深圳市健康人群流脑抗体水平调查及菌群变迁研究
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摘要
目的研究深圳市健康人群对流脑的易感性，为预防控制流脑提供依据；分析深圳市健康人群中脑膜炎奈瑟菌(以下简称Nm)的带菌率及性别、年龄分布，确定人群中Nm的感染情况；研究Nm的菌群结构和遗传关系，对流脑的流行趋势作出预测，分析Nm的耐药性，指导临床和预防用药。
方法采集健康人群血清样本800份，依据GB16884-1997的酶联免疫法(ELISA)，分别定量血清中A、C、Y和W135群Nm抗体的含量；采集1225份健康人的咽拭子，分离鉴定Nm，用血清学诊断和PCR法分群；用脉冲场凝胶电泳(PFGE)和多位点测序(MLST)进行分子分型；并用2006 CLSI／NCCLS推荐的纸片扩散法测定Nm的抗生素敏感性。
结果A、C、Y、W135群流脑抗体含量中位数分别是4.07，2.31，1.35，1.75μg／mL，健康人群C群和Y群流脑抗体水平的性别差异无统计学意义，A群和W135群流脑抗体水平有性别差异，各年龄组抗体水平的差异均有统计学意义；Nm带菌率为4.4％(55／1255)，其中B群49株(3.9％)，C群4株(0.3％)，29E 2株(0.2％)。在两次流脑疫情中各分离到1株B群Nm；用PFGE对57株Nm进行分型，可以得到52个PFGE型，按照85％相似度可以分为11个群(complex)，每群含有2～4株菌；对39株Nm进行MLST，发现36个不同的ST型和9个新的等位基因，58.3％(21／36)的ST型是首次发现，75.0％(27／36)的ST型是中国独有的；氨苄西林，头孢曲松，头孢噻肟，美洛培南，米诺环素，氯霉素，利福平敏感；所有菌株均对复方新诺明耐药，部分Nm对环丙沙星，萘啶酸耐药，有多重耐药现象。
结论45岁以上人群的A群和C群流脑抗体含量低，是流脑流行期的高危人群；人群中Y群和W135群流脑抗体水平较低，人群可能普遍易感；人群中主要携带B群Nm，PFGE显示菌株基因高度多态化，无优势菌株，B群Nm引起暴发的机会不大；MLST结果显示Nm基因的变化可能来自不同菌株间基因的水平传递，来自外源基因的可能性较小；Nm菌株对多数抗生素敏感，对磺胺类药物耐药情况严重。
Objective To learn the immune level of healthy people to N. meningitidis(Nm) in Shenzhen, China, to learn the population structure and genetic relationship among carried meningococci, in order to facilitate the prevention and control of meningococcal disease.
Methods ELISA was applied to measure the antibody against meningococcal serogroupA, C, Y, and W135(MenA, C, Y, and MenW135). Nm were isolated and identified from throat swabs culture according to GB16884-1997. The isolates were characterized by pulse field gel electrophoresis(PFGE) and multilocus sequence typing(MLST). The antibiotic susceptibility of Nm was tested by K-B assay.
Results The median concentration of anti-MenA, C, Y, and W135 were 4.07, 2.31, 1.35, 1.75μg/mL, respectively, there were statistical difference between different age groups. Nm were isolated from 55 swabs, giving an overall carriage rate of 4.4%, in which 34(3.9%) isolates were Group B, 4(0.3%) isolates were Group C, 2(0.2%) isolates were Group 29E. Moreover, 2 strains were isolated from patients and close contact. 52 PFGE patterns were obtained, which designated to 11 complexes according to 85% relateness(tolerance was 1.5%,optimization was 1.5%). 36 different STs were obtained by MLST, and 9 new allele number were assigned. 58.3%(21/36) STs were found for the first time, 75.0% (27/36) STs were unique to China. All isolates were sensitive to penicillin, ampicilin, ceftriaxone, cefotaxime, meropenem, minocycline, chloramphenicol succinate, rifampicin, while all isolates were resistant to compound sulfamethoxazole.
