Journal of Medical Microbiology (2006), 55, 751–757
DOI 10.1099/jmm.0.46451-0
Phenotypic characterization of Neisseria meningitidis strains isolated from invasive disease in Brazil from 1990 to 2001 Ana Paula S. Lemos,1 Angela P. Branda˜o,1,2,3 Maria Cecı´lia O. Gorla,1 Maria Vaneide Paiva,1 Vera Simonsen1 and Carmo Elias A. Melles1 Sec¸a˜o de Bacteriologia, Instituto Adolfo Lutz, Avenida Doutor Arnaldo, 355/9 u andar, Cerqueira Ce´sar, Sa˜o Paulo, SP, 01246-902, Brazil
1
Correspondence Angela P. Branda˜o
2
[email protected]
Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, RJ, 21045-900, Brazil
3
Fundac¸a˜o Nacional de Sau´de, Sa˜o Paulo, SP, 01220-000, Brazil
Received 2 December 2005 Accepted 14 February 2006
Phenotype characterization of 11 181 invasive Neisseria meningitidis isolates collected in Brazil from 1990 to 2001 was performed. Based on laboratory data, there were 7436 (67 %) serogroup B isolates, 3391 (30 %) C, 236 W135, 51 Y, four 29E, three X, one Z, and 59 of unknown serogroup. Phenotype B : 4,7 : P1.19,15 (54 %) remained the most common during the whole of the 12-year period. Two waves were observed within the serogroup C population: the most frequent phenotype C : 2b : P1.3 (47 %) was replaced after 1998 by non-typable isolates (C : NT : NST) (16 %).
INTRODUCTION The severity, permanent sequelae, and the fulminant and contagious nature of meningococcal disease (MD) make it a feared public health problem worldwide (Brandtzaeg, 1996; Tzeng & Stephens, 2000). Twelve serogroups of Neisseria meningitidis (Men) can be differentiated based on the chemical and immunological properties of their capsular polysaccharides. A Men isolate can also be further classified into serotype and serosubtype based on structural differences of PorB (class 2 or 3) and PorA (class 1) outer-membrane proteins (OMPs), respectively. Standard nomenclature lists serogroup : serotype : serosubtype (Popovic et al., 1998; Tzeng & Stephens, 2000). Most MD cases in the world are caused by serogroups A, B, C, W135 and Y (MenA, MenB, MenC, MenW and MenY, respectively) with geographical and temporal variation (Tzeng & Stephens, 2000). In Brazil, 68 332 MD cases were notified from 1990 to 2001, and the annual incidence rate was estimated to be around 1–3 cases per 100 000. For 35 % of the cases, the serogroup was determined, of which about 46 % were diagnosed by isolation of the causative strain (Ma´rcia L. Carvalho, personal communication; http://portal. saude.gov.br/portal/arquivos/pdf/doencas_meningo.pdf). Abbreviations: CRNM, Centro de Refereˆncia Nacional para Meningites (National Reference Centre for Meningitis Brazil); ET, electrophoretic type; MD, meningococcal disease; Men, Neisseria meningitidis; MenA, -B, -C, -29E, -W, -X, -Y, -Z, N. meningitidis serogroup A, B, C, 29E, W135, X, Y, Z, respectively; NST, non-serosubtypable; NT, nonserotypable; OMP, outer-membrane protein.
46451 G 2006 SGM
Printed in Great Britain
Free or conjugate serogroup-specific capsular polysaccharide vaccines are available against MenA, MenC, MenW and MenY. Since MenB polysaccharide is poorly immunogenic (Finne et al., 1983), research on vaccine candidates has targeted non-capsular antigens, mainly the PorA OMP, which is expected to provide serosubtype-specific protection (de Moraes et al., 1992; Peeters et al., 1996; Tzeng & Stephens, 2000). A bivalent MenB vaccine, consisting mainly of OMP-containing vesicles expressing the two serosubtypes most frequent in the country, is being studied in Brazil (Jessouroun et al., 2004; Sacchi et al., 2001). As the immunogenicity and epidemic features of MD may differ according to the serogroup, characterization of the strain from an invasive case is essential for the control of an epidemic. Surveillance of serosubtype distribution over time is also crucial if OMP vaccines are to be used to prevent MenB disease. This report describes the serogroup, serotype and serosubtype distribution of invasive Men isolates collected in Brazil during the 12-year period 1990–2001.
