Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

MENINGOCOCCAL DISEASE CAUSED BY Neisseria meningitidis SEROGROUP B SEROTYPE 4 IN SÃO PAULO, BRAZIL, 1990 TO 1996

Claudio Tavares Sacchi (¹), Ana Paula Silva de Lemos (¹), Maria Claudia C. Camargo (²),

Anne M. Whitney (³), Carmo Elias A. Melles (¹), Claude André Solari (⁴),

Carl E. Frasch (⁵) & Leonard W. Mayer (²).

SUMMARY

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.²¹ A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Keywords:Neisseria meningitidis: Ribotyping.

INTRODUCTION

Meningococcal disease (MD) remains a significant cau-se of mortality and morbidity throughout the world^1,18. During the last 10 years different methods have been used to study the epidemiology of MD^9,15,21,23. Neisseria meningitidis can be classified into serogroups, serotypes, and serosubtypes on the basis of antigenic differences in their capsular poly-saccharides, class 2 or 3 outer membrane proteins (OMPs), and class 1 OMPs, respectively¹². Antibodies against both proteins are bactericidal, making serotyping results useful not only for epidemiologic tracing of meningococcal disease, but also for identifying potential vaccine components^5,11.

Another technique used for epidemiological tracing of N. meningitidis is multilocus enzyme electrophoresis typing (MEE), or electrophoretic typing (ET typing). ET typing examines electrophoretic differences in enzymes required for growth of the bacteria. ET 5 complex strains, a group of approximately 22 closely related ETs, have become a significant element of meningococcal populations causing outbreaks in different parts of the world⁷.

Beginning in 1988, the incidence of MD in greater São Paulo began to surpass the upper 95% confidence limit of an 8-year average incidence (from 1979 to 1986), indicating a new epidemic. This epidemic, which extended to 1997, was different from previous epidemics in that it was caused by N. meningitidis serogroup B strains²¹.

In a previous study, N. meningitidis serogroup B isolates from MD cases recovered from 1989 to 1990 were characterized by serotyping and MEE^8,21. The increased incidence of MD was paralleled by an increased prevalence of B:4:P1.15 strains. By MEE, the B:4:P1.15 strains belong to ET 5, a member of ET 5 complex. Restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping), was adapted to further characterize those N. meningitidis ET 5 epidemic strains^22,25. The ribotyping analysis, using ClaI endonuclease, showed a single rRNA gene restriction pattern (Rb1) for all ET 5 strains, the MEE and ribotyping results were 100% concordant.

In an attempt to control the epidemic of serogroup B disease in greater São Paulo a Cuban produced outer-membrane-protein-based serogroup B meningococcal vaccine (VAMENGOC B/C), was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990¹⁷. The Cuban vaccine is based on OMPs from a Cuban epidemic B:4:P1.15 N. meningitidis strain, combined with serogroup C meningococcal capsular polysaccharide¹⁷.

During the period of 1991 to 1996, the incidence of MD in greater São Paulo area did not decrease and most of the serogrouped cultures (61%), were by serogroup B. In 1996 the incidence of MD reached 8.2 per 100,000 inhabitants per year, the highest incidence of meningococcal disease since the large epidemics in the 1970's caused by serogroups A or C.

The goal of this paper was to determine if changes have occurred in the B:4:P1.15 epidemic strains isolated since the vaccination trial as measured by serotyping and ribotyping that could explain the continued increase of MD cases by serogroup B.

MATERIALS AND METHODS

Meningococcal strains. All 834 N. meningitidis serogroup B strains recovered from blood or cerebrospinal fluid samples from patients with systemic disease in greater São Paulo from 1990 to 1996 received at the National Reference Center were serotyped. These strains represent 51% of all MD cases known to be due to serogroup B in greater São Paulo during this period. We randomly selected 438 strains to be analyzed by ribotyping. Eighty five serogroup B strains previously used for the 1990 direct comparison of MEE and ribotyping were included²⁵. Other strains are described in Table 1. Serotyping and MEE, of strains isolated from 1977 to 1990, and the equivalence between ET 5 and Rb1 are described elsewhere^8,21,25. From 1990, only serotyping and ribotyping were used in order to analyze large numbers of isolates.