Conclusion People seniored to 35 years old were the high risk population who need more attention in meningococcal disease. The result demonstrated diversity of MenB, without a dominant PFGE type, which means a little chance to breakout. It was speculated that Nm isolates in Shenzhen comprised heterogeneous clones, which mostly derive from clones identified in local area.
方法采集健康人群血清样本800份，依据GB16884-1997的酶联免疫法(ELISA)，分别定量血清中A、C、Y和W135群Nm抗体的含量；采集1225份健康人的咽拭子，分离鉴定Nm，用血清学诊断和PCR法分群；用脉冲场凝胶电泳(PFGE)和多位点测序(MLST)进行分子分型；并用2006 CLSI／NCCLS推荐的纸片扩散法测定Nm的抗生素敏感性。
结果A、C、Y、W135群流脑抗体含量中位数分别是4.07，2.31，1.35，1.75μg／mL，健康人群C群和Y群流脑抗体水平的性别差异无统计学意义，A群和W135群流脑抗体水平有性别差异，各年龄组抗体水平的差异均有统计学意义；Nm带菌率为4.4％(55／1255)，其中B群49株(3.9％)，C群4株(0.3％)，29E 2株(0.2％)。在两次流脑疫情中各分离到1株B群Nm；用PFGE对57株Nm进行分型，可以得到52个PFGE型，按照85％相似度可以分为11个群(complex)，每群含有2～4株菌；对39株Nm进行MLST，发现36个不同的ST型和9个新的等位基因，58.3％(21／36)的ST型是首次发现，75.0％(27／36)的ST型是中国独有的；氨苄西林，头孢曲松，头孢噻肟，美洛培南，米诺环素，氯霉素，利福平敏感；所有菌株均对复方新诺明耐药，部分Nm对环丙沙星，萘啶酸耐药，有多重耐药现象。
结论45岁以上人群的A群和C群流脑抗体含量低，是流脑流行期的高危人群；人群中Y群和W135群流脑抗体水平较低，人群可能普遍易感；人群中主要携带B群Nm，PFGE显示菌株基因高度多态化，无优势菌株，B群Nm引起暴发的机会不大；MLST结果显示Nm基因的变化可能来自不同菌株间基因的水平传递，来自外源基因的可能性较小；Nm菌株对多数抗生素敏感，对磺胺类药物耐药情况严重。
Objective To learn the immune level of healthy people to N. meningitidis(Nm) in Shenzhen, China, to learn the population structure and genetic relationship among carried meningococci, in order to facilitate the prevention and control of meningococcal disease.
Methods ELISA was applied to measure the antibody against meningococcal serogroupA, C, Y, and W135(MenA, C, Y, and MenW135). Nm were isolated and identified from throat swabs culture according to GB16884-1997. The isolates were characterized by pulse field gel electrophoresis(PFGE) and multilocus sequence typing(MLST). The antibiotic susceptibility of Nm was tested by K-B assay.
Results The median concentration of anti-MenA, C, Y, and W135 were 4.07, 2.31, 1.35, 1.75μg/mL, respectively, there were statistical difference between different age groups. Nm were isolated from 55 swabs, giving an overall carriage rate of 4.4%, in which 34(3.9%) isolates were Group B, 4(0.3%) isolates were Group C, 2(0.2%) isolates were Group 29E. Moreover, 2 strains were isolated from patients and close contact. 52 PFGE patterns were obtained, which designated to 11 complexes according to 85% relateness(tolerance was 1.5%,optimization was 1.5%). 36 different STs were obtained by MLST, and 9 new allele number were assigned. 58.3%(21/36) STs were found for the first time, 75.0% (27/36) STs were unique to China. All isolates were sensitive to penicillin, ampicilin, ceftriaxone, cefotaxime, meropenem, minocycline, chloramphenicol succinate, rifampicin, while all isolates were resistant to compound sulfamethoxazole.
