the spanish reference laboratory for meningococci routinely receives meningococci isolated from sterile sites for serogrouping , serotyping , and serosubtyping . from january 1995 to november 2000 ( just before the new c conjugate vaccine was routinely introduced ) , the laboratory received 2,975 meningococcal strains to be characterized by serotyping and serosubtyping with monoclonal antibodies ( 8) .
the b:2b : p1.2,5 and b:2b : p1.2 antigenic combinations were found in 18 isolates ( table 1 ) .
all these strains were suspected of belonging to the a4 lineage and were fully characterized by pfge and mlst as described previously ( 2,11 ) ; results were compared with those obtained among the c:2b : p1.2,5 epidemic strains .
two additional strains characterized as b:4:p1.2,5 were also included to determine if these antigenic combinations might be caused by similar genetics events .
nr , pulse types not related to those found in c:2b : p1.2,5 strains . 
fifteen ( 83.3% ) meningococci showed sequence types identified as representative of the a4 clonal lineage ; this lineage also represents the genotype of the c:2b : p1.2,5 epidemic strain .
the proportions of isolates belonging to the a4 clonal lineage were 75% and 85% , respectively , in both b:2b : p1.2 and b:2b : p1.2,5 strains .
seven of these 15 meningococci characterized as serogroup b belonging to the a4 lineage were isolated from patients who had never been immunized with the a+c polysaccharide vaccine .
three group b strains that were suspected by antigenic characterization of belonging to the a4 complex showed nonrelated sequence types ( table 1 ) . in a different study ( data not shown ) , most of the c:2b : p1.2,5 epidemic strains grouped in two closely related pattern profiles by pfge : pt7 and pt8 . table 1 shows the pfge pattern profiles of the b:2b : p1.2,5 strains .
some strains showed minor pattern profiles already present among c:2b : p1.2,5 isolates ( pt1 , pt4 , and pt38 , all of them closely related to pt7 and pt8 ) .
those strains showing pfge pattern profiles that we did not find among the c:2b : p1.2,5 epidemic strain belonged to lineages different than a4 ( table 1 ) . on the other hand
, the b:4:p1.2,5 strains showed the same sequence type , st33 , associated with et5 .  
the frequency of the c:2b : p1.2,5 and b:2b : p1.2,5 meningococci of the a4 lineage is shown in table 2 .
recombinant strains expressing serogroup b or c have been previously described as resulting from a capsule - switching genetic mechanism ( 9,10 ) . a similar event with w135 isolates has been recently described ( 12 ) .
however , the relevance of this phenomenon has not been fully described . in our surveillance analysis , the group b strains of the a4 lineage appeared before the vaccination campaigns ; this finding differs from results of an analysis conducted in canada after a similar surveillance ( 10 ) .
our findings show that those genetic variants are being produced in the meningococcal population at random and that a variant s appearance is not necessarily related to mass immunization campaigns .
in fact , these group b meningococci belonging to the a4 lineage were also isolated in some of the regions that used the vaccine on a small scale ( 13 ) .
however , the increased number of these b:2b : p1.2,5 strains from the a4 lineage during the study period might indicate a positive selection caused by mass immunization campaigns during 1996 and 1997 ( table 2 ) .
seven ( 50% ) of these strains were isolated from patients who did not receive a+c vaccine , indicating that the individual immune status should not be a critical factor for developing meningococcal disease with these serogroup b strains of the a4 lineage rather than other clonal lineages .
nasopharingeal competition might be an important factor in the spread of these group b strains , as has been suggested to explain the spread of serogroup b meningococci belonging to the et15 lineage ( 10 ) .
theoretically , however , the c:2b strains of the a4 lineage and the b:2b meningococci also belonging to the a4 lineage should have a very similar genetic background with the exception of a locus in the capsular operon ( 9 ) .
in fact , both types of strains showed not only the same or similar sequence type by mlst but also a very similar genetic profile by pfge ( data not shown ) .
nevertheless , those strains with a group c polysaccharide capsule maintained a major epidemic even after a mass immunization campaign ( table 2 ) and even when these group c strains were not common in the carrier population ( 14 ) . whether a similar observation can be made for the serogroup b strains belonging to the a4 clonal lineage is not clear ; the small increase in these serogroup b strains does not appear to be linked with increased epidemic potential .
thus , the capsular polysaccharide appears to be the only differing factor between these two types of meningococci .
once again , the high number of serogroup b strains in asymptomatic carrier population ( 14 ) associated with a natural immunity , might partially explain the different epidemic potential of the two genotypes .
however , the nature of the group c polysaccharide alone does not explain why c:2b : p1.2,5 strains were responsible for an important epidemic wave in spain in 19961997
. some other factors , such as the amount of polysaccharide , might explain differences in virulence ( 15 ) .
two meningococci characterized as b:4:p1.2,5 were included to analyze if some other antigenic combinations might appear as result of different genetic events .
this possibility was not confirmed in our study but should be more accurately analyzed in the future . in 2000 , a new increase in cases of group c meningococcal disease was detected in some regions of spain ( 6 ) . because of these data , spanish health authorities made the decision to include a new group c conjugate vaccine in the routine infant immunization schedule beginning in autumn 2000 .
how the two different vaccines against group c meningococci ( polysaccharide and conjugate ) influence the selection of serogroup b belonging to the a4 lineage strains in spain that have had a capsular - switching event would be an interesting future topic of research .
 a mass immunization campaign for 18-month to 19-year - olds was undertaken in spain in 19961997 because of an epidemic of serogroup c meningococcal disease associated with a c:2b : p1.2,5 strain belonging to the a4 lineage . 
 surveillance for the  capsule - switching  
 phenomenon producing b:2b : p1.2,5 isolates was undertaken . 
 of 2,975 meningococci characterized , b:2b : p1.2,5 and b:2b : p1.2 antigenic combinations were found in 18 isolates ; 15 meningococci were defined as serogroup b belonging to the a4 lineage . 