METHODS Meningococcal isolates. The data about the isolates were obtained from the Bacteriology Section of Instituto Adolfo Lutz, National Reference Centre for Meningitis (Centro de Refereˆncia National para Meningites, CRNM), a public health laboratory of the state of Sa˜o Paulo. Strains were voluntarily forwarded to the CRNM mainly by regional public health laboratories throughout Brazil. Serogroup identification. All Men isolates had the serogroup confirmed by slide agglutination (Popovic et al., 1998) with polyclonal
751
A. P. S. Lemos and others
1027
898
789
910
983
1096 1012 1016
837
819
879
915
1750 1500
Serogroup
6750
MD*
B (7436) C (3391) W135 (236) X (3) Y (51) Z (1) 29E (4) NK (59) All (11 181)
4500
1000 750
2250
500
No. of cases of MD*
No. of isolates
1250
250 0
0 90
91
92
93
94
95
96
97
98
99
00
01
Year
Table 1. Distribution of N. meningitidis serogroups by the major administrative regions of Brazil and by age group, from 1990 to 2001 2, Number of isolates was too small to calculate a percentage. No. and percentage of meningococcal isolates by serogroup and period 1990–1993 B
C
Major administrative region North No. 8 2 % 2 2 North east No. 520 77 % 83?5 12?4 Central west No. 99 103 % 45?6 47?5 South east No. 1384 712 % 62?7 32?2 South No. 274 275 % 48?4 48?6 All regions No. 2285 1169 % 63?1 32?3 Age group ¡2 years No. 413 197 % 64?3 30?7 3–5 years No. 320 170 % 62?1 33?0 6–14 years No. 378 186 % 63?1 31?1 ¢15 years No. 313 145 % 65?6 30?4 All ages No. 1424 698 % 63?8 31?3
1994–1996
Others Total
B
C
1997–2001
Others Total
B
C
Entire period, 1990–2001
Others Total
B
C
Others
All
0 2 26 4?2 15 6?9 112 5?1 17 3 170 4?7
10 2 623 100 217 100 2208 100 566 100 3624 100
3 2 364 86?1 113 60?8 1217 59?6 201 46 1898 61?4
0 2 57 13?5 66 35?5 793 38?8 226 51?7 1142 36?9
0 2 2 0?5 7 3?8 32 1?6 10 2?3 51 1?6
3 2 423 100 186 100 2042 100 437 100 3091 100
25 2 1033 87?6 254 73?8 1476 64?1 465 76?4 3253 72?8
6 2 137 11?6 84 24?4 739 32?1 114 18?7 1080 24?2
0 2 9 0?8 6 1?7 88 3?8 30 4?9 133 3
31 2 1179 100 344 100 2303 100 609 100 4466 100
36 81?8 1917 86?2 466 62?4 4077 62?2 940 58?3 7436 66?5
8 18?2 271 12?2 253 33?9 2244 34?2 615 38?2 3391 30?3
0 0 37 1?7 28 3?7 232 3?5 57 3?5 354 3?2
44 100 2225 100 747 100 6553 100 1612 100 11181 100
32 5?0 25 4?9 35 5?8 19 4?0 111 5?0
642 100 515 100 599 100 477 100 2233 100
489 59?1 259 57?7 365 67?0 318 57?2 1431 60?2
324 39?1 188 41?9 174 31?9 224 40?3 910 38?3
15 1?8 2 0?4 6 1?1 14 2?5 37 1?6
828 100 449 100 545 100 556 100 2378 100
953 73?6 475 71?4 635 73?1 596 73?5 2659 73?1
307 23?7 172 25?9 223 25?7 165 20?3 867 23?8
34 2?6 18 2?7 11 1?3 50 6?2 113 3?1
1294 100 665 100 869 100 811 100 3639 100
1855 67?1 1054 64?7 1378 68?5 1227 66?5 5514 66?8
828 30?0 530 32?5 583 29?0 534 29?0 2475 30?0
81 2?9 45 2?8 52 2?6 83 4?5 261 3?2
2764 100 1629 100 2013 100 1844 100 8250 100
%* Cum%*
0?4 19?9 6?7 58?6 14?4 100
33?5
33?5
19?7
53?2
24?4
77?6
22?4
100
*Percentage (%) and cumulative percentage (Cum%) of the total number of isolates from all regions (n=11 181) or of the total number of patients of known age (n=8250). 752
Journal of Medical Microbiology 55
Invasive meningococcal strains, Brazil, 1990–2001
Fig. 1. Yearly numbers of N. meningitidis isolates from invasive disease in Brazil, by serogroup, 1990–2001. The total number of isolates for each year is shown above the respective data points. DThe unknown serogroup (NK) includes 45 polyagglutinable, 10 autoagglutinable, and four non-serogroupable strains; *data available at http://portal.saude.gov.br/portal/ arquivos/pdf/doencas_meningo.pdf
goat or horse antisera prepared at CRNM against MenA, MenB, MenC, Men29E, MenW, MenX, MenY and MenZ, as described previously (Alkmin et al., 1994).