Conclusion People seniored to 35 years old were the high risk population who need more attention in meningococcal disease. The result demonstrated diversity of MenB, without a dominant PFGE type, which means a little chance to breakout. It was speculated that Nm isolates in Shenzhen comprised heterogeneous clones, which mostly derive from clones identified in local area.
引文
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15 Schouls, Leo M. van der Ende, Arie. Damen, et, al. Multiple-locus variable-number tandem repeat analysis of Neisseria meningitidis yields groupings similar to those obtained by multilocus sequence typing. Journal of Clinical Microbiology, 2006,44:1509-18.
16 田国忠，邵祝军，李马超等．可变数目串联重复序列技术用于中国C群脑膜炎奈瑟菌基因分型的研究．中华流行病学杂志，2006，27：595-599．
17 朱沛轩，胡绪敬，徐丽．B群脑膜炎奈瑟氏菌PCR-RFLP分型及其流行病学意义．中华微生物学和免疫学杂志，1993，13：148-51．
18 朱沛轩．限制性酶切片段长度多态性分析法在细菌分子流行病学研究中的应用．国外医学：流行病学．传染病学分册，1991，18：252-56．
19 Swaminathan B, Matar GM, Reeves MW, et, al. Molecular subtyping of Neisseria meningitidis serogroup B :comparison of five methods. J Clin Microbiol, 1996,34.
20 Gouleing JN, Stanley J, Olver W, et, al. Independent subsets of amplified fragments from the genome of Neisseria meningitidis identify the same invasive clones of ET 37 and ET5. J. Med. Microbiol, 2003,52:151-154.
21 Goulding JN, Hookey JV, Stanley J, et, al. Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease. J. Clin. Microbiol, 2000,38:4580-4585.
22 Ivano de Filippis, Ann Carolina Paulo Vicente. Multilocus sequence typing and repetitive element-based polymerase chain reaction analysis of Neisseria meningitidis isolates in Brazil reveal the emergence of iI new sequence types genetically related to the ST-32 and ST-41/44 complexes and high prevalence of strains related to hypervirulent lineages. Diagnostic Microbiology and Infectious Disease, 2005,53:161-167.
23 Woods CR, Koruth T, Estabrook MM, et, al. Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction genotyping. J Infect Dis, 1996,174:760-767.
24 Mimi Healy, Joe Huong, Traci Bittner, et, al. Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR. JOURNAL OF CLINICAL MICROBIOLOGY, 2005,43.
25 Newcombe J, Cartwright KAV, Palmer WH, et, al. PCR-SSCP of clinical specimens for non-culture-based sub-typing of the meningococcus in clinical specimens. Proceding of the Tenth International Pathogenic Neisseria Conference. Naltimore. USA, 1996,498-499.
26 Newcombe J, Dyer S, Blackman L, et, al. PCR-single-strand confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens. J. Clin. Microbiol, 1997,35:1809-1812.
27 Georgina Tzanakaki, Maria Tsosia, Vasiliki Vlachou, et, al. Evaluation of non-culture diagnosis og invasive meningococcal disease by polymerase chain reation(PgR). FEMS Immumology and Medical Microbiology, 2003,39:31-36.
28 张力，邵祝军，徐丽．鉴别脑膜炎奈瑟菌A、B、C、Y、W135多重聚合酶链反应诊断方法．中华流行病学杂志，2006，27：399-401．
29 张力，胡绪敬．PCR用于流脑诊断的国内外研究进展．疾病监测，2003，18：271-75．
30 Martin C. J. Maiden, Jane A. Bugaves, Edward Feil, et, al. Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Microbiology, 1998,3:3140-3145.
31 De' sire', e E. Bennett, Mary T. Multilocus restriction typing: a tool for Neisseria meningitidis strain discrimination. Journal of Medical Microbiology, 2003,52:781-787.
32 Hideyuki Takahashi, Toshiro Kuroki. Characterization of Neisseria meningitidis isolates collected from 1974 to 2003 in Japan by multilocus sequence typing. Journal of Medical Microbiology, 2004,53:657-662.
