Genetic structure of Neisseria meningitidis serogroup C epidemic strains in South Brazil

Sacchi, Claudio Tavares; Tondella, Maria Lúcia Cecconi; Gorla, Maria Cecília Outeiro; Lemos, Ana Paula Silva de; Melles, Carmo Elias A.; Paiva, Maria Vaneide de; Rodrigues, Dauri Santos; Andrade, Antonio Joaquim F.; Ribeiro, Marta Osório; Sperb, Alethea

doi:10.1590/S0036-46651995000400001

Abstracts

In the present study we report the results of an analysis, based on serotyping, multilocus enzyme electrophoresis (MEE), and ribotyping of N. meningitidis serogroup C strains isolated from patients with meningococcal disease (MD) in Rio Grande do Sul (RS) and Santa Catarina (SC) States, Brazil, as the Center of Epidemiology Control of Ministry of Health detected an increasing of MD cases due to this serogroup in the last two years (1992-1993). We have demonstrated that the MD due to N.meningitidis serogroup C strains in RS and SC States occurring in the last 4 years were caused mainly by one clone of strains (ET 40), with isolates indistinguishable by serogroup, serotype, subtype and even by ribotyping. One small number of cases that were not due to an ET 40 strains, represent closely related clones that probably are new lineages generated from the ET 40 clone referred as ET 11A complex. We have also analyzed N.meningitidis serogroup C strains isolated in the greater São Paulo in 1976 as representative of the first post epidemic year in that region. The ribotyping method, as well as MEE, could provide useful information about the clonal characteristics of those isolates and also of strains isolated in south Brazil. The strains from 1976 have more similarity with the actual endemic than epidemic strains, by the ribotyping, sulfonamide sensitivity, and MEE results. In conclusion, serotyping with monoclonal antibodies (C:2b:P1.3), MEE (ET 11 and ET 11A complex), and ribotyping by using ClaI restriction enzyme (Rb2), were useful to characterize these epidemic strains of N.meningitidis related to the increased incidence of MD in different States of south Brazil. It is mostly probable that these N.meningitidis serogroup C strains have poor or no genetic corelation with 1971-1975 epidemic serogroup C strains. The genetic similarity of members of the ET 11 and ET 11A complex were confirmed by the ribotyping method by using three restriction endonucleases.

N. meningitidis C; Ribotyping; Multilocus Enzyme Electrophoresis

No presente estudo, nós reportamos os resultados de uma análise, baseada na sorotipagem, multilocus enzimático (MEE) e ribotipagem de N. meningitidis sorogrupo C isoladas de paciente com doença meningocócica no Rio Grande do Sul (RS) e Santa Catarina (SC), onde o Centro de Controle Epidemiológico do Ministério da Saúde detectou um aumento do número de casos de doença meningocócica (DM) devido a este sorogrupo nos últimos 2 anos (1992-1993). Nós demonstramos que a DM devido a cepas de N. meningitidis sorogrupo C no RS e SC que ocorreram nos últimos 4 anos foi devido principalmente por um clone (ET 40), com isolados indistinguíveis por sorogrupo, sorotipo, subtipo e até por ribotipagem. Um pequeno número de casos que não foram devidos a cepas do ET 40 representaram um grupo geneticamente relacionado, que provavelmente é uma nova linhagem gerada do clone ET 40, referido como complexo ET 11 A. Nós também analisamos cepas de N. meningitidis sorogrupo C isoladas na grande São Paulo em 1976 como um grupo representativo do primeiro ano pós-epidêmico na região. A ribotipagem, bem como MEE, puderam fornecer informações sobre as características clonais das cepas isoladas no período pós-epidêmico e também no Sul do Brasil. As cepas de 1976 possuem mais similaridades com as cepas endêmicas atuais do que com as cepas epidêmicas (1992-1993) por ribotipagem, sensibilidade a sulfonamida e MEE. Em conclusão, sorotipagem com anticorpos monoclonais (C:2b:P1.3), MEE (complexo ET11 e ET11A) e ribotipagem usando a enzima de restrição ClaI, foram úteis em caracterizar estas cepas epidêmicas de N. meningitidis relacionadas com o aumento da incidência da DM em diferentes estados do sul do Brasil. É muito provável que estas cepas de N. meningitidis sorogrupo C possuam pouca ou nenhuma correlação genética com as cepas epidêmicas sorogrupo C de 1971-1975. A similaridade genética dos membros do complexo ET 11 e ET 11A foram confirmadas por ribotipagem usando-se 3 enzimas de restrição.

