Capsular Switching in Invasive Neisseria meningitidis, Brazil

During the 1990s, an epidemic of B:4 Neisseria meningitidis infections affected Brazil. Subsequent increase in C:4 disease suggested B→C capsular switching. This study identified B→C switches within the sequence type 32 complex. Substantial disease related to capsular switching emphasizes the need for surveillance of circulating meningococcal strains to optimize disease control.

T he species Neisseria meningitidis includes successful commensal strains and devastating human pathogens (1)(2)(3). Invasive strains produce a polysaccharide capsule, which is essential for virulence (3) and is a target for most licensed vaccines. Characteristic genomic fl uidity (e.g., facilitated by transformation, slipped-strand mispairing, or 2-component regulatory pathways) enables N. meningitidis to alter its antigenic profi le (1). Capsular switching, which occurs through transformation and horizontal gene transfer, enables N. meningitidis to escape from host defenses against the original serogroup. This event is common in the absence of vaccination and has implications for meningococcal vaccines that do not cover all serogroups (4)(5)(6).
Brazil experienced a prolonged epidemic of B:4 N. meningitidis infections during 1988-1999. In 1990 and 1994, mass vaccination was performed by using a vaccine consisting of a serogroup B outer membrane vesicle (B:4:P1.15) and a serogroup C polysaccharide. During 1993-1994, a total of 4 C:4 N. meningitidis isolates were identifi ed in samples from patients in Rio de Janeiro State (RJ). The presence of class3-PorB in these strains suggested the possibility of B→C capsular switching (7).

The Study
The study was performed at Oswaldo Cruz Institute, RJ, Brazil. The case defi nition for invasive meningococcal disease was isolation of N. meningitidis from a normally sterile body fl uid from an RJ resident. Patient characteristics were obtained from the epidemiologic records of the RJ Department of Health during 1988-2009 and analyzed with EpiInfo (version 3.5.3; Centers for Disease Control and Prevention, Atlanta, GA, USA). This study was approved by the Ethical Committee of the Evandro Chagas Research Institute of the Oswaldo Cruz Foundation (protocol 0070.0.009.000-10).
Meningococcal isolates were serogrouped by agglutination and serotyped and serosubtyped by dot blot. The C:4 phenotype was used as a marker of isolates that could have resulted from B:4 capsular switching. Of 41 C:4 isolates recovered from RJ patients during the study period, 35 were viable for further characterization. We also randomly selected 35 B:4 isolates (≈10%) from a list of B:4 isolates available in the laboratory collection from the same period by using SPSS-15 for Windows (SPSS Inc., Chicago, IL, USA).
Multilocus sequence typing (MLST) (9) was performed in combination with outer membrane protein (OMP) gene sequencing (10) of porA variable regions (VRs) 1 and 2, porB, and fetA VR. Serogroup-specifi c PCR (8) and MLST were repeated for isolates with a suspected capsular switch by using the same template DNA. The assignment to allele, sequence type (ST), clonal complex (CC), porA, porB, and fetA was performed by querying the Neisseria Sequence Typing Home Page (http://pubmlst.org/neisseria).
STs were considered part of the same clonal complex if they shared at least 4 alleles of the 7 MLST loci with the designated central genotype. OMP gene sequence results were expressed as porB:P1, porA-VR1, porA-VR2, and F.fetA-VR (10). Meningococcal capsular switching was presumed to have occurred when an ST within a serogroup not usually associated with that ST and more frequently associated with another serogroup was found. MLST and OMP genotypes were used to defi ne capsular switching in specifi c clones. MLST results showed that of the 35 B:4 isolates, 30 (86%) belonged to ST32CC and most were ST33 and ST639. There were 2 predominant OMP profi les: 3-1: P1.19.15: F5-1 (47%) and 3-79: P1:7-1.1: F5-1(33%), which are consistent with Brazil B epidemic clones. The other STs were single locus variants of ST32 or ST33. Of the 5 remaining isolates, 3 belonged to ST41/44CC, 1 to ST35CC, and 1 to ST3766 (no defi ned CC).
OMP genotyping data demonstrated capsular switching in 4 specifi c meningococcal clones ( Table 2)

Conclusions
This study demonstrated that after a prolonged epidemic of serogroup B ST32 complex N. meningitidis in Brazil, isolates genetically indistinguishable, but expressing the serogroup C capsule, emerged and persisted as a cause of invasive disease during 2000-2009. A plausible explanation for this clonal emergence is the maintenance of virulence after capsular switch and a lack of immunity in a portion of the population in RJ. One of the clones associated with the serogroup B epidemic, B:3-1:P1.19.15:F5-1:ST-33(32CC), against which mass vaccination was directed, resulted in only 2 serogroup C cases.
Capsular switching represents a mechanism by which meningococci escape protective immunity directed at specifi c capsular polysaccharides (4). The emergence and expansion of the serogroup W135 Hajj clone in the setting of vaccination against serogroups A and C illustrate this phenomenon (11). However, capsular switching does not always lead to clonal emergence. For example, the serogroup C clone that resulted from capsular switching within the serogroup B epidemic in Oregon has caused relatively few cases (5).
We report a substantial number of cases of meningococcal disease and case clusters during 2000-2009 that were associated with ST32/ET-5 serogroup C clones that had emerged in Brazil during 1993-1998. These strains might retain the attributes of the lineages of the ST32 complex B clones, including a propensity to cause outbreaks. Additionally, some capsular variants may have a selective advantage and might, at least in part, replace original strains circulating in the population (12). In studies of N. meningitidis ST11CC strains bearing serogroups C or W135 in Brazil (13,14) and serogroups C and B in Spain (15), a pattern of preserved hyperinvasiveness in the emerged W135 and B strains was also found. The ability of N. meningitidis to cause substantial disease after capsular switching highlights the need for surveillance of circulating meningococcal strains to delineate optimal disease control policies. The opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affi liated.