33 Christopher B. Sullivan, MatthewA. Diggle, and Stuart C. Clarke. Multilocus Sequence Typing. Molecular Biotechnology, 2005,29.
34 朱沛轩，胡绪敬，徐丽．A群脑膜炎奈瑟菌porA基因限制性片段长度多态性分型研究．中华流行病学杂志．中华流行病学杂志，1992，13：7-10．
35 Urwin R, Russell JE, Thompson EA, et, al. Distribution of surface protein variantsamong hyperinvasive meningococci: implications for vaccine design. Infect Immun, 2004,72:5955-62.
36 Claudio T. Sacchi, Anne M. Whitney, Michael W. Reeves, et ,al. Sequence Diversity of Neisseria meningitidis 16S rRNA Genes and Use of 16S rRNA Gene Sequencing as a Molecular Subtyping Tool. Journal of Clinical Microbiology, 2002,40:4520-4527.
37 Diggle MA, Clarke SC. Genotypic characterization of Neisseria meningitidis using Pyrosequencing. Mol. Biotechnol, 2004,28:139-145.
38 Aria Paula S. Lemos, Claudio T. Sacchi, Maria V. Paiva, et, al. Genetic relationships among serogrouop B serotype 4 Neisseria meningitidis strains. Rev. Inst. Med. trop. S. Paulo, 2001,43:119-124.
39 Maria Lucia Cecconi Tondella, Claudio Tavares Sacchi, Bianca Cruz Neves. Ribotyping as an Additional Molecular Marker for Studying Neisseria meningitidis Serogroup B Epidemic Strains. Journal of Clinical Microbiology, 1994,32:2745-2748.
40 Swiderek, H, H. Claus, M. Frosch, U. Vogel. Evaluation of custom-made DNA microarrays for multilocus sequence typing of Neisseria meningitidis. J. Med. Microbiol, 2005,295:39-45.
41 Claus, H, M. C. J. Maiden, D. J. Wilson. Genetic analysis of meningococci carried by children and young adults. J. Infect. Dis, 2005,191:1263-71.
2 Zhujun Shao, Wei Li, Jun Ren, et, al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet, 2006,367:419-423.
3 刘美真，杜志明，张莉萍，等．广东省2004年流脑监测结果分析．华南预防医学，2005，31：18-20．
4 胡绪敬．流行性脑脊髓膜炎的流行病学监测与预防．中国计划免疫，2001，7：300-303．
5 Levent Doganci, Mehmet Baysallar, Mehmet All Saracli, et, al. Neisseria meningitidis W135, Turkey. Emerging Infectious Diseases, 2004,10:936-937.
6 吕静，刘力，孙净，等．湖北省2000～2005年流行性脑脊髓膜炎监测分析．公共卫生与预防医学，2006，17：41-44．
7 毕诚，徐爱玲，王美芳．江苏省流脑流行前期人群抗体水平和带菌率调查．江苏预防医学，2002，13：6-9．
8 袁珩，罗隆泽，祝小平，等．四川省2001年流脑监测结果分析．疾病监测，2002，17：218-220．
9 刘美真，杜志明，陈经雕，等．广东省2002～2003年流脑监测结果分析．华南预防医学，2004，30：31-32．
10 李漫时，宋立志，肖作奎，等．山东省部分地区2003年健康人群A群C群流行性脑脊髓膜炎抗体水平监测．中国计划免疫，2005，11：195-197．
11 原灵，郭维植，董新平，等．福建省2005年流脑带菌率及免疫水平调查．海峡预防医学杂志，2006，12：28-29．
12 伏晓庆，尹建雯，高援，等．云南省2005年健康人群流脑带菌调查结果分析．实用预防医学，2006，13：341-342．
13 杨建英，杨枢敏，张广业，等．甘肃省2005年流脑发病高峰季节人群带菌监测结果分析．卫生职业教育，2005，23：82-83．
14 崔步云，张秋香，任泽萍，等．山西省健康人群A群流脑抗体及A群流脑多糖菌苗免疫效果血清观察．疾病监测，2004，19：333-335．
15 张宏，杭惠，须勤燕．苏州市流脑流行病学分析．现代预防医学，2006，33：1864-1866．
16 李美霞，周秀珍，张瑞丹，等．广州市正常人群流行性脑脊髓膜炎抗体水平调查．中华流行病学杂志，2002，23：93．
17 刘美真，杨华可，廖国东，等．2005年广东省健康人群流脑免疫水平和带菌状况调查．中国卫生检验杂志，2007，17：325-327．
18 Conterno LO, Silva Filho CR, R 竒geberg JU, et, al. Conjugate vaccines for preventing meningococcal C meningitis and septicaemia. Evid.-Based Child Health, 2007,2.
