Vandersea M W, Yother J. Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.[J]. J Exp Med,1995,181(3):973-983.
[161]Kolkman M A, van der Zeijst B A, Nuijten P J. Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae.[J]. J Biochem,1998,123(5):937-945.
[162]Garcia E, Llull D, Munoz R, et al. Current trends in capsular polysaccharide biosynthesis of streptococcus pneumoniae [J]. Res. Microbiol.,2000,151:429-435.
[163]Bentley S D, Aanensen D M, Mavroidi A, et al. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.[J]. PLoS Genet,2006, 2(3):e31.
[164]Jiang S, Wang L, Reeves P R. Molecular Characterization of Streptococcus pneumoniae Type 4,6B,8, and 18C Capsular Polysaccharide Gene Clusters[J]. Infection and Immunity,2001,69(3):1244-1255.
[165]Lalitha M K, Thomas K, Kumar R S, et al. Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera.[J]. J Clin Microbiol, 1999,37(1):263-265.
[166]Sorensen U B. Typing of pneumococci by using 12 pooled antisera.[J]. J Clin Microbiol,1993,31 (8):2097-2100.
[167]Brito D A, Ramirez M, de Lencastre H. Serotyping Streptococcus pneumoniae by Multiplex PCR[J]. J Clin Microbiol,2003,41(6):2378-2384.
[168]Gratten M, Montgomery J, Gerega G, et al. Multiple colonization of the upper respiratory tract of Papua New Guinea children with Haemophilus influenzae and Streptococcus pneumoniae.[J]. Southeast Asian J Trop Med Public Health, 1989,20(4):501-509.
[169]Lloyd-Evans N, O'Dempsey T J, Baldeh I, et al. Nasopharyngeal carriage of pneumococci in Gambian children and in their families.[J]. Pediatr Infect Dis J, 1996,15(10):866-871.
[170]Obaro S K, Adegbola R A, Banya W A, et al. Carriage of pneumococci after pneumococcal vaccination.[J]. Lancet,1996,348(9022):271-272.
[171]O'Brien K L, Millar E V, Zell E R, et al. Effect of pneumococcal conjugate vaccine on nasopharyngeal colonization among immunized and unimmunized children in a community-randomized trial.[J]. J Infect Dis, 2007,196(8):1211-1220.
[172]Maiden M C, Bygraves J A, Feil E, et al. Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms. [J]. Proc Natl Acad Sci U S A,1998,95(6):3140-3145.
[173]Enright M C, Spratt B G. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease.[J]. Microbiology,1998,144 (Pt 11):3049-3060.
[174]Pai R, Gertz R E, Beall B. Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates[J]. J Clin Microbiol, 2005,44(1):124-131.
[175]Lawrence E R, Griffiths D B, Martin S A, et al. Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups[JJ. J Clin Microbiol,2003,41(2):601-660.
[176]Morais L, Carvalho M G, Roca A, et al. Sequential multiplex PCR for identifying pneumococcal capsular serotypes from South-Saharan African clinical isolates.[J]. J Med Microbiol,2007,56(Pt 9):1181-1184.
[177]O'halloran D M, Cafferkey M T. Multiplex PCR for Identification of Seven Streptococcus pneumoniae Serotypes Targeted by a 7-Valent Conjugate Vaccine[J]. J Clin Microbiol,2005,43(7):3487-3490.
[178]Dias C A, Teixeira L C M, Carvalho M D G R, et al. Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children[J]. Journal of Medical Microbiology,2007,56:1185-1188.
[179]Rivera-Olivero I A, Blommaart M, Bogaert D, et al. Multiplex PCR reveals a high rate of nasopharyngeal pneumococcal 7-valent conjugate vaccine serotypes co-colonizing indigenous Warao children in Venezuela.[J]. J Med Microbiol, 2009,58(Pt 5):584-587.
[180]Turner P, Hinds J, Gould K, et al. Conventional techniques for detecting nasopharyngeal pneumococcal carriage significantly underestimate the serotype carriage[C]. Tel Aviv, Isreal:2010.
[181]Billal D S, Hotomi M, Suzumoto M, et al. Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.[J]. Eur J Pediatr,2008,167(4):401-407.
[182]Saha S K, Darmstadt G L, Baqui A H, et al. Identification of Serotype in Culture Negative Pneumococcal Meningitis Using Sequential Multiplex PCR: Implication for Surveillance and Vaccine Design[J]. PLOS one,2008,3(10): e3576.
[183]Butler J C, Breiman R F, Campbell J F, et al. Pneumococcal polysaccharide vaccine efficacy. An evaluation of current recommendations.[J]. JAMA,1993, 270(15):1826-1831.
[184]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[185]Whitney C G, Pilishvili T, Farley M M, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease:a matched case-control study.[J]. Lancet,2006,368(9546):1495-1502.
[186]Pelton SI, Huot H, Finkelstein JA, et al., Emergence of 19A as Virulent and Multidrug Resistant Pneumococcus in Massachusetts Following Universal Immunization of Infants With Pneumococcal Conjugate Vaccine[J]. Pediatr Infect Dis J.2007;26(6):468-472.
[187]Mahjoub-Messai F, Doit C, Koeck JL, et al., Population Snapshot of Streptococcus pneumoniae Serotype 19A Isolates before and after Introduction of Seven-Valent Pneumococcal Vaccination for French Children[J]. J Clin Microbiol,2009,47(3):837-840.
[188]Konradsen H B. Validation of serotyping of Streptococcus pneumoniae in Europe.[J]. Vaccine,2005,23(11):1368-1373.
[189]Finch R G, Phillips I. Serological grouping of streptococci by a slide coagglutination method.[J]. J Clin Pathol,1977,30(2):168-170.
[190]Rajalakshmi B, Kanungo R. Cost-effective method of serotyping streptococcus pneumoniae using staphylococcal co-agglutination. [J]. Indian J Med Microbiol,2001,19(4):197-200.
[191]MK Lalitha, R Pai, TJ John, et al. Serotyping of Streptococcus pneumoniae by agglutination assays:a cost-effective technique for developing countries[J]. Bull World Health Organ.,1996,74(4):387-390.
[192]Arai S, Konda T, Wada A. Use of antiserum-coated latex particles for serotyping streptococcus pneumoniae [J]. Microbiol Immunol.,2001,45(2):159-162.
[193]A S, K L M, J J T, et al. Modified latex agglutination test for rapid detection of Streptococcus pneumoniae and haemophilus influenzae in cerebrospinal fluid and direct serotyping of Streptococcus pneumoniae [J]. European journal of clinical microbiology & infectious diseases,1996,15(6):472-477.
[194]Slotved H C, Kaltoft M, Skovsted I C, et al. Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).[J]. J Clin Microbiol, 2004,42(6):2518-2522.
[195]Yu J, Lin J, Jr Benjamin W H, et al. Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies. [J]. J Clin Microbiol,2005,43(1):156-162.
[196]Lin J, Kaltoft M S, Brandao A P, et al. Validation of a multiplex pneumococcal serotyping assay with clinical samples.[J]. J Clin Microbiol,2006,44(2):383-388.
[197]Yu J, Carvalho M G, Beall B, et al. A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.[J]. J Med Microbiol,2008,57(Pt 2): 171-178.
[198]Batt S L, Charalambous B M, Mchugh T D, et al. Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.[J]. J Clin Microbiol,2005,43(6):2656-2661.
[199]Lawrence E R, Arias C A, Duke B, et al. Evaluation of Serotype Prediction by cpsA-cpsB Gene Polymorphism in Streptococcus pneumoniae [J]. J Clin Microbiol,2000,38(4):1319-1323.
[200]Moreno J, Hernandez E, Sanabria O, et al. Detection and Serotyping of Streptococcus pneumoniae from Nasopharyngeal Samples by PCR-Based Multiplex Assay[J]. J Clin Microbiol,2005,43(12):6152-6154.
[201]Billal D S, Hotomi M, Tasnim S, et al. Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction.[J]. ORL J Otorhinolaryngol Relat Spec,2006,68(3):135-138.
[202]Kong F, Wang W, Tao J, et al. A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy-or wzx-specific PCR[J]. Journal of Medical Microbiology,2005,54: 351-356.
[203]Kong F, Gilbert G L. Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes[J]. Journal of Medical Microbiology,2003,52:1047-1058.
[204]Kong F, Brown M, Sabananthan A, et al. Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes[J]. J Clin Microbiol,2006,44(5):1887-1891.
[205]Wang H, Kong F, Jelfs P, et al. Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.[J]. Appl Environ Microbiol,2004,70(3):1483-1486.
[206]Zhao Z, Kong F, Gilbert G L. Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.[J]. Clin Vaccine Immunol,2006,13(1):145-149.
[207]Wang Q, Wang M, Kong F, et al. Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.[J]. J Microbiol Methods,2007,68(1):128-136.
[1]Siber G R, Klugman K P, Makela P H. Pneumococcal vaccines:the impact of conjugate vaccine [M]. Washington, DC:ASM Press,2008.
[2]Park I H, Pritchard D G, Cartee R, et al. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae.[J]. J Clin Microbiol, 2007,45(4):1225-1233.
[3]Bratcher P E, Kim K H, Kang J H, et al. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization.[J]. Microbiology,2010,156(Pt 2):555-560.
[4]Butler J C, Breiman R F, Campbell J F, et al. Pneumococcal polysaccharide vaccine efficacy. An evaluation of current recommendations.[J]. JAMA,1993, 270(15):1826-1831.
[5]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[6]Black S, Shinefield H, Fireman B, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group.[J]. Pediatr Infect Dis J,2000, 19(3):187-195.
[7]Whitney C G, Pilishvili T, Farley M M, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease:a matched case-control study.[J]. Lancet,2006,368(9546):1495-1502.
[8]Emergence of 19A as Virulent and Multidrug Resistant Pneumococcus in Massachusetts Following Universal Immunization of Infants With Pneumococcal Conjugate Vaccine[J].
[9]Population Snapshot of Streptococcus pneumoniae Serotype 19A Isolates before and after Introduction of Seven-Valent Pneumococcal Vaccination for French Children[J].
