Molecular surveillance of meningococcal meningitis in Africa
Vaccine, (September 2007)DOI: 10.1016/j.vaccine.2007.04.033
Abstract
aWHO Collaborating Centre for Reference and Research on Meningococci, Norwegian Institute of Public Health, Oslo, Norway bIMTSSA, WHO Collaborating Centre for Reference and Research on Meningococci, Le Pharo, BP 46, 13998 Marseille, France Analysis of meningococcal strains recovered from outbreaks and epidemics in the African meningitis belt, using molecular methods, has demonstrated for nearly 20 years the dominance among serogroup A organisms of a single clone complex, the sequence type 5 (ST-5) complex. However, a shift was observed since the mid-1990s when ST-5 gradually was replaced by ST-7 in all countries of the region. Since 2001, outbreaks caused by serogroup W135 strains belonging to the ST-11 complex became an additional problem. Monitoring of the clones responsible for meningococcal disease provides important insights on the biology and epidemiology of this most serious pathogen. Keywords: Meningococcal disease; Molecular epidemiology; Sequence type; Serogroup A; Serogroup W135 In the countries of the African “meningitis belt” in sub-Saharan Africa, meningococcal meningitis outbreaks occur every year during the dry season and large epidemics emerge approximately every 5–10 years. In that part of the world, most of meningococcal disease cases are caused by N. meningitidis serogroup A, but strains belonging to serogroup C and more recently serogroup W135 also have been involved. Large epidemics occur when a new strain is introduced in the population. The annual pilgrimage to Mecca has been clearly linked in several instances to dissemination of new strains in the region 2. Serogroup determination of patient isolates with polyclonal sera or monoclonal antibodies is the first essential step to characterize meningococcal strains because the results will dictate the control strategies that can be used. In addition to serogrouping, strains of N. meningitidis can be differentiated on the basis of immunological specificities of several major outer membrane proteins and of their lipopolysaccharides 3. The major outer membrane porins, PorA and PorB, are suitable for typing as they are stable within a strain, but vary between isolates. PorB defines the serotype, while PorA defines the serosubtype of a strain. Murine monoclonal antibodies specific for PorA and PorB have been developed, permitting to serosubtype, and to a lesser extent serotype, a large proportion of isolates recovered from patients with meningococcal disease. The PorA molecule has two main variable loops (loops 1 and 4) and the monoclonal antibodies developed for typing usually recognize linear epitopes that have been assigned to each of the two loops. Thus, the serosubtype of an isolate often includes two independent designations based on the antigenic variation in each of the variable regions. The lipopolysaccharides present in the cell wall of the bacteria are used to determine the immunotype of a strain. Several epitopes or incomplete epitopes can however be present on the same organism, rending immunotyping not very useful for classification purposes. Conventionally, a meningococcal strain is described by its serogroup, serotype and dual serosubtype separated by semicolon, and sometimes followed by its immunotype 3, as for example: A:21:P1.20,9:L10,11. Many of the DNA-based approaches developed in the past two decades for genetic typing of bacteria have been applied to the characterization of meningococcal strains. These methods include restriction enzyme analysis, ribotyping, insertion sequence analyses, pulsed-field gel electrophoresis (PFGE), sequencing of individual genes that may be related to virulence, and PCR-based techniques, such as random amplification of polymorphic DNA, amplified fragment length polymorphism and variable number of tandem repeat analysis. Some of these methods, especially PFGE, are highly discriminative and are attractive for outbreak investigation and local epidemiology. PFGE allows the electrophoretic separation of large DNA molecules in agarose gel by alternating the direction of the electric field applied to the gel. After digestion of the complete meningococcal chromosome with a restriction endonuclease that cuts infrequently, a fingerprint pattern based on the whole chromosome of the strain can be resolved by PFGE. The technique has been extremely valuable when applied to serogroup A strains, allowing to discriminate between different epidemic waves caused by closely