19 孙银燕，胡绪敬．脑膜炎奈瑟氏菌ACPS-BOMPC偶联物的免疫原性、安全性和稳定性．中华微生物学和免疫学杂志，2002，22：170．
20 MacLennan JM, HeathPT, DeeksJJ. et al. safety, immunogenicity, and induction of immunologic memory by a serogroup C meningococcal conjugate caccine in infants. JMAA, 2000,283:2795.
21 胡绪敬．流脑流行的监测与预防．中国公共卫生，2005，20：638-640．
1 Georgina Tzanakaki, Maria Tsosia, Vasiliki Vlachou, et, al. Evaluation of non-culture diagnosis og invasive meningococcal disease by polymerase chain reation(PCR). FEMS Immumology and Medical Microbiology, 2003, 39:31-36.
2 Tanja Popovic, Gloria Ajello, and Richard Facklam. Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. World Health Organization, 1999:1-22.
3 MaidenMCJ, Stuart J, M. Carriage of serogroupC meningococci 1 Year after meningococcal conjugate polysaccharide vaccination. Lancet, 2002,359:1829.
4 Persistence of W135 Neisseria meningitidis carriage in returning Hajj Pilgrims: risk for early and late transmission to household contacts. Emerging Inf Dis, 2003,9:123.
5 Caugant DA, Hoiby EA, Rosenqvist E, et, al. Transmission of Neisseria meningitidis among asymptomatic military recruits and antibody analysis. Epidemiol Infect, 1999,109:241-253.
6 Stevens DS. Uncloaking the meningococcus: dynamics of carriage and disease. Lancet, 1999,353.
7 吕静，刘力，孙净，等．湖北省2000～2005年流行性脑脊髓膜炎监测分析．公共卫生与预防医学，2006，17：41-44．
8 毕诚，徐爱玲，王美芳．江苏省流脑流行前期人群抗体水平和带菌率调查．江苏预防医学，2002，13：6-9．
9 袁珩，罗隆泽，祝小平，等．四川省2001年流脑监测结果分析．疾病监测，2002，17：218-220．
10 刘美真，杨华可，廖国东，等．2005年广东省健康人群流脑免疫水平和带菌状况调查．中国卫生检验杂志，2007，17：325-327．
11 原灵，郭维植，董新平，等．福建省2005年流脑带菌率及免疫水平调查．海峡预防医学杂志，2006，12：28-29．
12 游旅，田克诚，姚光海，等．2005年贵州省健康人群和病例密切接触者流脑病原菌带菌状况调查．疾病监测，2006，21：120-122．
13 伏晓庆，尹建雯，高援，等．云南省2005年健康人群流脑带菌调查结果分析．实用预防医学，2006，13：341-342．
14 杨建英，杨枢敏，张广业，等．甘肃省2005年流脑发病高峰季节人群带菌监测结果分析．卫生职业教育，2005，23：82-83．
15 刘敏，孙立华，门兆杰，等．辽宁省流脑流行病学分析．疾病监测，2003，18：173-174．
16 王联君．流脑健康人群带菌率调查及控制措施探讨．中国公共卫生管理，2006，22：155-157．
17 I. D. Pavlopouloul, G. L. Daikos2, H. Alexandrou, et, al. Carriage of Neisseria meningitidis by Greek children: risk factors and strain characteristics. European Society of Clinical Microbiology and Infectious Diseases, 2004,10.
