Molecular detection and speciation of pathogenic Leptospiraspp. in blood from patients with culture-negative leptospirosis

Siriphan Boonsilp,Vanaporn Wuthiekanun,Wirongrong Chierakul,Janjira Thaipadungpanit,Sharon J Peacock,Direk Limmathurotsakul,Premjit Amornchai,Nicholas P Day

doi:10.1186/1471-2334-11-338

Abstract

BackgroundPathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.MethodsWe evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed.Results39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species).ConclusionMolecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.

Highlights

Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR
Parallel blood samples for culture and PCR (5 ml collected into each of heparin and EDTA blood tubes, respectively) were taken on Primer design Primer design was based on three criteria. These were that primers would: (i) anneal to DNA belonging to Leptospira spp. associated with disease in humans or animals, but not to saprophytes which can be laboratory contaminants; (ii) amplify a gene fragment small enough to be sequenced in a single reaction; and (iii) amplify a gene fragment containing the maximal number of polymorphic sites between species for the size of product, maximizing the ability of the region to discriminate between different but closely related Leptospira species
An analysis was performed to determine the discriminatory power of the 443-nucleotide rrs fragment to speciate the 14 Leptospira spp. compared with the nearly whole length rrs gene for the 184 sequence traces by comparing average (%) nucleotide distances

Summary

Introduction

Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospirosis is an acute febrile illness caused by pathogenic species belonging to the genus Leptospira [1,2]. This zoonotic disease has a worldwide distribution but is most common in tropical and subtropical regions and has the greatest impact on public health in developing countries [1,2,3,4]. In a recent study conducted by us to assess the diagnostic accuracy of a real-time PCR assay for human leptospirosis in Thailand, culture was positive in only 39/133 (29%)

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