Abstract
BackgroundRapid 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.Methodology and FindingsAn 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]).ConclusionTyping 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.
Highlights
Leptospirosis is a zoonotic disease caused by pathogenic species of Leptospira that can be carried naturally by most mammalian species [1,2,3]
Using the sequences in phylogenetic analysis we identified the predominant Leptospira species in both countries as L. interrogans
The objective of this study was to develop an assay based on a published multilocus sequence typing (MLST) scheme that lowers the limit of detection (LoD) to enable rapid provision of typing data directly from patient specimens whilst minimising costs for subsequent sequencing [22]
Summary
Leptospirosis is a zoonotic disease caused by pathogenic species of Leptospira that can be carried naturally by most mammalian species [1,2,3]. Annual worldwide case number was estimated at around 1 million with the majority of cases and death occurring in tropical regions [6]. Despite these relatively high numbers the epidemiology of leptospirosis is not well understood. Epidemics in humans and animals are increasingly reported and are often related to natural events like floods [3, 7]. In these settings rapid typing is essential to identify potential clusters and transmission pathways. 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
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