18 邓志爱，张欣强，周秀珍，等．广州市外来建筑工人流脑病原菌带菌状况调查．疾病监测，2005，20：404-406．
19 Simmons G, Jones N, Calder L. Equivalence of ceftriaxone and rifampicin in eliminating nasopharyngeal carriage of serogroup B Neisseria meningitidis. The Journal Of Antimicrobial Chemotherapy, 2000,45:909-11.
1 Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed- field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol, 1995,33.
2 Zhujun Shao, Wei Li, Jun Ren, et, al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet, 2006,367:419-423.
3 Tanja popovic, Susanna Schmink, Nancy A. Rosenstein, et al. Evaluation of Pulsed-Field Gel Electrophoresis in Epidemiological Investigations of Meningococcal Disease Outbreaks Caused by Neisseria meningitidis Serogroup C. Journal of Clinical Microbiology, 2001,39:75-85.
4 Ulrich Vogel, Giovanna Morell,Kerstin Zurth, et al. Necessity of Molecular Techniques To Distinguish between Neisseria meningitidis Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts Journal of Clinical Microbiology. Journal of Clinical Microbiology, 1998,36:2465-2470.
5 Raymond S. W. Tsang, Lori Kiefer, Dennis K. S. Law, et, al. Outbreak of Serogroup C Meningococcal Disease Caused by a Variant of Neisseria meningitidis Serotype 2a ET-15 in a Community of Men Who Have Sex with Men. Journal of Clinical Microbiology, 2003,41:4411-4414.
6 L. Arreaza, S. Berron, S.Fernandez, et, al. Investigation for a more virulent variant among the C:2b:Pl. 2, 5 Spanish meningococcal epidemid strains by molecular epidemiology. J. Med. Microbiol, 2000,49:1079-1084.
7 Zhujun Shao, Wei Li, Jun Ren, et, al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet, 2006,367:419-23.
1 Martin C. J. Maiden, Jane A. Bygraves, Edward Feil, et, al. Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci, 1998,95.
2 Zhujun Shao, Wei Li, Jun Ren, et, al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet, 2006,367:419-423.
3 Chien-Shun Chiou, Jui-Cheng Liao, Tsai-Ling Liao, et, al. Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan. BMC Infectious Diseases, 2006,6:1-8.
4 Hideyuki Takahashi, Toshiro Kuroki. Characterization of Neisseria meningitidis isolates collected from 1974 to 2003 in Japan by multilocus sequence typing. Journal of Medical Microbiology, 2004,53:657-662.
1 Clinical and Laboratory Standards Institute. Performance standards for antimierobial susceptibility testing; sixteenth informational supplement. CLSI document M100-S16, 2006,26:1-78.
2 李德好，胡绪敬，徐丽，等．脑膜炎奈瑟氏菌对常用抗菌药物敏感性监测．疾病监测，1996，11：405．
3 徐丽，邵祝军，李马超，等．脑膜炎奈瑟菌对12种抗菌药物体外敏感性检测．中国计划免疫，2006，12：53-55．
4 吕静，刘力，孙净，等．湖北省2000～2005年流行性脑脊髓膜炎监测分析．公共卫生与预防医学，2006，17：41-44．
5 原灵，郭维植，董新平，等．福建省2005年流脑带菌率及免疫水平调查．海峡预防医学杂志，2006，12：28-29．
6 陈民钧．美国临床实验室标准化研究所2005年版有关药敏试验标准化更新要点．中华检验医学杂志，2005，28：449-451．
1 胡绪敬．流脑流行的监测与预防．中国公共卫生，2005，20：638-640．
2 Claus, H, Maiden, M. C, Maag, R, et, al. Many carried meningococci lack the genes, required for capsule synthesis and transport. Microbiology, 2002,148:1813-1819.
3 Caugant DA, Bovre K, Gaustad P, et,al. Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. J Gen Microbiol, 1986,132:641-652.