MICROBIOLOGY

Genetic structure of Neisseria meningitidis serogroup C epidemic strains in South Brazil

Estrutura genética de cepas epidêmicas de Neisseria meningitidis sorogrupo C do Sul do Brasil

Claudio Tavares Sacchi^I; Maria Lúcia Cecconi Tondella^I; Maria Cecília Outeiro Gorla^I; Ana Paula Silva de Lemos^I; Carmo Elias A. Melles^I; Maria Vaneide de Paiva^I; Dauri Santos Rodrigues^II; Antonio Joaquim F. Andrade^III; Marta Osório Ribeiro^IV; Alethea Sperb^V

^IBacteriology Division, Adolfo Lutz Institute, São Paulo, Brazil

^IIBacteriology Division, Central Laboratory of Santa Catarina State, Brazil

^IIICenter of Epidemiological Surveillance of Parana State, Brazil

^IVBacteriology Division, Central Laboratory of Rio Grande do Sul State, Brazil

^VCenter for Epidemiological Surveillance of Rio Grande do Sul State, Brazil

^{Correspondence to} Correspondence to: Claudio T. Sacchi Bacteriology Division, Adolfo Lutz Institute Av. Dr. Arnaldo 351 01246-902 São Paulo, Brazil Phone: 55.11.8510111. Fax:55.11.8513505 Electronic mail address: sacchi@usp.br

SUMMARY

In the present study we report the results of an analysis, based on serotyping, multilocus enzyme electrophoresis (MEE), and ribotyping of N. meningitidis serogroup C strains isolated from patients with meningococcal disease (MD) in Rio Grande do Sul (RS) and Santa Catarina (SC) States, Brazil, as the Center of Epidemiology Control of Ministry of Health detected an increasing of MD cases due to this serogroup in the last two years (1992-1993). We have demonstrated that the MD due to N.meningitidis serogroup C strains in RS and SC States occurring in the last 4 years were caused mainly by one clone of strains (ET 40), with isolates indistinguishable by serogroup, serotype, subtype and even by ribotyping. One small number of cases that were not due to an ET 40 strains, represent closely related clones that probably are new lineages generated from the ET 40 clone referred as ET 11A complex. We have also analyzed N.meningitidis serogroup C strains isolated in the greater São Paulo in 1976 as representative of the first post epidemic year in that region. The ribotyping method, as well as MEE, could provide useful information about the clonal characteristics of those isolates and also of strains isolated in south Brazil. The strains from 1976 have more similarity with the actual endemic than epidemic strains, by the ribotyping, sulfonamide sensitivity, and MEE results. In conclusion, serotyping with monoclonal antibodies (C:2b:P1.3), MEE (ET 11 and ET 11A complex), and ribotyping by using ClaI restriction enzyme (Rb2), were useful to characterize these epidemic strains of N.meningitidis related to the increased incidence of MD in different States of south Brazil. It is mostly probable that these N.meningitidis serogroup C strains have poor or no genetic corelation with 1971-1975 epidemic serogroup C strains. The genetic similarity of members of the ET 11 and ET 11A complex were confirmed by the ribotyping method by using three restriction endonucleases.

Keywords:N. meningitidis C; Ribotyping; Multilocus Enzyme Electrophoresis.