Strain G:S:Sba ET/Rb typeb Characteristic Reference H44/76 B:15:P1.7,16 ET5/Rb1 Epidemic in Norway 13 H355/75 B:15:P1.15 ET5/Rb1 Epidemic in Norway 12 CU385/83 B:4:P1.15 ET5/Rb1 Epidemic in Cuba 29 BB393 B:15:P1.3 ND/Rb1 Epidemic in Chile 25 N.44/89 B:4:P1.15 ET5/Rb1 Epidemic in Brazil 16 N.150/88 B:4:P1.15 ET5/Rb1 Epidemic in Brazil 16 N.1002/90 C:2b:P1.3 ET49/Rb2 Epidemic in Brazil 23 N.610/93 B:4:P1.15 ND/Rb1 Epidemic in Brazil this work N.300/94 B:4:P1.9 ND/Rb3 Epidemic in Brazil this work H276 (870227) B:4:P1.10 ND/Rb1 Reference strain for serosubtype P1.10 24 Z4008 (882066) B:4:P1.9 ND/Rb8 Reference strain for serosubtype P1.4 24 M981 B:4:- ND/Rb2 Reference strain for serotype 4 12

Strain	G:S:Sba	ET/Rb typeb	Characteristic	Reference
H44/76	B:15:P1.7,16	ET5/Rb1	Epidemic in Norway	13
H355/75	B:15:P1.15	ET5/Rb1	Epidemic in Norway	12
CU385/83	B:4:P1.15	ET5/Rb1	Epidemic in Cuba	29
BB393	B:15:P1.3	ND/Rb1	Epidemic in Chile	25
N.44/89	B:4:P1.15	ET5/Rb1	Epidemic in Brazil	16
N.150/88	B:4:P1.15	ET5/Rb1	Epidemic in Brazil	16
N.1002/90	C:2b:P1.3	ET49/Rb2	Epidemic in Brazil	23
N.610/93	B:4:P1.15	ND/Rb1	Epidemic in Brazil	this work
N.300/94	B:4:P1.9	ND/Rb3	Epidemic in Brazil	this work
H276 (870227)	B:4:P1.10	ND/Rb1	Reference strain for serosubtype P1.10	24
Z4008 (882066)	B:4:P1.9	ND/Rb8	Reference strain for serosubtype P1.4	24
M981	B:4:-	ND/Rb2	Reference strain for serotype 4	12

Table 1

Control strains of N. meningitidis

Not all strains were typed by MEE/ribotyped. In reference 13 it was shown that ET 5 is 100% equivalent to Rb1.

a G:S:Sb = serogroup:serotype:serosubtype

b ND = Non determinated

Serogrouping and Serotyping. All N. meningitidis strains were serogrouped as described previously²¹. Serotyping and serosubtyping were done by dot-blotting of whole cell suspensions²⁸. MAbs for serotypes 8, 15, 16 and serosubtypes P1.2, P1.3, P1.16 were provided by Dr. W. D. Zollinger. MAbs for serosubtype P1.9 was provided by Dr. J. T. Poolman, and MAbs for serotypes 2a, 2b, 4, 17 and serosubtypes P1.4, P1.7, P1.23, and P1.15 were produced at Adolfo Lutz Institute²⁰.

Ribotyping. Cells were harvested from tryptic soy agar (Difco) containing 1% (v/v) horse serum into 10 ml of 0.1 M NaCl, 0.05 M EDTA, and 0.05 M Tris, pH 8. Cells were lysed with sodium dodecyl-sulphate (SDS), and high molecular weight DNA was extracted as previously described⁴. Approximately 5mg of DNA was digested with ClaI endonuclease and blotted²⁵. DNA from the pKK3535² plasmid was labeled with digoxigenin-11-dUTP and detected with Lumi-Phos 530 (Genius 1 Kit Boehringer Mannheim Biochemical, Indianapolis), Haemophilus influenzae biogroup aegyptius strain 3031 EcoRI fragments were used as molecular weigh marker in each gel¹⁴. H.44/76 DNA digested with ClaI was also included as a control of Rb1 pattern in each gel.