4 Davis E. Yakubu, Fariborz J. R. Abadi, T. Hguh Pennington. Molecular typing method for Neisseria meningitides. J. Med.Microbiol, 1999,48.
5 G. tzanakaki, R. Urwin, M. Musilek, et, al. Phenotypic and Genotypic Approaches to Characterization of Isolates of Neisseria meningitidis from Patients and Their Close Family Contacts. Journal of Clinical Microbiology, 2001,39:1235-1240.
6 Tanja popovic, Susanna Schmink, Nancy A. Rosenstein, et al. Evaluation of Pulsed-Field Gel Electrophoresis in Epidemiological Investigations of Meningococcal Disease Outbreaks Caused by Neisseria meningitidis Serogroup C. Journal of Clinical Microbiology, 2001,39:75-85.
7 Ulrich Vogel, Giovanna Morel1, Kerstin Zurth, et al. Necessity of Molecular Techniques To Distinguish between Neisseria meningitidis Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts Journal of Clinical Microbiology. Journal of Clinical Microbiology, 1998,36:2465-2470.
8 Raymond S. W. Tsang, Lori Kiefer, Dennis K. S. Law, et, al. Outbreak of Serogroup C Meningococcal Disease Caused by a Variant of Neisseria meningitidis Serotype 2a ET-15 in a Community of Men Who Have Sex with Men. Journal of Clinical Microbiology, 2003,41:4411-4414.
9 L. Arreaza, S.Berron, S.Fernandez, et, al. Investigation for a more virulent variant among the C:2b:Pl. 2,5 Spanish meningococcal epidemid strains by molecular epidemiology. J.Med. Microbiol, 2000,49:1079-1084.
10 Zhujun Shao, Wei Li, Jun Ren, et, al. Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China. Lancet, 2006,367:419-23.
11 Susanna Schmink, Michael W. Reeves, Brian Plikaytis, et, al. Random Amplified Polymorphic DNAAssay as a Rapid Tool in Screening for Neisseria meningitidis Serogroup C Isolates of Electrophoretic Type 24. 2001,39:1622-1625.
12 Aldert Bart, Ilse G.A. Schuurmen, Mark Achtman, et, al. Randomly Amplified Polymorphic DNAGenotyping of Serogroup A Meningococci Yields Results Similar to Those Obtained by Multilocus Enzyme Electrophoresis and Reveals New Genotypes. Journal of Clinical Microbiology, 1998,36:1746-1749.
13 Siamak P. Yazdankhah, Bj?rn-Arne Lindstedt, Dominique A. Use of Variable-Number Tandem Repeats To Examine Genetic Diversity of Neisseria meningitidis American Society for Microbiology. American Society for Microbiology, 2005,43:1669-1706.
14 Liao JC. Li CC. Chiou CS. Use of a multilocus variable-number tandem repeat analysis method for molecular subtyping and phylogenetic analysis of Neisseria meningitidis isolates. BMC Microbiology, 2006,6:44.
15 Schouls, Leo M. van der Ende, Arie. Damen, et, al. Multiple-locus variable-number tandem repeat analysis of Neisseria meningitidis yields groupings similar to those obtained by multilocus sequence typing. Journal of Clinical Microbiology, 2006,44:1509-18.
16 田国忠，邵祝军，李马超等．可变数目串联重复序列技术用于中国C群脑膜炎奈瑟菌基因分型的研究．中华流行病学杂志，2006，27：595-599．
17 朱沛轩，胡绪敬，徐丽．B群脑膜炎奈瑟氏菌PCR-RFLP分型及其流行病学意义．中华微生物学和免疫学杂志，1993，13：148-51．
18 朱沛轩．限制性酶切片段长度多态性分析法在细菌分子流行病学研究中的应用．国外医学：流行病学．传染病学分册，1991，18：252-56．
19 Swaminathan B, Matar GM, Reeves MW, et, al. Molecular subtyping of Neisseria meningitidis serogroup B :comparison of five methods. J Clin Microbiol, 1996,34.
