An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of <i>Leptospira</i> from Clinical Samples

<div><p>Background</p><p>Rapid typing of <i>Leptospira</i> 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.</p><p>Methodology and Findings</p><p>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 <i>Leptospira</i> 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 <i>L</i>. <i>interrogans</i> (10/14 and 7/8 from UK and Lao PDR, respectively). All other species were detected in samples from only one country (Lao PDR: <i>L</i>. <i>borgpetersenii</i> [1/8]; UK: <i>L</i>. <i>kirschneri</i> [1/14], <i>L</i>. <i>santarosai</i> [1/14], <i>L</i>. <i>weilii</i> [2/14]).</p><p>Conclusion</p><p>Typing information of pathogenic <i>Leptospira</i> 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 <i>Leptospira</i> 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.</p></div

Maximum likelihood phylogenetic tree based on concatenated MLST sequences.

<p>All species defining branches are fully supported by 500 bootstraps. Bar represents substitutions per site. Tips are labelled with <i>strain_ST</i> (WHO panel) or <i>sample ID_ST</i> (clinical samples) and coloured by species: marine = <i>L</i>. <i>interrogans</i>, red = <i>L</i>. <i>kirschneri</i>, green: <i>L</i>. <i>noguchii</i>, yellow: <i>L</i>. <i>santarosai</i>, pink: <i>L</i>. <i>weilii</i>, light blue: <i>L</i>. <i>borgpetersenii</i>. P = 2015, C = 2014, both UK; L = Lao PDR, 2014.</p

MLST nested primers for all seven loci designed to accompany the MLST scheme by Boonsilp <i>et al</i>., 2013 [22].

<p>MLST nested primers for all seven loci designed to accompany the MLST scheme by Boonsilp <i>et al</i>., 2013 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004996#pntd.0004996.ref022" target="_blank">22</a>].</p

Results of nested MLST PCR for clinical specimens by sample type and full/ partial allelic profiles.

<p>Results of nested MLST PCR for clinical specimens by sample type and full/ partial allelic profiles.</p

Species assigned to clinical specimens based on phylogenetic analysis.

<p>Species assigned to clinical specimens based on phylogenetic analysis.</p

Occurrence of <i>E</i>. <i>coli</i> in different African bat species.

<p>Occurrence of <i>E</i>. <i>coli</i> in different African bat species.</p

Distribution of virulence associated genes among <i>E</i>. <i>coli</i> from African fruit bats.

<p>Distribution of virulence associated genes among <i>E</i>. <i>coli</i> from African fruit bats.</p

Sampling sites of African bats in the Republic of Congo.

<p>Parc National d’Odzala-Kokoua (PNOK); Industrie forestière d’Ouesso (IFO).</p

Rodent and eulipotyphlan partial cytochrome b sequences

FASTA file comprising 248 sequences generated during this study

Virulence gene pattern and characteristic features of 39 <i>E</i>. <i>coli</i> and one <i>E</i>. <i>albertii</i> strain.

<p>Strains of the EcoR group B2 are indicated in red, D in green, B1 in orange, A in dark blue, AxB1 in grey and ABD in light blue; genes absent among strains (<i>afa/dra</i>, <i>bmaE</i>, <i>gafD</i>, <i>iha</i>, <i>nfaE</i>, <i>tsh</i>, <i>eitA</i>, <i>eitC</i>, <i>ireA</i>, <i>iucD</i>, <i>iutA</i>, <i>sitD epi</i>, <i>neuC</i>, <i>ompT</i>, <i>cnf1/2</i>, <i>hlyF</i>, <i>sat</i>, <i>stx1</i>, <i>stx2</i>, <i>cvaA</i>, <i>cvi/cva</i>, <i>etsB</i>, <i>gimB</i>, <i>puvA)</i> are not shown. Abbreviations: ST = sequence type; STC = ST complex; In = intestines; Li = liver; Lu = lung; Ki = kidney; M = <i>Myonycteris</i>; Ei = <i>Eidolon</i>; Ep = <i>Epomops;</i> H = <i>Hypsignathus</i>; R = <i>Rousettus</i>; RC = Republic of Congo; PNOK = Park National d’Odzala Kokoua (Odzala National Park); IFO = Industrie Forestière d’Ouesso. ** Newly assigned STs are indicated by a diamond.</p