Novel TaqMan® PCR for detection of Leptospira species in urine and blood: pit-falls of in silico validation

J Microbiol Methods. 2012 Oct;91(1):184-90. doi: 10.1016/j.mimet.2012.06.009. Epub 2012 Jun 29.

Abstract

In the acute phase of leptospirosis, the diagnosis can be established with high sensitivity by testing blood and urine samples with polymerase chain reaction (PCR). However, only few real-time PCR assays have been validated for diagnostic use. The diagnostic accuracy of a novel TaqMan® PCR (LipL32 real-time PCR) targeting the lipl32 gene (or hap-1) and a previously described TaqMan® PCR (16S real-time PCR) targeting the rrs gene coding for 16S rRNA was evaluated when applied to both urine and blood specimens from humans suspected of leptospirosis. Applied to at least two blood cultures LipL32 real-time PCR had a sensitivity of 86%, and a specificity of 100%; and 16S real-time PCR had a sensitivity of 100%, and a specificity of 97%. Applied to urine samples, patients that were positive by the reference methods were also positive by both real-time PCR assays (n=4). For LipL32 real-time PCR the specificity was 100%, while for 16S real-time PCR it was only 91.5% due to unexpected cross-reactions with other bacteria. The analytical sensitivity was close to the theoretical limit-of-detection for both assays detecting all described human pathogenic species. We report a specific real-time PCR assay for detection of Leptospira, i.e., LipL32 real-time PCR that has been validated for diagnostic application in both urine and blood specimens from humans. We further show that a previously described 16S real-time PCR no longer can be recommended for diagnostic use due to a low specificity.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Blood / microbiology*
  • Child
  • Child, Preschool
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Female
  • Genes, Bacterial
  • Humans
  • Leptospira / genetics
  • Leptospira / isolation & purification*
  • Leptospirosis / diagnosis*
  • Leptospirosis / microbiology
  • Male
  • Molecular Diagnostic Techniques / methods*
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Urine / microbiology*
  • Young Adult

Substances

  • DNA, Bacterial

Associated data

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