Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case … (original) (raw)
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1999
Multilocus sequence typing and antigen gene sequencing were used to investigate an outbreak of meningococcal disease in a university in the United Kingdom. The data obtained showed that five distinct Neisseria meningitidis strains belonging to the ET-37 complex were present in the student population during the outbreak. Three of these strains were not associated with invasive disease, and two distinct strains caused invasive disease, including several fatalities. The initial case of the disease cluster was caused by a strain distinct from that responsible for at least two subsequent cases and two cases remote from the university, which were epidemiologically linked to the outbreak. These observations were consistent with pulsed-field gel electrophoresis data, but the sequence data alone were sufficient to resolve the strains involved in the disease cluster. Interpretation of the nucleotide sequence data was more straightforward than interpretation of the fingerprint patterns, and the sequence data provided information on the genetic differences among the isolates.
Multilocus sequence typing for global surveillance of meningococcal disease.
The global surveillance of bacterial pathogens is particularly important for bacteria with diverse and dynamic populations that cause periodic epidemics or pandemics. The isolate characterization methods employed for surveillance should: (1) generate unambiguous data; (2) be readily implemented in a variety of scenarios and be reproducible among laboratories; (3) be scalable and preferably available in a high throughput format; and (4) be cost effective. Multilocus sequence typing (MLST) was designed to meet these criteria and has been implemented effectively for a wide range of microorganisms. The 'Impact of meningococcal epidemiology and population biology on public health in Europe (EU-MenNet)' project had amongst its objectives: (1) to disseminate meningococcal MLST and sequence-based typing throughout Europe by establishing a centre for training and data generation, and (2) to produce a comprehensive Europe-wide picture of meningococcal disease epidemiology for the first time. Data produced from the project have shown the distribution of a relatively small number of STs, clonal complexes and PorA types that account for a large proportion of the disease-associated isolates in Europe. The project demonstrates how molecular typing can be combined with epidemiological data via the Internet for global disease surveillance.
1997
Subspecific typing of clinical meningococcal strains is important in the investigation of outbreaks and for disease surveillance. Serogrouping, typing, and subtyping of strains currently require isolation of a meningococcus from one or more clinical specimens. However, the increasing widespread practice of preadmission administration of parenteral antibiotics has resulted in a decrease in the frequency of positive cultures obtained from blood and cerebrospinal fluid. Confirmation of meningococcal disease can be obtained by meningococcus-specific PCR from both cerebrospinal fluid (H. Ni et al., Lancet 340:1432-1434, 1992) and peripheral blood (J. Newcombe et al., J. Clin. Microbiol. 34:1637-1640, 1996) specimens. However, current PCR protocols do not yield epidemiologically useful typing information. We report here the use of PCR-singlestranded confirmational polymorphism (PCR-SSCP) analysis to amplify and type meningococcal DNA present in clinical specimens. PCR-SSCP analysis with the VR1 region of the Neisseria meningitidis porA gene as the target produced unique banding patterns for each serosubtype. Direct PCR-SSCP of clinical specimens can therefore provide typing data that can be used to investigate the epidemiology of clusters of cases and outbreaks and for disease surveillance in situations in which culture of patient specimens proves negative.
Journal of Medical Microbiology, 2003
A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88·5 % and, using dot-blot DNA detection, the sensitivity increased to 96·4 %. The detection limit for meningococcus in cerebrospinal fluid was 2310 2 c.f.u. ml À1 . Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96·4 % using dot-blot DNA detection.
Molecular typing of meningococci: recommendations for target choice and nomenclature.
The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).
Canadian Journal of Microbiology, 2003
We compared multilocus enzyme electrophoresis (MEE) and ribosomal DNA fingerprinting (ribotyping) for subtyping 44 strains of Neisseria meningitidis serogroup C that were isolated in Los Angeles County, California, between December 1985 and July 1986. The isolates were divided into six enzyme types (ETs) by MEE, but 36 of the isolates were clustered in one ET, 3. The same isolates were divided into 17 ribotypes by use of restriction endonucleases ClaI, EcoRI, and XhoI. Twenty of the 36 ET 3 isolates were grouped in a single ribotype, J. The rate of infection with ribotype J strains was higher in the southern part of the study area than in the northern part. Isolates from each of eight pairs (each isolate pair was cultured from the same patient from the same or different sites) were found identical by MEE, but ribotyping revealed a difference in one pair. In this study, ribotyping showed a greater discriminating capacity than MEE for subtyping N. meningitidis serogroup C, but the epidemiologic relevance of this increased sensitivity needs further assessment.
Isolation, Culture, and Identification of Meningococci from Clinical Specimens
Meningococcal Disease, 2001
Neisseria meningitidis is a major cause of morbidity and mortality in childhood in industrialized nations and the organism is responsible for epidemics of disease in Africa and in Asia. Because of its public health impact, meningococcal disease is of interest and importance to clinicians, clinical microbiologists, public health physicians, epidemiologists, and research scientists. In Meningococcal Disease we have brought together a series of review and methods-based chapters that provide essential information for diagnosis in the clinical microbiology laboratory, isolate characterization, clinical management, and control of meningococcal disease.
Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes
Epidemiology and Infection, 1992
SUMMARYThe genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence IS1106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15: P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion, were found to be derived from two distinct lineages within the B15: P1.16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningoccus do not necessarily reflect true genetic relationships.
Meningococcus genome informatics platform: a system for analyzing multilocus sequence typing data
Nucleic acids research, 2009
The Meningococcus Genome Informatics Platform (MGIP) is a suite of computational tools for the analysis of multilocus sequence typing (MLST) data, at http://mgip.biology.gatech.edu. MLST is used to generate allelic profiles to characterize strains of Neisseria meningitidis, a major cause of bacterial meningitis worldwide. Neisseria meningitidis strains are characterized with MLST as specific sequence types (ST) and clonal complexes (CC) based on the DNA sequences at defined loci. These data are vital to molecular epidemiology studies of N. meningitidis, including outbreak investigations and population biology. MGIP analyzes DNA sequence trace files, returns individual allele calls and characterizes the STs and CCs. MGIP represents a substantial advance over existing software in several respects: (i) ease of use-MGIP is user friendly, intuitive and thoroughly documented; (ii) flexibility--because MGIP is a website, it is compatible with any computer with an internet connection, can be used from any geographic location, and there is no installation; (iii) speed--MGIP takes just over one minute to process a set of 96 trace files; and (iv) expandability--MGIP has the potential to expand to more loci than those used in MLST and even to other bacterial species.