Source of epidemiological data. The data pertaining to meningococcal disease among people residing in the greater São Paulo were obtained from the System for Epidemiological Vigilance Alexandre Vranjac, State of São Paulo where they were consolidated and analyzed.

RESULTS

Incidence and serotype prevalence. The incidence of MD, including meningitis and septicemia, in greater São Paulo has increased since 1988 and reached its highest level in 1996 with 8.2 cases per 100,000 inhabitants (Table 2). The incidence of serogroup B MD also reached its highest level at the same year with 1.8 cases per 100,000 inhabitants. Table 3 shows the number of strains of the predominant serogroup B serotypes and serosubtypes found in the greater São Paulo area since 1990. Serotype B:4 strains have been prevalent since 1987 and have accounted over 80% of all cases since 1990 (Fig. 1). The relative frequency of serotype 4 increased from 20% during 1977-1981 to 80% in 1988, just before the 1989 epidemic. During 1990 to 1996 the frequency of serosubtype P1.15 increased from 68% to 85%, and the combination of serotype 4 and serosubtype P1.15 (B:4:P1.15) was the most frequent. After 1990 the proportion of B:4:P1.15 strains increased gradually from 60% to 75% of all group B isolates in 1996. Over the years (1990 to 1996), the proportion of NT strains was almost constant at 10%, and that of nt strains decrease from 30% to 7% in 1996.

Figure 1 - Prevalence of Neisseria meningitidis B:4 (thick line), B:4:P1.15 (thin line), and Rb1 (column) in greater São Paulo during the period of 1977 to 1996.

Year MD* Serogroup B CI Cases CI Cases Laboratory collection (%) Studied by Serotyping Number of strains studied by Ribotypi ng % of studied strains related to % of ET 5 strains % of Rb 1 strains % of B:4:P1:15 strains Cases Collection 1996 8.2 1337 1.8 290 87(30) 81 28 93 ND 81 75 1995 8.0 1278 1.7 272 130(48) 62 23 48 nd 77 74 1994 6.8 1088 1.5 245 100(41) 50 20 50 nd 76 62 1993 5.3 842 1.4 224 126(56) 58 26 46 nd 79 70 1992 5.3 833 1.3 215 99(47) 50 23 50 nd 86 76 1991 6.0 926 1.1 167 104(62) 52 31 50 nd 65 52 1990 6.2 941 1.4 215 188(87) 85 39 45 80 79 66 Total 7245 1628 834(51) 438 27 52 78 68

Year	MD*		Serogroup B
	CI	Cases	CI	Cases	Laboratory collection (%) Studied by Serotyping	Number of strains studied by Ribotypi ng	% of studied strains related to		% of ET 5 strains	% of Rb 1 strains	% of B:4:P1:15 strains
							Cases	Collection
1996	8.2	1337	1.8	290	87(30)	81	28	93	ND	81	75
1995	8.0	1278	1.7	272	130(48)	62	23	48	nd	77	74
1994	6.8	1088	1.5	245	100(41)	50	20	50	nd	76	62
1993	5.3	842	1.4	224	126(56)	58	26	46	nd	79	70
1992	5.3	833	1.3	215	99(47)	50	23	50	nd	86	76
1991	6.0	926	1.1	167	104(62)	52	31	50	nd	65	52
1990	6.2	941	1.4	215	188(87)	85	39	45	80	79	66
Total		7245		1628	834(51)	438	27	52		78	68

Table 2

Incidence of meningococcal disease in greater São Paulo from 1990 to 1996, and distribution by year of serogroup B isolates used in this study

* Abreviations: MD, Meningococcal disease; CI, Coefficient of incidence by 100,000/inhabitants; ET 5 strains and Rb1 strains, groups of genetic related strains previously defined7,8,17,22,25; ND, Non determinated.

