Neisseria meningitidis

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Molecular methods for the detection and characterization of

Neisseria meningitidis Mathew A Diggle and Stuart C Clarke†

CONTENTS Meningococcal disease Laboratory confirmation of meningococcal disease Molecular methods for the characterization of Neisseria meningitidis Expert commentary Five-year view Key issues References Affiliations

Neisseria meningitidis remains a common global cause of morbidity and mortality. The laboratory confirmation of meningococcal disease is, therefore, very important for individual patient management and for public health management. Through surveillance schemes, it provides long-term epidemiologic data that can be used to inform vaccine policy. Traditional methods, such as latex agglutination and the enzyme-linked immunosorbent assay, are still used, but molecular methods are now also established. In this review, molecular methods for the laboratory confirmation and characterization of meningococci are described. PCR is an invaluable tool in modern biology and can be used to predict the group, type and subtype of meningococci. It is now also used in a fluorescence-based format for increased sensitivity and specificity. The method also provides the amplified DNA for other techniques, such as multilocus sequence typing. Other methods for the discrimination of meningococci have also played and continue to play an important part in epidemiology. For example, pulsed-field gel electrophoresis is highly discriminatory, whilst multilocus enzyme electrophoresis provided the basis for the description of global meningococcal clones and formed the foundation for multilocus sequence typing. Other less commonly used methods, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and pyrosequencing, may increasingly find their way into microbiology reference laboratories. Nevertheless, nucleotide sequencing and laboratory automation have aided the introduction of many methods and provide data that are digitally based and, therefore, highly accurate and portable. Expert Rev. Mol. Diagn. 6(1), 79–87 (2006) Meningococcal disease

†

Author for correspondence Portsmouth City Primary Care Trust, Portsmouth, UK [email protected] KEYWORDS: bacteria, DNA sequencing, genome, genotyping, meningococcal disease meningococci, multilocus sequence typing, Neisseria meningitidis, nucleotide, pyrosequencing, typing

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Neisseria meningitidis (the meningococcus) remains an important cause of morbidity and mortality, causing meningitis and septicemia worldwide [1,2]. Only five (namely A, B, C, W135 and Y) of the 13 recognized serogroups are considered important [2]. Serogroup C meningococcal disease became a particular problem in the UK during the 1990s and was associated with a high prevalence of the ET37/ST11 complex [3,4]. It has also been epidemic in other European countries [5]. Meningococcal disease remains common in the meningitis belt of sub-Saharan Africa where serogroup A is most common [6], whilst serogroup B is prevalent in New Zealand [7]. However, recent problems have arisen with the 10.1586/14737159.6.1.79

spread of a serogroup W135 clone [8–10], which emphasizes the need for accurate methods of strain characterization as well as collaborative disease surveillance. Laboratory confirmation of meningococcal disease

The laboratory confirmation of meningococcal disease is, therefore, very important for individual patient management and for public health management. It provides long-term epidemiologic data, through surveillance schemes, which can be used to inform vaccine policy. Although the laboratory confirmation of meningococcal disease has changed relatively little, with classic methods such as latex agglutination and enzyme-linked immunosorbent

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assay (ELISA) still widely used, molecular methods are becoming well established. In patients with suspected meningococcal disease, specimens may be taken from sterile sites such as blood, cerebrospinal fluid and the conjunctiva. Isolation of the infecting organism in cases of meningococcal disease may be attained, but the possibility of carriage must be remembered when samples are taken from a nonsterile site. Neisseriae are normally cultured on a medium, such as Columbia agar with 5% horse blood or heated Columbia blood agar, and are incubated at 37oC in air containing 5–10% CO2. Commercial and noncommercial carbohydrate tests may be used for genus and species identification and often rely on the fact that N. meningitidis utilize glucose and maltose. The serogroup of the meningococcus can be determined using latex agglutination and coagglutination with serogroup-specific antisera. Commercially available reagents are available for the identification of serogroups A, C, Y, W135 and B/Escherichia coli by latex agglutination, whilst reference coagglutination reagents are available for the confirmation of these serogroups and also the identification of other serogroups, such as X and Z1 [11]. Typing of microorganisms traditionally involves the subdivision of a single species utilizing a standard set of characteristics. This has included methods such as biochemical characterization, antibiotic susceptibility, phage typing and antigen characterization. These typing methods are usually based upon phenotypic markers and biochemical pathways, some of which may be species specific. Meningococci can be further characterized by serotyping and serosubtyping. These are denoted by the meningococcal class 2 and 3 porin proteins (porB) and class 1 porin protein (porA), respectively. Serotyping and serosubtyping is traditionally performed using a whole-cell ELISA typing method [12,13]. However, the infecting meningococcus may not be isolated in all cases of disease. Therefore, nonculture methods must be employed, such as latex agglutination and PCR [11,14]. For the former, the sensitivity of the assay has been improved through the use of ultrasound-enhanced latex immunoagglutination (USELAT) [15]. For PCR, numerous assays are now available, which will be discussed further. Molecular methods for the characterization of Neisseria meningitidis