RESUMO

No presente estudo, nós reportamos os resultados de uma análise, baseada na sorotipagem, multilocus enzimático (MEE) e ribotipagem de N. meningitidis sorogrupo C isoladas de paciente com doença meningocócica no Rio Grande do Sul (RS) e Santa Catarina (SC), onde o Centro de Controle Epidemiológico do Ministério da Saúde detectou um aumento do número de casos de doença meningocócica (DM) devido a este sorogrupo nos últimos 2 anos (1992-1993). Nós demonstramos que a DM devido a cepas de N. meningitidis sorogrupo C no RS e SC que ocorreram nos últimos 4 anos foi devido principalmente por um clone (ET 40), com isolados indistinguíveis por sorogrupo, sorotipo, subtipo e até por ribotipagem. Um pequeno número de casos que não foram devidos a cepas do ET 40 representaram um grupo geneticamente relacionado, que provavelmente é uma nova linhagem gerada do clone ET 40, referido como complexo ET 11 A. Nós também analisamos cepas de N. meningitidis sorogrupo C isoladas na grande São Paulo em 1976 como um grupo representativo do primeiro ano pós-epidêmico na região. A ribotipagem, bem como MEE, puderam fornecer informações sobre as características clonais das cepas isoladas no período pós-epidêmico e também no Sul do Brasil. As cepas de 1976 possuem mais similaridades com as cepas endêmicas atuais do que com as cepas epidêmicas (1992-1993) por ribotipagem, sensibilidade a sulfonamida e MEE. Em conclusão, sorotipagem com anticorpos monoclonais (C:2b:P1.3), MEE (complexo ET11 e ET11A) e ribotipagem usando a enzima de restrição ClaI, foram úteis em caracterizar estas cepas epidêmicas de N. meningitidis relacionadas com o aumento da incidência da DM em diferentes estados do sul do Brasil. É muito provável que estas cepas de N. meningitidis sorogrupo C possuam pouca ou nenhuma correlação genética com as cepas epidêmicas sorogrupo C de 1971-1975. A similaridade genética dos membros do complexo ET 11 e ET 11A foram confirmadas por ribotipagem usando-se 3 enzimas de restrição.

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Recebido para publicação em 01/02/1995.

Aceito para publicação em 28/06/1995.