Variable Data for year of recovery 1990 1991 1992 1993 1994 1995 1996 No. % No. % No. % No. % No. % No. % No. % Serotypes 2a 1 1 1 1 0 0 0 0 0 0 1 1 0 0 2b 5 2 5 5 1 1 1 1 0 0 6 4 1 1 4 150 80 77 74 84 85 105 83 84 84 110 84 75 86 8 6 3 2 2 3 3 4 3 2 2 2 2 1 1 15 6 3 2 2 3 3 1 1 0 0 1 1 2 2 17 0 0 0 0 3 3 1 1 3 3 2 2 0 0 NT 20 11 17 16 5 5 14 11 11 11 8 6 8 10 Total 188 100 104 100 99 100 126 100 100 100 130 100 87 100 Serosubtypes P1.2 3 2 2 2 1 1 0 0 1 1 1 1 1 1 P1.3 0 0 0 0 2 2 2 2 1 1 7 5 1 1 P1.7 0 0 0 0 0 0 0 0 5 5 6 4 2 2 P1.9 0 0 0 0 2 2 2 2 5 5 6 4 1 1 P1.15 127 68 67 64 78 79 97 76 80 80 93 73 73 85 P1.16 0 0 0 0 1 1 3 3 1 1 0 0 1 1 P1.23 0 0 0 0 3 3 3 3 0 0 4 3 2 2 nt 58 30 35 34 12 12 19 14 7 7 13 10 6 7 Total 188 100 104 100 99 100 126 100 100 100 130 100 87 100

Variable	Data for year of recovery
	1990		1991		1992		1993		1994		1995		1996
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
Serotypes
2a	1	1	1	1	0	0	0	0	0	0	1	1	0	0
2b	5	2	5	5	1	1	1	1	0	0	6	4	1	1
4	150	80	77	74	84	85	105	83	84	84	110	84	75	86
8	6	3	2	2	3	3	4	3	2	2	2	2	1	1
15	6	3	2	2	3	3	1	1	0	0	1	1	2	2
17	0	0	0	0	3	3	1	1	3	3	2	2	0	0
NT	20	11	17	16	5	5	14	11	11	11	8	6	8	10
Total	188	100	104	100	99	100	126	100	100	100	130	100	87	100
Serosubtypes
P1.2	3	2	2	2	1	1	0	0	1	1	1	1	1	1
P1.3	0	0	0	0	2	2	2	2	1	1	7	5	1	1
P1.7	0	0	0	0	0	0	0	0	5	5	6	4	2	2
P1.9	0	0	0	0	2	2	2	2	5	5	6	4	1	1
P1.15	127	68	67	64	78	79	97	76	80	80	93	73	73	85
P1.16	0	0	0	0	1	1	3	3	1	1	0	0	1	1
P1.23	0	0	0	0	3	3	3	3	0	0	4	3	2	2
nt	58	30	35	34	12	12	19	14	7	7	13	10	6	7
Total	188	100	104	100	99	100	126	100	100	100	130	100	87	100

Table 3

Relative distribution of serotypes and serosubtypes among serogroup B meningococci recovered from patients with meningococcal disease in greater São Paulo between 1990 and 1996 (n = 834)

Ribotyping and Serotyping. The results of 438 N. meningitidis isolates analyzed by ribotyping are presented in Figure 1. Because of the equivalency of the methods, ET 5/Rb1 is used to describe strains originally defined as ET 5 by MEE^7,8, and Rb1 by ribotyping²⁵. In a previous study, we demonstrated that the increased incidence of MD, during the period between 1978 to 1990, was paralleled by an increased prevalence of a single group B clone, B:4:P1.15, of the ET 5 complex.

DISCUSSION

One possible explanation for a rise in the number of meningococcal infections is the introduction of a new strain of N. meningitidis in a susceptible population, as occurred in the Netherlands (B:2b:P1.2) in 1966, and Norway (B:15:P1.7,16) in 1974^6,13,19,24.

In greater São Paulo during 1988 to 1989, about 78% and 56% of the strains were B:4 and B:4:P1.15 respectively. The increased spread of the phenotype B:4:P1.15 could account for the increase of MD in those years²¹. After the 1989-90 vaccination trial, B:4 and B:4:P1.15 strains continued to be the most frequent recovered phenotype. The incidence of MD by serogroup B strains during 1990 to 1995 was around 1.4 per 100,000 inhabitants, and during 1996, this coefficient rose to 1.8 per 100,000 inhabitants, the highest rate since the 1970's epidemics (Fig. 1).

The case control study designed to estimate the protective efficacy of the Cuban vaccine in the campaign in greater São Paulo, indicated the vaccine may be effective in preventing serogroup B meningococcal disease in older children and adults¹⁷. The bactericidal assay on sera from Brazilian children who received two doses of vaccine also revealed age-dependent differences in serological response¹⁶.