Molecular biology has led to the development of new methods that, when united with our improved understanding of the meningococcus, enable the molecular characterization of this important pathogen (TABLE 1). This allows meningococci to be characterized in far greater detail than previously possible, since phenotypic methods are often based on biochemical and antigenic/serologic markers. However, with most molecular methods it is usually subtle changes in DNA sequence that are detected (FIGURE 1). PCR

During the past decade, PCR has developed into an invaluable tool and is considered a key advance in molecular biology. The method is used in many areas that utilize molecular techniques in

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research and nonresearch environments, including microbiology, animal and human genetics and clinical diagnostics. There are a number of methods available for the amplification and detection of specific DNA sequences. Traditional PCR involves the amplification of DNA in a commercial thermocycler followed by visualization of PCR products on a gel-based system. Although some assays have low sensitivity, lack specificity, are costly and are laborious, a number of technologies are now available to exploit the PCR method beyond its basic concept. Real-time detection of PCR products is now possible [16], and there is a move towards automation [17]. New chemistries have also been developed commercially to provide real-time PCR methodology based on fluorescent probe technology. Therefore, these are more sensitive and specific than gel-based systems [18,19]. New PCR chemistries also allowed further applications of PCR such as single nucleotide polymorphism (SNP) analysis, whilst standard PCR can provide amplicons for microarray analysis. There are three chemistries currently used for the real-time detection of PCR products, namely hybridization, hydrolysis and hairpin probes [20]. Hybridization probes are small oligonucleotide probes that hybridize to a specific nucleotide sequence target. Hydrolysis probes (commercially known as TaqMan® probes) rely on the 5´→3´ exonuclease activity of Taq polymerase, which degrades a hybridized nonextendible DNA probe during the extension step of the PCR. Hairpin probes, otherwise known as molecular beacons, possess a duplex region adjacent to a single-stranded target capture region that, when denatured, hybridizes to the target nucleotide sequence. These three chemistries allow the elimination of post-PCR processing, while allowing real-time analysis of PCR products produced during amplification. Molecular beacons are molecules with an internally quenched fluorophore whose fluorescence is restored on binding to a target DNA sequence. The beacons are designed in a way that allows the loop portion of the molecule to be the probe sequence for the target DNA. The PCR method has been applied to the laboratory confirmation of meningococcal disease, and a number of assays have been described. These are relatively quick and can be sensitive and specific; a typical test can take only a few hours. PCR-based detection and characterization is especially useful when treatment has been given and culture has proven negative, although the success of PCR depends largely on the gene target used [21–24]. The detection of the insertion element IS1106 can be used to confirm the presence of N. meningitidis in suspected cases, and was developed in both standard and PCR ELISA formats [23,25]. However, as insertion sequences are genetically mobile and spread between species and even genera, the IS1106 PCR can lead to false positives [25]. Therefore, a species-specific PCR assay was developed to detect the ctrA gene [26]. A fluorescence-based PCR method has since been developed to provide improved sensitivity [24,27]. Further PCR assays have been developed to characterize the meningococcus. Assays that exploit the nucleotide sequence differences between the siaD genes of serogroups B, C, Y and W135 have been described by detecting restriction sites or by nucleotide sequencing (TABLE 2) [28–31].

Expert Rev. Mol. Diagn. 6(1), (2006)

Characterization of Neisseria meningitidis

Capsule (siaD)

Outer membrane

Genome (MLST)

Figure 1. Schematic representation of the various facets of meningococcal genotyping. MLST: Multilocus sequence typing.