non-serosubtypable (NST) isolates were re-examined after the inclusion of new mAbs into the typing panel. Several strains still remaining NT or NST were further submitted to porA and porB gene typing, and the data have been reported previously (Sacchi et al., 1998b, 2001).
Type and subtype determination. Isolates were serotyped and Electrophoretic type (ET) determination. Several epidemic
serosubtyped by a whole-cell dot-blotting assay (Wedege et al., 1990). Some mAbs were introduced during the study period, and in 2001, the full sets consisted of mAbs for serotypes 1, 2a, 2b, 2c, 4, 5, 7, 9, 10, 11, 14, 15, 17, 19, 21 and 22, and to serosubtypes P1.1, P1.2, P1.3, P1.4, P1.5, P1.7, P1.9, P1.10, P1.12, P1.14, P1.15, P1.16, P1.19 and P1.22-1. Many of the partially, non-serotypable (NT), and
strains isolated in different regions of Brazil from 1990 to 2001 were analysed by multilocus enzyme electrophoresis (MLEE) to determine their clonal characteristics. The methods and results have been reported previously (Barroso et al., 1996; Sacchi et al., 1992a, b, 1994, 1995).
Table 2. Serotype distribution of invasive N. meningitidis isolates by serogroup, Brazil, 1990–2001 2, No isolate;
NT,
non-serotypable;
ND,
not submitted to typing.
Serotype
No. (%) of isolates by serogroup and period B 1990–1996 No.
1 2a 2b 4 4,1 4,7 4,10 4,14 4,19 4,19,10 4,21 7 10 14 15 17 17,7 17,10 19 19,1 19,7 19,7,1 19,10 19,14 21 NT ND
All
2 17 50 2783 2 58 72 2 18 2 2 1 7 3 169 61 2 9 81 2 3 2 51 2 2 496 300 4183
(%) (0?4) (1?2) (66?5) (1?4) (1?7) (0?4) (0?05) (0?02) (0?2) (0?1) (4?0) (1?5) (0?2) (1?9) (0?1) (1?2) (0?05) (11?9) (7?2) (100)
http://jmm.sgmjournals.org
C
1997–2001
1990–2001
1990–1996
No.
(%)
No.
(%)
No.
1 23 26 51 1 2494 71 5 2 2 3 30 7 38 146 11 28 6 38 36 25 1 49 61 5 97 2 3253
(0?03) (0?7) (0?8) (1?6) (0?03) (76?7) (2?2) (0?2)
1 40 76 2834 1 2552 143 5 18 2 3 31 14 41 315 72 28 15 119 36 28 1 100 63 5 593 300 7436
(0?01) (0?5) (1?0) (38?1) (0?01) (34?3) (1?9) (0?1) (0?2) (0?03) (0?04) (0?4) (0?2) (0?6) (4?2) (1?0) (0?4) (0?2) (1?6) (0?5) (0?4) (0?01) (1?3) (0?8) (0?1) (8?0) (4?0) (100)
2 419 1529 77 2 1 2 2 1 2 1 2 1 2 4 6 2 1 8 2 2 2 3 2 1 158 97 2311
(0?1) (0?9) (0?2) (1?2) (4?5) (0?3) (0?9) (0?2) (1?2) (1?1) (0?8) (0?03) (1?5) (1?9) (0?2) (3?0) (100)
(%) (18?1) (66?2) (3?3) (0?04) (0?1) (0?04) (0?04) (0?04) (0?2) (0?3) (0?04) (0?3)
(0?1) (0?1) (0?04) (6?8) (4?2) (100)
1997–2001 No. 2 89 353 6 2 96 2 2 2 2 5 1 2 2 5 2 3 2 2 1 1 2 4 4 5 382 119 1080
(%) (8?2) (32?7) (0?6) (0?2) (8?9)
(0?5) (0?1) (0?2) (0?5) (0?3) (0?2) (0?1) (0?1) (0?4) (0?4) (0?5) (35?4) (11?0) (100)
1990–2001 No. 2 508 1882 83 2 97 2 2 1 2 6 1 1 2 9 6 3 1 10 1 1 2 7 6 6 540 216 3391
(%) (15?0) (55?5) (2?4) (0?1) (2?9) (0?1) (0?03) (0?2) (0?03) (0?03) (0?1) (0?3) (0?2) (0?1) (0?03) (0?3) (0?03) (0?03) (0?2) (0?2) (0?2) (15?9) (6?4) (100)
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A. P. S. Lemos and others
Table 3. Serosubtype distribution of invasive N. meningitidis isolates by serogroup, Brazil, 1990–2001 2, No isolate;
NST,
non-serosubtypable;
ND,
not submitted to typing.