1. ACHTMAN, M. & PLUSCHKE, G. - Clonal analysis of descent and virulence among selected Escherichia coli. Ann. Rev. Microbiol., 40: 185-210, 1986.
2. ALTWEGG, M. & MAYER, L.W. - Bacterial molecular epidemiology based on a non-radioactive probe complementary to ribosomal RNA. Res. Microbiol., 140: 325-333, 1989.
3. APICELLA, M.A. - Lipopolysaccharide-derived serotype polysaccharides from Neisseria meningitidis group B. J. infect. Dis., 140: 62-72, 1979.
4. ASHTON, F.E.; RYAN, J.A.; BORCZYK, A.L. et al. - Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococccal group C disease in Canada. J. clin. Microbiol., 29: 2489-2493, 1991.
5. BRENNER, D.J.; MCWHORTER, A.C; KNUTSON, J.K.L. & STEIGERWALT, A.G. - Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. clin. Microbiol., 15: 1133-1140, 1982.
6. CAUGANT, D.A.; BOL, P.; HOIBY, E.A.; ZANEN, H.C. & FROHOLM, L.O. - Clones of serogroup B Neisseria meningitidis causing systemic disease in the Netherlands, 1958-1986. J. infect. Dis., 162: 867-874, 1990.
7. CAUGANT, D.A.; FROHOLM, L.O.; BOVRE, K. et al. - Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. Proc. nat. Acad. Sci. (Wash.), 83: 4927-4931, 1986.
8. CAUGANT, D.A.; MOCCA, L.F.; FRASCH, C.E. et al. - Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, outer membrane protein pattern. J. Bact., 169: 2781-2792, 1987.
9. CAUGANT, D.A.; FROHOLM, L.O.; SELANDER, R.K. & BOVRE, K. - Sulfonamide resistence in Neisseria meningitidis isolates of clones of the ET-5 complex. APMIS., 97: 425-428, 1989.
10. CAUGANT, D.A.; FROHOLM, L.O.; SACCHI, C.T. & SELANDER, R.K. - Genetic structure and epidemiology of serogroup B Neisseria meningitidis. In: INTERNATIONAL PATHOGENIC Neisseria CONFERENCE, 7, Berlin, 1990. Proceedings. Berlin, Walter de Gruyter, 1991. p.37-42.
11. CRUZ, C.; PAVEZ, G.; AGUILAR, E. et al. - Serotype-specific outbreak of group B meningococcal disease in Iquique, Chile. Epidem. Infect., 105: 119-126, 1990.
12. FRASCH, C.E. & CHAPMANS, S.S. - Classification of Neisseria meningitidis group B into distinct serotypes. I. Serological typing by a microbactericidal method. Infect. Immun., 5: 98-102, 1972a.
13. FRASCH, C.E. & CHAPMANS, S.S. - Classification of Neisseria meningitidis group B into distinct serotypes. II. Extraction of type-specific antigens for serotyping by preciptin techniques. Infect. Immun., 6: 127-133, 1972b.
14. IRINO, K.; GRIMONT, F.; CASIN, I.; GRIMONT, P.A.D. & THE BRAZILIAN PURPURIC FEVER STUDY GROUP - rRNA gene restriction patterns of Haemophilus influenzas biogroup aegyptius strains associated with Brazilian purpuric fever. J. clin. Microbiol., 26: 1535-1538, 1988.
15. JORDENS, J.Z. & PENNINGTON, T.H. - Characterization of N. meningitidis isolates by ribosomal RNA gene restriction patterns and restriction endonuclease digestions of chromosomal DNA. Epidem. Infect., 107: 253-262, 1991.
16. LIMA Jr., G. - Discours de Monsieur le Ministre de la Santé de Brezil. Seminaire international sur les vaccinations en Afrique. Lyon, Fondation Merieux, 1974. p. 124-128.
17. MACHADO, M. - L'epidemie de meningite cerebro-spinale au Brezil. Med. Hyg., 34: 483-485, 1976.
18. MANDRELL, R.E. & ZOLLINGER, W.D. - Lipopolysaccharide serotyping of Neisseria meningitidis by hemaglutination inhibition. Infect. Immun., 16: 471-475, 1977.
19. MORAIS, J.S.; MUNFORD, R.S.; RISI, J.B.; ANTEZANA, E. & FELDMAN, R.A. - Epidemic disease due to serogroup C Neisseria meningitidis in Săo Paulo, Brazil. J. infect. Dis., 129: 568-571, 1974.
20. MUNFORD, R.S.; PATTON, C.M. & GORMAN, G.W. - Epidemiologic studies of serotype antigens common to groups B and C Neisseria meningitidis. J. infect. Dis., 131: 286-290, 1975.
21. NI, H.; KNIGHT, A.I.; CARTWRIGHT, K.A.V. & MCFADDEN, J.J. - Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes. Epidem. Infect., 109-227-239, 1992.
22. OCHMAN, H.; WHITTAM, T.S.; CAUGANT, D.A. & SELANDER, R.K. - Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella. J. gen. Microbiol., 129: 2715-2726, 1983.
23. OLYHOEK, T.; CROWE, B.A. & ACHTMAN, M. - Clonal population structure of Neisseria meningitidis serogroup A isolated from epidemics and pandemics between 1915 and 1983. Rev. infect. Dis., 9: 665-692, 1987.
24. PELTOLA, H. - Meningococcal disease: still with us. Rev. infect. Dis., 5: 71-91, 1983.
25. SACCHI, C.T.; ZANELLA, R.C.; CAUGANT, D.A. et al. - Emergence of a new clone of serogroup C Neisseria meningitidis in Săo Paulo, Brazil. J. clin. Microbiol., 30: 1282-1286, 1992.
26. SACCHI, C.T.; PESSOA, L.L.; RAMOS, S.R. et al. - Ongoing group B Neisseria meningitidis epidemic in Săo Paulo, Brazil, due to increased prevalence of a single clone of the ET-5 complex. J. clin. Microbiol., 30: 1734-1738, 1992.
27. SACCHI, C.T.; TONDELLA, M.L.C.; LEMOS, A.P.S. et al. - Characterization of epidemic Neisseria meningitidis serogroup C strains in several Brazilian States. J. clin. Microbiol., 32: 1783-1787, 1994.
28. SCHWARTZ, B.; MOORE, P.S. & BROOME, C.V. - Global epidemiology of meningococcal disease. Clin. Microbiol. Rev.,2: S118-S124, 1989.
29. SELANDER, R.K.; CAUGANT, D.A.; OCHMAN, H. et al. - Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. environ. Microbiol., 51: 873-874, 1986.
30. STRATHDEE, C.A.; TYLER, S.D.; RYAN, J.A.; JOHNSON, W.M. & ASHTON, F.E. - Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada. J. clin. Microbiol., 31: 2506-2508, 1993.
31. TONDELLA, M.L.C.; SACCHI, C.T. & NEVES, B.C. - Ribotyping as an additional molecular marker of Neisseria meningitidis serogroup B epidemic strains. J. clin. Microbiol., 32: 2745-2748, 1994.
32. WOODS, T.C.; HELSEL, L.O.; SWAMINATHAN, B. et al. - Characterization of N. meningitidis serogroup C by multilocus enzyme electrophoresis and ribosomal DNA restriction profiles (Ribotyping). J. clin. Microbiol., 30: 132-137, 1992.