PCR assays for the confirmation of serogroup A meningococci have also been described [32,33]. PCR-based methodology can also be used for serotype and serosubtype characterization, but these usually require DNA sequencing to be performed in addition to PCR [34–39]. Moreover, some of these assays lack the sensitivity required to detect the small amounts of DNA present in clinical samples and therefore use nested PCR in order to increase the sensitivity of the assay [30,31,37,39,40]. Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) uses cultured meningococci, and the chromosomal DNA is extracted within agarose plugs. In addition, the DNA is digested with restriction endonucleases that cut the chromosome into 10–40 fragments that are resolved into fingerprint patterns by gel electrophoresis, which is controlled by current and voltage programming [41,42]. The method is highly discriminatory and characterizes the complete genome. Therefore, cross-genome comparisons can be made using PFGE. However, there are problems with standardization due to inter-gel variation, particularly between different laboratories. Consequently, it can be difficult when comparing similar strains from different laboratories, although patterns produced are interpreted


relatively simply and quickly. Even so, due to its high level of discrimination, it is particularly useful in outbreaks of meningococcal disease [41]. Multilocus enzyme electrophoresis

This method has long been considered the gold standard for the molecular characterization of N. meningitidis [43,44]. Multilocus enzyme electrophoresis (MLEE) was first described in 1966 as a molecular approach to the study of genetic variation in eukaryotic systems [45], and microbiologists have been able to incorporate MLEE as a highly useful tool. MLEE is fundamentally based around the different electrophoretic mobilities of constitutive enzymes from different amino acid changes. These enzymes were chosen on the basis of their powers to discriminate and type most strains and, therefore, allow enhanced population and evolutionary studies. Although only a small number of variants are detected at each locus, analysis of 20 or more loci can provide a high level of resolution [43]. MLEE does have many attractive features for global epidemiology and epidemic-associated characterization [44,46]. MLEE has been used for the phenotyping of meningococci, to identify specific clones and to study the genetic diversity of the meningococcus. Although the correlation between the

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electrophoretic migration of individual enzymes and the genotype may be disrupted by horizontal gene transfer [47], the use of multiple enzymes make MLEE a fairly robust typing method for meningococci [43,48]. The electrophoretic pattern gained by MLEE is known as an electrophoretic type (ET) and major meningococcal clones have been designated on this basis, such as ET-37, ET-15 and ET-5 [43,44,49–51]. Nowadays, MLEE is considered labor intensive compared with other currently available methods. Moreover, it can be subjective, relying on uncharacterized genomic differences between isolates while producing results that are difficult to compare between different laboratories. Given the current emphasis on global epidemiology, due to the ability of meningococci to spread between countries and continents, other methods have been developed that not only maintain the same level of discrimination but are also easy to compare between laboratories. Multilocus sequence typing

Multilocus sequence typing (MLST) was developed as a method to replace MLEE. It allows the differentiation of strains that appear identical by standard phenotypic typing methods and, importantly, allows data to be compared between laboratories, as it produces nucleotide sequence data of approximately 500-bp segments from seven housekeeping genes. The results generated are digital and, therefore, highly portable between laboratories, thereby allowing global comparisons to be made as data can be shared via the worldwide web [52]. The method can also be performed on purified chromosomal DNA and therefore has the advantage over MLEE that noninfective samples can be transported by mail [53]. MLST was first validated on meningococci by Maiden and colleagues [52]. It was applied to a collection of 107 N. meningitidis isolates from invasive disease and healthy carriers that had been previously characterized by MLEE.

Table 1. Methods for the molecular characterization of

Neisseria meningitidis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Much interest has been generated in the area of matrixassisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry in recent years, and it has become a tool of choice for molecule analysis [58]. Essentially, MALDI-TOF is a technique for measuring molecular masses precisely and is sufficiently accurate to detect the difference in mass between distinct nucleotides [59]. Therefore, SNPs can be detected, which allows strains to be characterized. This has been exploited for the differentiation of meningococci within the ET-37 complex [60]. Otherwise, the use of this technique has not been reported for its application towards the characterization of meningococci. Pyrosequencing

Method

Complexity

Cost

PCR

Minimal

Low

Pulsed-field gel electrophoresis

Moderate

Low

Multilocus enzyme electrophoresis

Moderate

Medium

Amplified fragment-length polymorphism

Moderate

Medium

Multilocus sequence typing

Moderate

Medium

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Moderate

Medium

Pyrosequencing

Moderate

Medium

DNA microarray

High

High

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MLST has now been validated, with varying success, for a number of important bacteria and fungi [54,55]. This method was originally evaluated on N. meningitidis because it provides a good example in which genetic recombination events are considered common. Originally, MLST for meningococci centered on ten loci from isolates that had been previously characterized, but the number of meningococcal MLST housekeeping genes was subsequently reduced to seven loci, on the basis of its discriminatory power (TABLE 2). MLST can be used for analyzing the population biology of bacteria since nucleotide sequence differences allow the evolution of strains to be determined [52,54]. The method can be used for routine characterization of strains and disease surveillance. Further discrimination can be provided if MLST is combined with antigen gene sequencing for the investigation of case clusters of meningococcal disease [38,56]. As such, MLST is useful for immediate and long-term public health surveillance and has been utilized for the characterization of meningococci during case clusters or outbreaks in certain settings [38,56]. In some countries, MLST is incorporated into enhanced surveillance schemes so that clusters of disease can be identified and changes in strain distribution can be monitored [57].