Problems faced in development of an improved group B meningococcal vaccine are the diversity of group B serotypes and serosubtypes, and how to choose representative strains for evaluation in experimental vaccines. Among the major OMPs, the porins, classes 1, 2 and 3 proteins, are important targets for bactericidal antibodies, therefore considered vaccine candidates¹¹. OMP vaccines are prepared from one particular strain; since many of the major OMP show a high degree of variation among different strains, large proportion of the protective antibodies induced are expected to be strain-specific.

During the vaccination period, (1989-1990), most isolates were the same serotype and serosubtype (54%) as the vaccine strain (B:4:P1.15). An additional 3% were P1.15 and 15% were serotype 4. In Cuba, over 95% of serogroup B strains were 4:P1.15 what could explain why the efficacy of the same vaccine in Cuba was higher¹⁷.

Serological methods have been used extensively for routine epidemiological studies and played an important part in the development of meningococcal vaccines. The current reagent panel of MAbs does not completely identify all the diversity found in the porins. Many isolates remain non - or only partially serotyped or serosubtyped, consequently many of the antigenic characteristics of the meningococcal porins are still not known. More detailed information on exposure of other epitopes on N. meningitidis would allow better assessment of vaccine potential protective immunogenicity.

The total effects on the N. meningitidis circulating in the greater São Paulo population after the administration of the Cuban vaccine to 2.4 million persons in 1989-90 are not understood. Although, we do not know what would have occurred if the vaccine had not been administered, the proportion of isolates that were B:4:P1.15 ET5/Rb1 has continued to increase since 1990. We conclude that the same meningococcal clone (B:4:P1.15 ET5/Rb1) is still responsible for the high incidence of MD in São Paulo, in spite of the vaccination trial.

RESUMO

Doença meningocócica causada por Neisseria meningitidis sorogrupo B sorotipo 4 em São Paulo, Brasil, 1990 a 1996

Epidemia de doença meningocócica por sorogrupo B tem ocorrido na Grande São Paulo desde 1988. Uma vacina cubana, baseada em proteína de membrana externa de uma cepa de Neisseria meningitidis sorogrupo B: sorotipo 4: sorosubtipo P1.15 (B:4:P1.15), foi administrada em cerca de 2.4 milhões de crianças entre 3 meses a 6 anos de idade durante 1989 e 1990. A administração da vacina teve pouco ou nenhum efeito na evolução da epidemia. Para detectar mudança clonal que poderia explicar o contínuo aumento da doença depois da vacinação, foram sorotipadas 834 cepas isoladas entre 1990 e 1996 de pacientes com doença sistêmica. Cepas B:4:P1.15 isoladas desde 1977, têm sido o fenótipo mais prevalente desde 1988. Essas cepas ainda são prevalentes na região e foram responsáveis por cerca de 68% dos 834 casos por sorogrupo B nos últimos 7 anos. Foram analisados 438 (52%) dessas cepas por RFLP do rRNA gene (ribotipagem), e o perfil mais frequente foi o Rbl (68%). Concluímos que o clone B:4:P1.15 - Rbl foi o mais prevalente e responsável pelo contínuo aumento da doença meningocócica na Grande São Paulo durante os últimos 7 anos apesar da campanha de vacinação.

Correspondence to: Claudio T. Sacchi. Bacteriology Division, Adolfo Lutz Institute, Av. Dr. Arnaldo 351, 01246-902 São Paulo, SP, Brasil. Phone [phone] Extention 2055. E-mail:[e-mail]

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(1) Bacteriology Division and National Reference Center for

) Bacteriology Division and National Reference Center for N. meningitidis, Adolfo Lutz Institute, São Paulo, Brazil.

(2) Center for Epidemiological Vigilance of São Paulo State Alexandre Vranjac, São Paulo, SP, Brazil.

(3) Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

(4) Bacteriology Department, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.

(5) Center of Biologics Evaluation and Research, Bethesda, Maryland, USA.

Publication Dates

• Publication in this collection
  01 Dec 1998
• Date of issue
  Mar 1998