Serosubtype
No. (%) of isolates by serogroup and period B 1990–1996
P1.1 P1.2 P1.3 P1.3,7 P1.4 P1.5 P1.5,2 P1.5,10 P1.7 P1.7,1 P1.7,2 P1.7,3 P1.7,15 P1.7,16 P1.9 P1.10 P1.12 P1.14 P1.15 P1.16 P1.19 P1.19,15 P1.22-1 P1.22-1,3 P1.22-1,14 NST ND
All
C
1997–2001
1990–2001
1990–1996
1997–2001
1990–2001
No.
(%)
No.
(%)
No.
(%)
No.
(%)
No.
(%)
No.
(%)
2 40 76 2 9 2 1 2 314 8 2 2 1 5 123 1 2 67 2033 158 2 30 2 2 2 985 328 4183
(0?05) (1?0) (1?8)
5 7 83 2 10 31 28 4 20 317 1 2 1 126 86 8 25 22 1208 31 8 972 1 3 62 191 3 3253
(0?2) (0?2) (2?6)
7 47 159 2 19 31 29 4 334 325 1 2 2 131 209 9 25 89 3241 189 8 1002 1 3 64 1176 331 7436
(0?1) (0?6) (2?1)
1 352 834 4 2 18 2 2 3 2 2 2 2 2 6 143 2 6 56 15 2 2 2 2 2 771 100 2311
(0?04) (15?2) (36?1) (0?2)
1 20 113 2 2 46 88 6 1 28 2 1 2 2 6 64 2 3 23 2 2 54 2 2 9 492 121 1080
(0?1) (1?9) (10?5)
2 372 947 4 2 64 90 6 4 28 2 1 2 2 12 207 2 9 79 15 2 54 2 2 9 1263 221 3391
(0?1) (11?0) (27?9) (0?1)
(0?2) (0?02) (7?5) (0?2)
(0?02) (0?1) (2?9) (0?02) (1?6) (48?6) (3?8) (0?7)
(0?05) (23?5) (7?8) (100)
(0?3) (1?0) (0?9) (0?1) (0?6) (9?7) (0?03) (0?03) (3?9) (2?6) (0?2) (0?8) (0?7) (37?1) (1?0) (0?2) (29?9) (0?03) (0?1) (1?9) (5?9) (0?1) (100)
RESULTS AND DISCUSSION
(0?3) (0?4) (0?4) (0?1) (4?5) (4?4) (0?01) (0?03) (1?8) (2?8) (0?1) (0?3) (1?2) (43?6) (2?5) (0?1) (13?5) (0?01) (0?04) (0?9) (15?8) (4?5) (100)
(0?8) (0?1) (0?1)
(0?3) (6?2) (0?3) (2?4) (0?6)
(33?4) (4?3) (100)
(4?3) (8?1) (0?6) (0?1) (2?6) (0?1) (0?2) (0?6) (5?9) (0?2) (0?3) (2?1)
(5?0)
(0?8) (45?6) (11?2) (100)
(1?9) (2?7) (0?2) (0?1) (0?8) (0?03) (0?1) (0?4) (6?1) (0?1) (0?3) (2?3) (0?4) (1?6)
(0?3) (37?2) (6?5) (100)
For the period 1990 to 2001 there were 11 181 Men isolates from invasive MD in the CRNM database (8250 of known age, ranging from birth to 88 years; 5898 male, 503 of unspecified gender; male : female ratio, 1?23). Isolates were recovered from cerebrospinal fluid (91?9 %), blood (8 %), and synovial, pleural or pericardial fluids (0?1 %). No further information about the site of infection was available.