similar organisms 4. Since the mid-1980s, genetic methods indexing overall bacterial diversity have been developed which allow to better understand the biology of N. meningitidis: these methods have proved extremely valuable for the study of the global epidemiology of meningococcal disease. For many years, multilocus enzyme electrophoresis (abbreviated MEE or MLEE) was the only method to permit large-scale analysis of N. meningitidis strains causing disease in various parts of the world and the only one to document the intercontinental spread of particularly pathogenic clones 5. The method assesses the diversity of randomly chosen, multiple genes encoding essential metabolic functions. These so-called housekeeping genes are expected to be subject to stabilising selection to preserve the metabolic functions of the proteins they encode. Indexing variation in housekeeping genes provided data pertinent to establish reliable evolutionary relationships among isolates. In MLEE, genetic variation is assessed indirectly by examination of the gene products (normally metabolic enzymes) after starch gel electrophoresis and specific histochemical staining 6. Genetic variants at multiple loci are determined and assigned an arbitrary allele number. The allele numbers for all loci examined by the scheme are combined into an allelic profile, designated as an electrophoretic type (ET). Bacteria with identical ETs are assumed to be members of the same clone. Closely related genotypes are grouped into clonal complexes by their similarities to a central genotype, which is believed to correspond to a common ancestor of the bacteria. As a result, MLEE analyses have often been designated as clonal analyses. This valuable method was rather cumbersome, however, and it became obsolete after the development in the late 1990s of the multilocus sequence typing (MLST), which exploits advances in high-throughput DNA sequencing technology to directly and accurately measure gene variations 7. MLST uses nucleotide variation in internal fragments (about 500 bases) of housekeeping genes to directly identify alleles. As MLEE, MLST permits to characterize each strain by its multilocus genotype, designated in that case as a sequence type (ST). MLST uses fewer loci than MLEE (7 versus 15–20), but the degree of resolution of MLST is much higher at each locus, in that all polymorphisms are detected. There is a very good congruence between the results obtained by MLEE and MLST, which has allowed extrapolation from the data from earlier MLEE studies and cross-references to the MLEE nomenclature. While both techniques produce equivalent data, the fundamental advantage of MLST data is that they are more easily compared across studies and laboratories, through a centralised curated MLST database available via the Internet (www.pubmlst.org/neisseria). Clonal analyses have been performed for many years at the two WHO Collaborating Centers for Reference and Research on Meningococci in Marseille (France) and Oslo (Norway) and applied to strain collections obtained from patients in countries of the meningitis belt. Despite high levels of genetic diversity and evidence of extensive horizontal genetic exchange in meningococcal populations, the diversity of the species is highly structured, with groups of related genotypes persisting for decades and achieving global spread. Consequently, the majority of cases of meningococcal disease reported since the second half of the 20th century have been caused by a limited number of clone complexes, the so-called hyper-invasive lineages, which appeared to have an increased propensity to cause invasive disease. Associated with the epidemiological data, information on the role of different clones and clonal complexes in relation to endemic disease and outbreaks allows a better understanding of the disease patterns and may permit to develop hypotheses on the evolution of meningococcal disease in the African belt, helping to prepare the most adapted responses. The first large clonal analyses of meningococcal strains from the African meningitis belt were performed using MLEE by Achtman, who showed that successive epidemic waves were associated with the spread of different clone complexes, described as the ‘subgroups’ of serogroup A 8. Three clonal complexes, subgroups I, IV-1, and III (now designated ST-1, ST-4, and ST-5, respectively), which are strictly associated with the serogroup A capsular PS, have successively caused the majority of meningococcal disease in Africa for the past 40 years. Organisms of subgroup I (ST-1 