Correspondence to:

Claudio T. Sacchi

Bacteriology Division, Adolfo Lutz Institute

Av. Dr. Arnaldo 351

01246-902 São Paulo, Brazil

Phone: 55.11.8510111. Fax:55.11.8513505

Electronic mail address:

sacchi@usp.br

Publication Dates

Publication in this collection
21 Sept 2006
Date of issue
Aug 1995

History

Accepted
28 June 1995
Received
01 Feb 1995

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ACHTMAN, M. & PLUSCHKE, G. - Clonal analysis of descent and virulence among selected Escherichia coli. Ann. Rev. Microbiol., 40: 185-210, 1986.

[2] 2. ALTWEGG, M. & MAYER, L.W. - Bacterial molecular epidemiology based on a non-radioactive probe complementary to ribosomal RNA. Res. Microbiol., 140: 325-333, 1989.

[3] 3. APICELLA, M.A. - Lipopolysaccharide-derived serotype polysaccharides from Neisseria meningitidis group B. J. infect. Dis., 140: 62-72, 1979.

[4] 4. ASHTON, F.E.; RYAN, J.A.; BORCZYK, A.L. et al. - Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococccal group C disease in Canada. J. clin. Microbiol., 29: 2489-2493, 1991.

[5] 5. BRENNER, D.J.; MCWHORTER, A.C; KNUTSON, J.K.L. & STEIGERWALT, A.G. - Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. clin. Microbiol., 15: 1133-1140, 1982.

[6] 6. CAUGANT, D.A.; BOL, P.; HOIBY, E.A.; ZANEN, H.C. & FROHOLM, L.O. - Clones of serogroup B Neisseria meningitidis causing systemic disease in the Netherlands, 1958-1986. J. infect. Dis., 162: 867-874, 1990.

[7] 7. CAUGANT, D.A.; FROHOLM, L.O.; BOVRE, K. et al. - Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. Proc. nat. Acad. Sci. (Wash.), 83: 4927-4931, 1986.

[8] 8. CAUGANT, D.A.; MOCCA, L.F.; FRASCH, C.E. et al. - Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, outer membrane protein pattern. J. Bact., 169: 2781-2792, 1987.

[9] 9. CAUGANT, D.A.; FROHOLM, L.O.; SELANDER, R.K. & BOVRE, K. - Sulfonamide resistence in Neisseria meningitidis isolates of clones of the ET-5 complex. APMIS., 97: 425-428, 1989.

[10] 10. CAUGANT, D.A.; FROHOLM, L.O.; SACCHI, C.T. & SELANDER, R.K. - Genetic structure and epidemiology of serogroup B Neisseria meningitidis. In: INTERNATIONAL PATHOGENIC Neisseria CONFERENCE, 7, Berlin, 1990. Proceedings. Berlin, Walter de Gruyter, 1991. p.37-42.

[11] 11. CRUZ, C.; PAVEZ, G.; AGUILAR, E. et al. - Serotype-specific outbreak of group B meningococcal disease in Iquique, Chile. Epidem. Infect., 105: 119-126, 1990.

[12] 12. FRASCH, C.E. & CHAPMANS, S.S. - Classification of Neisseria meningitidis group B into distinct serotypes. I. Serological typing by a microbactericidal method. Infect. Immun., 5: 98-102, 1972a.

[13] 13. FRASCH, C.E. & CHAPMANS, S.S. - Classification of Neisseria meningitidis group B into distinct serotypes. II. Extraction of type-specific antigens for serotyping by preciptin techniques. Infect. Immun., 6: 127-133, 1972b.

[14] 14. IRINO, K.; GRIMONT, F.; CASIN, I.; GRIMONT, P.A.D. & THE BRAZILIAN PURPURIC FEVER STUDY GROUP - rRNA gene restriction patterns of Haemophilus influenzas biogroup aegyptius strains associated with Brazilian purpuric fever. J. clin. Microbiol., 26: 1535-1538, 1988.