Pyrosequencing is a relatively new technique that enables the real-time synthesis of single-stranded DNA and, hence, the identification of nucleotides. It has uses in nucleotide sequencing and the detection or identification of SNPs [61], and thus can be used for detecting, identifying and typing bacteria. The method involves the amplification of target DNA using PCR, followed by the conversion of doublestranded DNA to single-stranded DNA template [61]. Oligonucleotide primers are then hybridized to the complementary nucleotide sequence of interest. The pyrosequencing assay itself catalyses the synthesis of complementary nucleotides that, through a cascade reaction, releases chemicals that result in the generation of light, which is detected by a charge-coupled device camera and converted into presentable data in a pyrogram [61]. The nucleotide sequence is determined from this pyrogram. The technology is simple and relatively robust



and the technology can be adapted to create dedicated tools for specific applications. Pyrosequencing has now been used for the characterization of a number of bacterial species, including the meningococcus [62]. Serogrouping and serosubtyping can be performed successfully using pyrosequencing and the procedure is rapid and accurate [62]. Single-stranded conformational polymorphism analysis

Single-stranded conformational polymorphism (SSCP) analysis was developed in order to obtain typing information on the meningococci present in clinical samples, such as blood and cerebrospinal fluid. Initially, the gene used for SSCP was the variable region 1 of porA [63]. SSCP analysis of post-PCR products allows detection of single-nucleotide point mutations within target DNA. This has been used to demonstrate the identities and nonidentities of meningococci between clinical samples and clinical specimens. This sort of method is not unlike a number of SNP methods available for identifying single-base variations in a DNA sequence, which are usually represented by two or three different bases at a single position and can occur in a population with an allele frequency greater than 1%. SNPs can be considered highly abundant within bacterial genomes and, along with the tools available to determine the exact variant present in a DNA target sequence, these are useful molecular typing tools. Amplified fragment-length polymorphism analysis

Amplified fragment-length polymorphism (AFLP) is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. Fingerprints are produced without prior sequence knowledge using a limited set of generic primers. This technique can be considered a combination of the robustness and reliability of restriction fragment-length polymorphism (RFLP) and the relative power of the PCR technique [64]. The genomic fingerprinting process can be separated into three appropriate steps: first, the restriction of the DNA and ligation of oligonucleotide adapters; second, selective amplification of sets of restriction fragments; and third, gel analysis of the amplified fragments using semiautomated scoring software of AFLP images [65]. Additionally, the PCR primers can be labeled fluorescently and the dye-labeled fragments can be separated by capillary electrophoresis, which is currently considered one of the most suitable methods for fragment analysis. Using the capillary electrophoresis system with appropriate size standards enables fluorescent AFLP (FAFLP) with a usual fragment accuracy of ±1 bp. FAFLP can be used as a meningococcal genotyping tool with a high resolution and accurate size determination [64,66,67]. Expert commentary

Culture has a traditional role in the diagnosis and laboratory confirmation of meningococcal disease. The characterization of meningococci, albeit limited, has been possible using traditional phenotypic methods such as latex agglutination and ELISAs [11–13,68–70]. Even so, these methods have provided


Table 2. Genes used for the characterization of Neisseria meningitidis. Gene(s)

Use

SiaD (serogroups B, C, Y and W135)

Grouping

MynA (serogroup A)

Grouping

PorB

Typing

PorA

Typing

Housekeeping genes (abcZ, adk, aroE, fumC, gdh, pdhC, pgm)