64 % were MenB, 34 % MenC, and the remaining 2 %, MenW, MenX, MenZ and Men29E (Ma´rcia L. Carvalho, personal communication). A statistically significant positive correlation was observed between the annual number of MD cases (http://portal.saude.gov.br/portal/ arquivos/pdf/doencas_meningo.pdf) and isolates referred to CRNM (Pearson’s correlation coefficient=0?59; P=0?0432), despite the differences in the magnitude of their values (cases : strains ratio, 6?1) (Fig. 1).
Submissions of MenB isolates remained predominant and relatively constant over time (mean±SD, 620±55 strains per year) (Fig. 1). MenC isolates, second in frequency (mean±SD, 283±82 strains per year), reached a peak in 1995, falling gradually over the next 3 years, and increasing slightly after 1999. MenB and MenC comprised 67 and 30 % of all isolates, respectively (MenB : MenC ratio, 2?19). This ratio is in agreement with the numbers of MD cases by serogroup reported in Brazil for the same period, of which
The distribution pattern of isolates correlated with the population density and income across the Brazilian regions (http://www.ibge.gov.br/home/estatistica/populacao/censo2000/ tabelagrandes_regioes211.shtm). Thus, the southeast, which is the highest-income, most populated region, forwarded most of the isolates (n=6553; 58?6 %), whereas the north ranked last in population density, income and isolation (n=44, 0?4 %) (Table 1). As estimates of disease and
754
Journal of Medical Microbiology 55
Invasive meningococcal strains, Brazil, 1990–2001
Percentage of isolates by year
(a)
Typed: 566
429
443
538
601
620
658
684
629
618
654
665
Total: 662 100
503
532
588
609
628
661
684
629
618
655
667
80
(1829)
B : 4,7 : P1.15
(1115)
B : 4,7 : P1.19,15 (918) The 3 above* (3862)
60
40
B : 4 : P1.7
(296)
B : 4,7 : P1.7,1
(296)
B : 15 : P1.16
(103)
B : 15 : P1.7,16
(99)
B : 4 : NST
20
0 90
(b)
B : 4 : P1.15
91
92
93
94
95
96
97
98
99
00
Typed: 282
319
189
281
349
452
339
294
136
151
156
222
Total: 295
344
230
300
350
453
339
294
186
179
199
222
(509)
B : 4,7 : NST
(68)
B : NT : NST
(335)
01
100 (569)
Percentage of isolates by year
C : 2b : NST 80
60
40
C : 2b : P1.3
(935)
The 2 above
(1504)
C : 2a : P1.2
(315)
C : 2b : P1.10
(206)
C : NT : NST
(496)
20
0 90
91
92
93
94
95
96
97
98
99
00
01
Year
Fig. 2. Proportions of the most frequent PorB and PorA N. meningitidis phenotypes of serogroup B (a) and serogroup C (b) isolated from invasive disease in Brazil, 1990–2001. The number of isolates for each year is shown above the respective data points. *The sum (%) of B : 4 : P1.15, B : 4,7 : P1.15 and B : 4,7 : P1.19,15; Dthe sum (%) of C : 2b : NST and C : 2b : P1.3.
isolates are likely to be less accurate in lower-income areas due to more limited access to health services and laboratory facilities, this may help explain the scarce number of isolates in northern Brazil. The predominant serogroups differed with place and time. In northern and northeastern regions, MenB accounted for more than 80 % of all isolates. In the south (1990–1996), central west (1990–1993) and southeast (44 % in 1995), the serogroup distribution differed due to emerging MenC strains, which contributed about 50 % of all isolates. The distribution of isolates differed with the age group, as shown in Table 1. The meningococcal strains were isolated mainly from children under 5 years of age, which is in http://jmm.sgmjournals.org
agreement with the age distribution of MD. According to Tzeng & Stephens (2000), the attack rate and case-fatality ratio among children can be twenty times that of adults, and in epidemic outbreaks there is a shift in disease to older children, adolescents and adults. Over the 12-year period, 77?6 % of the 8250 cases of known age occurred in individuals