[15] 15. JORDENS, J.Z. & PENNINGTON, T.H. - Characterization of N. meningitidis isolates by ribosomal RNA gene restriction patterns and restriction endonuclease digestions of chromosomal DNA. Epidem. Infect., 107: 253-262, 1991.

[16] 16. LIMA Jr., G. - Discours de Monsieur le Ministre de la Santé de Brezil. Seminaire international sur les vaccinations en Afrique. Lyon, Fondation Merieux, 1974. p. 124-128.

[17] 17. MACHADO, M. - L'epidemie de meningite cerebro-spinale au Brezil. Med. Hyg., 34: 483-485, 1976.

[18] 18. MANDRELL, R.E. & ZOLLINGER, W.D. - Lipopolysaccharide serotyping of Neisseria meningitidis by hemaglutination inhibition. Infect. Immun., 16: 471-475, 1977.

[19] 19. MORAIS, J.S.; MUNFORD, R.S.; RISI, J.B.; ANTEZANA, E. & FELDMAN, R.A. - Epidemic disease due to serogroup C Neisseria meningitidis in Săo Paulo, Brazil. J. infect. Dis., 129: 568-571, 1974.

[20] 20. MUNFORD, R.S.; PATTON, C.M. & GORMAN, G.W. - Epidemiologic studies of serotype antigens common to groups B and C Neisseria meningitidis. J. infect. Dis., 131: 286-290, 1975.

[21] 21. NI, H.; KNIGHT, A.I.; CARTWRIGHT, K.A.V. & MCFADDEN, J.J. - Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes. Epidem. Infect., 109-227-239, 1992.

[22] 22. OCHMAN, H.; WHITTAM, T.S.; CAUGANT, D.A. & SELANDER, R.K. - Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella. J. gen. Microbiol., 129: 2715-2726, 1983.

[23] 23. OLYHOEK, T.; CROWE, B.A. & ACHTMAN, M. - Clonal population structure of Neisseria meningitidis serogroup A isolated from epidemics and pandemics between 1915 and 1983. Rev. infect. Dis., 9: 665-692, 1987.

[24] 24. PELTOLA, H. - Meningococcal disease: still with us. Rev. infect. Dis., 5: 71-91, 1983.

[25] 25. SACCHI, C.T.; ZANELLA, R.C.; CAUGANT, D.A. et al. - Emergence of a new clone of serogroup C Neisseria meningitidis in Săo Paulo, Brazil. J. clin. Microbiol., 30: 1282-1286, 1992.

[26] 26. SACCHI, C.T.; PESSOA, L.L.; RAMOS, S.R. et al. - Ongoing group B Neisseria meningitidis epidemic in Săo Paulo, Brazil, due to increased prevalence of a single clone of the ET-5 complex. J. clin. Microbiol., 30: 1734-1738, 1992.

[27] 27. SACCHI, C.T.; TONDELLA, M.L.C.; LEMOS, A.P.S. et al. - Characterization of epidemic Neisseria meningitidis serogroup C strains in several Brazilian States. J. clin. Microbiol., 32: 1783-1787, 1994.

[28] 28. SCHWARTZ, B.; MOORE, P.S. & BROOME, C.V. - Global epidemiology of meningococcal disease. Clin. Microbiol. Rev.,2: S118-S124, 1989.

[29] 29. SELANDER, R.K.; CAUGANT, D.A.; OCHMAN, H. et al. - Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. environ. Microbiol., 51: 873-874, 1986.

[30] 30. STRATHDEE, C.A.; TYLER, S.D.; RYAN, J.A.; JOHNSON, W.M. & ASHTON, F.E. - Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada. J. clin. Microbiol., 31: 2506-2508, 1993.

[31] 31. TONDELLA, M.L.C.; SACCHI, C.T. & NEVES, B.C. - Ribotyping as an additional molecular marker of Neisseria meningitidis serogroup B epidemic strains. J. clin. Microbiol., 32: 2745-2748, 1994.

[32] 32. WOODS, T.C.; HELSEL, L.O.; SWAMINATHAN, B. et al. - Characterization of N. meningitidis serogroup C by multilocus enzyme electrophoresis and ribosomal DNA restriction profiles (Ribotyping). J. clin. Microbiol., 30: 132-137, 1992.

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