title: An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of Leptospira from Clinical Samples.
abstract: BACKGROUND
  Rapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST) data direct from patient specimens while minimising costs for subsequent sequencing.
  
  
  RESULTS
  An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK) and the Lao Peoples Democratic Republic (Lao PDR). Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine) PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50%) and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%). No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25%) and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively). All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14]).
  
  
  CONCLUSIONS
  Typing information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.|European Programme for Public Health Microbiology (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden.
authors.forename: Sabrina
authors.identifier: http://orcid.org/0000-0002-4835-3695
authors.initials: S
authors.lastname: Weiss
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.
authors.forename: Angela
authors.initials: A
authors.lastname: Menezes
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.|Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Vientiane, Lao PDR.
authors.forename: Kate
authors.initials: K
authors.lastname: Woods
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Vientiane, Lao PDR.
authors.forename: Anisone
authors.initials: A
authors.lastname: Chanthongthip
authors.affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Vientiane, Lao PDR.|Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.|Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland.
authors.forename: Sabine
authors.initials: S
authors.lastname: Dittrich
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.
authors.forename: Agatha
authors.initials: A
authors.lastname: Opoku-Boateng
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.
authors.forename: Maimuna
authors.initials: M
authors.lastname: Kimuli
authors.affiliation: Public Health England (PHE), National Infection Service (NIS), London, United Kingdom.
authors.forename: Victoria
authors.initials: V
authors.lastname: Chalker