Multi Locus Sequence Typing

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2005, p. 6007–6014 0095-1137/05/$08.00 0 doi:10.1128/JCM.43.12.6007–6014.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 12

Multilocus Sequence Typing of Neisseria meningitidis Directly from Clinical Samples and Application of the Method to the Investigation of Meningococcal Disease Case Clusters
AndrewBirtles,1 Katie Hardy,2 Stephen J. Gray,1 Suzanne Handford,1 Edward B. Kaczmarski,1 Valerie Edwards-Jones,3 and Andrew J. Fox1*
Health Protection Agency, Meningococcal Reference Unit, North West Regional Laboratory, Manchester,1 Health Protection Agency, West Midlands Regional Laboratory, Birmingham,2 and Department of Biological Sciences, Manchester Metropolitan University, Manchester,3 UnitedKingdom
Received 25 June 2005/Returned for modification 25 August 2005/Accepted 27 September 2005

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 PCRbased methodologies without an organism being isolated. A nested/seminested multilocus sequencetyping (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 hadcorresponding 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), eachfrom 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/seminestedformat for strain characterization directly from clinical specimens provides an important tool for surveillance and management of meningococcal infection. Meningococcal infection is a major public health problem in the United Kingdom (20, 28, 30) and elsewhere (1, 2, 9, 21, 31, 44), with a wide spectrum of disease, from benign meningococcemia and meningococcal meningitis to rapidly fulminant fatalsepticemia. During the 1990s, the discrepancy between the numbers of laboratory-confirmed cases of meningococcal infection and the numbers of cases reported to the Office of National Statistics for England and Wales (28) widened progressively. The impact of improved clinician awareness and the growing practice of early antibiotic treatment, before hospital admission or immediately upon arrival at theemergency department, led to a reduction in the numbers of culture- and therefore laboratory-confirmed cases (8, 32). In order to improve the ascertainment of meningococcal infection and to resolve this discrepancy, methods (including both serological and nucleic acid amplification methods) for the detection of meningococcal infection directly from clinical specimens, such as blood and cerebrospinalfluid (CSF), were developed and implemented in England, Wales, and Northern Ireland by the Meningococcal Reference Unit (MRU) of the Health Protection Agency (HPA) (5, 6, 10, 11, 16, 17). The development of nucleic acid-based detection using PCR assays was highly successful and had a major impact on the laboratory confirmation of meningococcal infection and improved case ascertainment (20). The...