20 Gouleing JN, Stanley J, Olver W, et, al. Independent subsets of amplified fragments from the genome of Neisseria meningitidis identify the same invasive clones of ET 37 and ET5. J. Med. Microbiol, 2003,52:151-154.
21 Goulding JN, Hookey JV, Stanley J, et, al. Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease. J. Clin. Microbiol, 2000,38:4580-4585.
22 Ivano de Filippis, Ann Carolina Paulo Vicente. Multilocus sequence typing and repetitive element-based polymerase chain reaction analysis of Neisseria meningitidis isolates in Brazil reveal the emergence of iI new sequence types genetically related to the ST-32 and ST-41/44 complexes and high prevalence of strains related to hypervirulent lineages. Diagnostic Microbiology and Infectious Disease, 2005,53:161-167.
23 Woods CR, Koruth T, Estabrook MM, et, al. Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction genotyping. J Infect Dis, 1996,174:760-767.
24 Mimi Healy, Joe Huong, Traci Bittner, et, al. Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR. JOURNAL OF CLINICAL MICROBIOLOGY, 2005,43.
25 Newcombe J, Cartwright KAV, Palmer WH, et, al. PCR-SSCP of clinical specimens for non-culture-based sub-typing of the meningococcus in clinical specimens. Proceding of the Tenth International Pathogenic Neisseria Conference. Naltimore. USA, 1996,498-499.
26 Newcombe J, Dyer S, Blackman L, et, al. PCR-single-strand confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens. J. Clin. Microbiol, 1997,35:1809-1812.
27 Georgina Tzanakaki, Maria Tsosia, Vasiliki Vlachou, et, al. Evaluation of non-culture diagnosis og invasive meningococcal disease by polymerase chain reation(PgR). FEMS Immumology and Medical Microbiology, 2003,39:31-36.
28 张力，邵祝军，徐丽．鉴别脑膜炎奈瑟菌A、B、C、Y、W135多重聚合酶链反应诊断方法．中华流行病学杂志，2006，27：399-401．
29 张力，胡绪敬．PCR用于流脑诊断的国内外研究进展．疾病监测，2003，18：271-75．
30 Martin C. J. Maiden, Jane A. Bugaves, Edward Feil, et, al. Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Microbiology, 1998,3:3140-3145.
31 De' sire', e E. Bennett, Mary T. Multilocus restriction typing: a tool for Neisseria meningitidis strain discrimination. Journal of Medical Microbiology, 2003,52:781-787.
32 Hideyuki Takahashi, Toshiro Kuroki. Characterization of Neisseria meningitidis isolates collected from 1974 to 2003 in Japan by multilocus sequence typing. Journal of Medical Microbiology, 2004,53:657-662.
33 Christopher B. Sullivan, MatthewA. Diggle, and Stuart C. Clarke. Multilocus Sequence Typing. Molecular Biotechnology, 2005,29.
34 朱沛轩，胡绪敬，徐丽．A群脑膜炎奈瑟菌porA基因限制性片段长度多态性分型研究．中华流行病学杂志．中华流行病学杂志，1992，13：7-10．
35 Urwin R, Russell JE, Thompson EA, et, al. Distribution of surface protein variantsamong hyperinvasive meningococci: implications for vaccine design. Infect Immun, 2004,72:5955-62.
36 Claudio T. Sacchi, Anne M. Whitney, Michael W. Reeves, et ,al. Sequence Diversity of Neisseria meningitidis 16S rRNA Genes and Use of 16S rRNA Gene Sequencing as a Molecular Subtyping Tool. Journal of Clinical Microbiology, 2002,40:4520-4527.
37 Diggle MA, Clarke SC. Genotypic characterization of Neisseria meningitidis using Pyrosequencing. Mol. Biotechnol, 2004,28:139-145.
38 Aria Paula S. Lemos, Claudio T. Sacchi, Maria V. Paiva, et, al. Genetic relationships among serogrouop B serotype 4 Neisseria meningitidis strains. Rev. Inst. Med. trop. S. Paulo, 2001,43:119-124.
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