complex) were responsible for outbreaks in the African continent at the beginning of the 1960s, both in North Africa and in countries of the African meningitis belt. In the late 1970s they were responsible for epidemics in Nigeria and Rwanda 8. Subgroup I strains have been identified since 1968 in South Africa, where they gave rise in 1991 to an outbreak among refugees from Mozambique; they still were the predominant serogroup A clone in South Africa in 1996 5. Although already present in West Africa from the 1960s, in the early 1980s meningococci of subgroup IV-1 (ST-4 complex) replaced subgroup I in West Africa and were responsible for epidemics in The Gambia and Mali 8. Strains of subgroup III (ST-5 complex), probably carried by pilgrims from South Asia, caused a severe epidemic involving nearly 2000 cases during the 1987 annual Haj pilgrimage to Mecca, Saudi Arabia and were then introduced, for the first time, into the African continent by returning pilgrims 9. In 1988 and 1989, major epidemics occurred in Ethiopia, Sudan, Chad and Kenya. In the following years, the other countries of the meningitis belt, as well as African countries outside that traditional meningitis region, such as Burundi, Zambia, Cameroon, Uganda, and Rwanda were reached by outbreaks caused by subgroup III strains. In 1996, the sub-Saharan region of Africa was reached by a new subgroup III epidemic of unprecedented scale with over 150,000 reported cases and 16,000 deaths, affecting principally Burkina Faso and Nigeria 10. Until the mid-1990s, essentially all strains of the ST-5 complex causing disease in the meningitis belt were true ST-5, the ancestral clone of the complex. In 1996, a new clone of the ST-5 complex, ST-7, differing from ST-5 at the pgm locus, was identified for the first time in Nigeria 11. ST-7 then gradually replaced ST-5 in the countries of the African meningitis belt and was responsible for outbreaks in Chad (1998), Sudan (1999), Ethiopia (2000–2003) and Niger (2003). In Niger, while the 1995 and 1996 outbreaks were due to ST-5, they were followed in 1999, 2000 and 2001 by outbreaks involving a mixed population of ST-5 and ST-7 and since 2002 only ST-7 strains were isolated. A similar shift occurred in Burkina Faso, where ST-7 was seen first in 2001. Since 2002, ST-5 has totally disappeared from Sub-Saharan Africa, being replaced by ST-7 12. In 2003, a new variant of the ST-5 complex, ST-2859, emerged in Niger and Burkina Faso causing sporadic cases. Preliminary data indicate that the 2006 epidemic in Burkina Faso was due to ST-2859. Microevolution through mutation and recombination can explain the genetic changes resulting in the emergence of new variants. The reasons for these replacements between very similar organisms within the same complex, however, are not yet fully understood 12, but immune selection driven by herd immunity and/or slight transmission fitness differences between the strains is likely to be responsible for these changes. Among the other ST-complexes, the ST-11 complex, associated to serogroup W135, has been responsible for an increasing number of sporadic cases since 2000 and for the first W135 epidemic ever seen, which occurred in Burkina Faso in 2002, resulting in more than 14,000 cases 13. Even though such strains had already been isolated in Africa in the early 1990s, it is probable that the annual Hajj of 2000 and 2001 amplified the spread of this clone, resulting in an increase in the number of cases caused by serogroup W135 strain belonging to ST-11 complex in the African continent 14. Increased notification of cases caused by W135 ST-11 strains in many countries of the region was a great concern, because of the limited availability of PS vaccines against serogroup W135 and, consequently, the necessity of laboratory confirmation of the organism responsible for the epidemic in order to appropriately manage vaccine stocks. Apart from these two major clonal complexes, few other clones, such as ST-2881, ST-181 and ST-751, have been sporadically detected. The identification of outbreaks caused by serogroup X, another serogroup previously considered of low pathogenic potential, may also be worrying 15 and 16. In spite of the improved understanding of the epidemiology of meningococcal disease that has been provided by clonal analyses, it is still not possible to predict reliably the occurrence of epidemics. Epidemiological surveillance on a global scale, including clonal analyses of the disease-causing organisms, needs to be pursued to be able to establish appropriate preventive measures.