title: Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.
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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand.
authors.forename: Siriphan
authors.initials: S
authors.lastname: Boonsilp
authors.forename: Janjira
authors.initials: J
authors.lastname: Thaipadungpanit
authors.forename: Premjit
authors.initials: P
authors.lastname: Amornchai
authors.forename: Vanaporn
authors.initials: V
authors.lastname: Wuthiekanun
authors.forename: Wirongrong
authors.initials: W
authors.lastname: Chierakul
authors.forename: Direk
authors.initials: D
authors.lastname: Limmathurotsakul
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Day
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Peacock
mesh_terms: D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000235:genetics; D005188:False Negative Reactions; D006801:Humans; D007919:Leptospira / Q000145:classification / Q000235:genetics*; D007922:Leptospirosis / Q000097:blood* / Q000453:epidemiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013785:Thailand / Q000453:epidemiology
chemical_list: D004269:DNA, Bacterial