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Open Access Highly Accessed Research article

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

Siriphan Boonsilp16, Janjira Thaipadungpanit12*, Premjit Amornchai1, Vanaporn Wuthiekanun1, Wirongrong Chierakul13, Direk Limmathurotsakul14, Nicholas P Day15 and Sharon J Peacock167

Author Affiliations

1 Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand

2 Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand

3 Department of Clinical Tropical Medicine,Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

4 Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

5 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, UK

6 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

7 Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK

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BMC Infectious Diseases 2011, 11:338  doi:10.1186/1471-2334-11-338

Published: 13 December 2011

Abstract

Background

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. 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.

Methods

We 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.

Results

39/418 (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).

Conclusion

Molecular 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.