[10]Konradsen H B. Validation of serotyping of Streptococcus pneumoniae in Europe.[J].Vaccine,2005,23(11):1368-1373.
[11]Finch R G, Phillips I. Serological grouping of streptococci by a slide coagglutination method.[J]. J Clin Pathol,1977,30(2):168-170.
[12]Rajalakshmi B, Kanungo R. Cost-effective method of serotyping streptococcus pneumoniae using staphylococcal co-agglutination.[J]. Indian J Med Microbiol, 2001,19(4):197-200.
[13]Mk L, R P, Tj J, et al. Serotyping of Streptococcus pneumoniae by agglutination assays:a cost-effective technique for developing countries[J]. Bull World Health Organ.,1996,74(4):387-390.
[14]Lalitha M K, Thomas K, Kumar R S, et al. Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera.[J]. J Clin Microbiol, 1999,37(1):263-265.
[15]Arai S, Konda T, Wada A. Use of antiserum-coated latex particles for serotyping streptococcus pneumoniae[J]. Microbiol Immunol.,2001,45(2):159-162.
[16]A S, K L M, J J T, et al. Modified latex agglutination test for rapid detection of Streptococcus pneumoniae and haemophilus influenzae in cerebrospinal fluid and direct serotyping of Streptococcus pneumoniae[J]. European journal of clinical microbiology & infectious diseases,1996,15(6) 472-477.
[17]Slotved H C, Kaltoft M, Skovsted I C, et al. Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).[J]. J Clin Microbiol,2004, 42(6):2518-2522.
[18]Yu J, Lin J, Jr Benjamin W H, et al. Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies.[J]. J Clin Microbiol, 2005,43(1):156-162.
[19]Lin J, Kaltoft M S, Brandao A P, et al. Validation of a multiplex pneumococcal serotyping assay with clinical samples.[J]. J Clin Microbiol,2006,44(2):383-388.
[20]Yu J, Carvalho M G, Beall B, et al. A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.[J]. J Med Microbiol,2008,57(Pt 2): 171-178.
[21]Maiden M C, Bygraves J A, Feil E, et al. Multilocus sequence typing:a portable approach to the identification of clones within populations of pathogenic microorganisms.[J]. Proc Natl Acad Sci U S A,1998,95(6):3140-3145.
[22]Enright M C, Spratt B G. A multilocus sequence typing scheme for Streptococcus pneumoniae:identification of clones associated with serious invasive disease.[J]. Microbiology,1998,144 (Pt 11):3049-3060.
[23]Batt S L, Charalambous B M, Mchugh T D, et al. Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.[J]. J Clin Microbiol,2005,43(6):2656-.2661.
[24]Lawrence E R, Arias C A, Duke B, et al. Evaluation of Serotype Prediction by cpsA-cpsB Gene Polymorphism in Streptococcus pneumoniae [J]. JOURNAL OF CLINICAL MICROBIOLOGY,2000,38(4):1319-1323.
[25]Lawrence E R, Griffiths D B, Martin S A, et al. Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups[J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2003,41(2):601-660.
[26]Brito D A, Ramirez M, de Lencastre H. Serotyping Streptococcus pneumoniae by Multiplex PCR[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2003,41(6): 2378-2384.
[27]Pai R, Gertz R E, Beall B. Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,44(1):124-131.
[28]Moreno J, Herna'ndez E, Sanabria O, et al. Detection and Serotyping of Streptococcus pneumoniae from Nasopharyngeal Samples by PCR-Based Multiplex Assay[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,43 (12):6152-6154.
[29]O'halloran D M, Cafferkey M T. Multiplex PCR for Identification of Seven Streptococcus pneumoniae Serotypes Targeted by a 7-Valent Conjugate Vaccine[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2005,43(7):3487-3490.
[30]Billal D S, Hotomi M, Tasnim S, et al. Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction.[J]. ORL J Otorhinolaryngol Relat Spec,2006,68(3):135-138.
[31]Billal D S, Hotomi M, Suzumoto M, et al. Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.[J]. Eur J Pediatr,2008,167(4):401-407.
[32]Dias C A, Teixeira L C M, Carvalho M D G R, et al. Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children[J]. Journal of Medical Microbiology,2007,56:1185-1188.
[33]Kong F, Wang W, Tao J, et al. A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy-or wzx-specific PCR[J]. Journal of Medical Microbiology,2005,54: 351-356.
[34]Kong F, Gilbert G L. Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes[J]. Journal of Medical Microbiology,2003,52:1047-1058.
[35]Kong F, Brown M, Sabananthan A, et al. Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes[J]. JOURNAL OF CLINICAL MICROBIOLOGY,2006,44(5):1887-1891.
[36]Wang H, Kong F, Jelfs P, et al. Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.[J]. Appl Environ Microbiol,2004,70(3):1483-1486.
[37]Zhao Z, Kong F, Gilbert G L. Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.[J]. Clin Vaccine Immunol,2006,13(1):145-149.
[38]Wang Q, Wang M, Kong F, et al. Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.[J]. J Microbiol Methods,2007,68(1):128-136.