Multilocus sequence typing

important information for the international epidemiology of meningococcal disease through capsule and porin analysis, as well as data for more localized case clusters and epidemiologic surveillance. Moreover, they provided the information for developing vaccine policy. However, as PCR and nucleotide sequencing has become more accessible, the feasibility of genotypic methods has become a reality. Fluorescence-based PCR is now considered to be the gold standard for the laboratory confirmation of meningococcal disease. Therefore, due to the urgency with which meningococcal disease needs to be confirmed, and the importance of gaining information that is useful for public health management, genetic methods are becoming increasingly useful for the confirmation and typing of meningococci. Such methods are now widely used in reference laboratories and enable detection, serogrouping, serotyping and sequence typing [24,28,29,38–40]. Decreasing costs for PCR, nucleotide sequencing and laboratory automation have aided the introduction of the methods that are complemented by the need for computer-based analysis software [17,71–75]. Genotypic methods are, in practice, far superior to phenotypic methods [76]. Although a number of methods are available (some of which have been described previously) for the characterization of meningococci, many of these methods are gel based and rely on the analysis of fragment sizes. Although progress has now been made in developing systems that are highly controlled so that the method is reproducible within and between laboratories, the results are not usually produced in a digital format, are not easily portable and are therefore not available on a global basis. MLST removes such problems since the method produces digitally based nucleotide sequence and is therefore highly accurate and portable. Data comparisons between laboratories are easily performed and can take place on a global scale. Therefore, MLST has rapidly become the method of choice for the molecular characterization of meningococci. Prospective enhanced surveillance of those meningococci circulating in the UK was initiated in the UK before the implementation of the meningococcal serogroup C conjugate vaccines towards the end of 1999 [77–79]. This surveillance has utilized MLST to enable the monitoring of meningococci for the emergence of new strains and for potential capsule switch from serogroup C to serogroup B [79–82]. Grouping PCRs have

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been available for some time [28,29], although nucleotide sequence-based methods are now also available [30,31]. Typing can be achieved by porB PCR [34] and subtyping can be achieved by porA PCR [35,37,39,83], which is useful because meningococci can appear identical by phenotypic methods, may appear different by porA nucleotide sequencing (genosubtyping), but may actually be similar by MLST. Five-year view

As 60–80% of all cases of meningococcal disease are laboratory confirmed, but only approximately half are culture confirmed [69,84], genotypic methods now play an important role in improving the typing information available. In the future, microarrays may become utilized by reference laboratories if appropriate methodology is developed. Methods such as MLST may then be performed by microarray rather than traditional sequencing [85,86]. Microarrays are DNA segments representing a collection of genes or the various combinations of a single gene to be assayed. These are amplified by PCR and mechanically spotted at high density on glass microscope slides using simple xyz-stage robotic systems. This results in a microarray containing thousands of elements. These microarrays can easily be constructed within a relatively short period of time. Using fluorescence-based technology, hybridization of complement DNA segments upon the microarray with target DNA can be detected and immediately identified. Although microarray expression analysis has become one of the most widely used functional genomics tools, it has not yet been successfully applied to the molecular characterization of meningococci [87]. Although, in theory, MLST could be applied by microarray, it has not yet been successful [88]. References Papers of special note have been highlighted as: • of interest •• of considerable interest 1

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Protein arrays may also be available in the near future [89–91]. This would allow the analysis of expressed gene products, which would enable virulence characteristics to be used as markers for strain characterization. However, much work is required before protein arrays can be used as a routine tool. The most likely way forward is whole-genome analysis. The ability of whole-genome sequencing has advanced rapidly over the past decade and nanotechnology promises further advances in terms of speed and cost. Ultimately, the routine molecular characterization of meningococci could encompass the whole genome. For example, recent work in the USA described an accurate and robust method for the sequencing of 25 million bases in 4 h [32]. The method provided a 100-fold increase in throughput over current Sanger sequencing technology. As for MLST, this would have the benefit of generating digital data in a portable format. Although such data would be generated in an unprecedented volume, they will be accompanied by complementary advances in computer technology. Key issues • Methods for the molecular characterization of Neisseria meningitidis have been developed over the past decade. • Methods for the molecular characterization of N. meningitidis are now rapid, accurate and reproducible. • Multilocus sequence typing is now considered the gold standard for the molecular characterization of N. meningitidis. • Sequence-based typing produces unambiguous data that are highly discriminatory and have the advantage of being in a digital (and therefore portable) format.

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Affiliations •

Mathew A Diggle, PhD Stobhill Hospital, Scottish Meningococcus & Pneumococcus Reference Laboratory, Glasgow, UK

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Stuart C Clarke, PhD Stobhill Hospital, Scottish Meningococcus & Pneumococcus Reference Laboratory, Glasgow, UK; Portsmouth City Primary Care Trust, Portsmouth, UK; Division of Infection & Immunity, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow, UK [email protected]

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