title: Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters.
abstract: Infections associated with Neisseria meningitidis are a major public health problem in England, Wales, and Northern Ireland. Currently, over 40% of cases are confirmed directly from clinical specimens using PCR-based methodologies without an organism being isolated. A nested/seminested multilocus sequence typing (MLST) system was developed at the Health Protection Agency Meningococcal Reference Unit to allow strain characterization beyond the serogroup for cases confirmed by PCR only. This system was evaluated on a panel of 20 meningococcus-positive clinical specimens (3 cerebrospinal fluid and 17 blood samples) from different patients containing various concentrations of meningococcal DNA that had corresponding N. meningitidis isolates. In each case, the sequence type generated from the clinical specimens matched that produced from the corresponding N. meningitidis isolate; the sensitivity of the MLST system was determined to be less than 12 genome copies per PCR. The MLST system was then applied to 15 PCR meningococcus-positive specimens (2 cerebrospinal fluid and 13 blood samples), each from a different patient, involved in three case clusters (two serogroup B and one serogroup W135) for which no corresponding N. meningitidis organisms had been isolated. In each case, an MLST sequence type was generated, allowing the accurate assignment of individual cases within each of the case clusters. In summary, the adaptation of the N. meningitidis MLST to a sensitive nested/seminested format for strain characterization directly from clinical specimens provides an important tool for surveillance and management of meningococcal infection.
authors.affiliation: Health Protection Agency, Manchester Medical Microbiology Partnership, P.O. Box 209, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WZ, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Birtles
authors.forename: Katie
authors.initials: K
authors.lastname: Hardy
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Gray
authors.forename: Suzanne
authors.initials: S
authors.lastname: Handford
authors.forename: Edward B
authors.initials: EB
authors.lastname: Kaczmarski
authors.forename: Valerie
authors.initials: V
authors.lastname: Edwards-Jones
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Fox
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D008585:Meningitis, Meningococcal / Q000382:microbiology; D008589:Meningococcal Infections / Q000382:microbiology*; D009345:Neisseria meningitidis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial