Characterization of Neisseria meningitidis isolates from recent outbreaks in Ethiopia and comparison with those recovered during the epidemic of 1988 to 1989

Abstract

The objectives of this study were to collect and characterize epidemic meningococcal isolates from Ethiopia from 2002 to 2003 and to compare them to 21 strains recovered during the previous large epidemic of 1988 to 1989. Ninety-five patients in all age groups with clinical signs of meningitis and a turbid cerebrospinal fluid (CSF) sample were included in the study of isolates from 2002 to 2003. Seventy-one patients (74.7%) were confirmed as having Neisseria meningitidis either by culture (n = 40) or by porA PCR (n = 31) of their CSF. The overall case fatality rate (CFR) was 11.6%; the N. meningitidis-specific CFR was 4.2%. All 40 strains were fully susceptible to all antibiotics tested except sulfonamide, were serotyped as A:4/21:P1.20,9, and belonged to sequence type 7 (ST-7). The strains from 1988 to 1989 were also equally susceptible and were characterized as A:4/21:P1.20,9, but they belonged to ST-5. Antigenic characterization of the strains revealed differences in the repertoire of lipooligosaccharides and Opa proteins between the old and the recent strains. PCR analysis of the nine lgt genes revealed the presence of the lgtAHFG genes in both old and recent strains; lgtB was present in only some of the strains, but no correlation with sequence type was observed. Further analysis showed that in addition to their pgm alleles, the Ethiopian ST-5 and ST-7 strains also differed in their tbpB, opa, fetA, and lgtA genes. The occurrence of new antigenic structures in strains sharing the same serogroup, PorA, and PorB may help explain the replacement of ST-5 by ST-7 in the African meningitis belt.

FIG. 1.

Map of Ethiopia, with cities of collaborating institutions indicated. The figure was prepared using ArcView 9.1 software (ESRI, Redlands, CA) and geographic data available from the European Joint Research Centre Digital Map Archive (http://dma.jrc.it).

FIG. 2.

Coomassie brilliant blue-stained 12% SDS-PAGE gels with OM extracts from N. meningitidis isolates listed in Table 4. Lane 1, Mk 499/03; lane 2, Mk 502/03; lane 3, Mk 804/03; lane 4, Mk 365/02; lane 5, Mk 686/02; lane 6, Mk 689/02; lane 7, Mk 691/02; lane 8, Mk 802/02; lane 9, Eth 2; lane 10, Eth 9; lane 11, Eth 12; lane 12, Eth 18; lane 13, Eth 35; lane 14, Eth 38. Unk.; unknown protein bands; Std, standard; MW, molecular weight (in thousands).

FIG. 3.

Silver-stained LOS bands of OM extracts separated in 16% TSDS-PAGE gels. 7880, L10 prototype strain (24). Lanes 1 to 14 contain OM extracts from same strains shown in Fig. 2. Lane 15, strain 44/76 (L3,7,9); lane 16, strain N 144/95 (L8); lane 17, strain Sudan 433/88 (L13) (42).

FIG. 4.

Typical patterns of tbpB alleles previously found in subgroup III strains digested with the restriction enzyme ApoI (lanes 1 to 4) or SspI (lanes 5 to 8). Lanes 1 and 5, strain Z1054 (control allele 1); lanes 2 and 6, strain Eth 4 (allele 1); lanes 3 and 7, strain Eth 12 (new allele); lanes 4 and 8, strain Mk 502/03 (allele 55). Std, standard.

FIG. 5.

Topology of FetA from strain H44/76 in the outer membrane (based on a figure by Pettersson et al. [40]), with the top part showing the surface-exposed loops and their numbers and with the VR marked with a box. Regions where the fetA07 allele encodes different amino acids than the fetA11 allele are marked with circles; in the VR, the fetA07 and fetA11 alleles encode similar amino acid sequences. *, in this position, fetA07 encodes an insertion of two additional amino acids compared to fetA11.