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Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization - PubMed

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization

Sven Poppert et al. J Clin Microbiol. 2005 Jul.

Abstract

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

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Figures

FIG. 1.
FIG. 1.

ROC analysis of the eubacterial 16S rRNA gene PCR. (A) Distribution of the crossing points of the individual measurements; (B) ROC analysis. neg., negative; pos., positive.

FIG. 2.
FIG. 2.

FISH detection of pneumococci in a CSF sample from a patient. The sample was stained by an S. pneumoniae-specific Cy3-labeled probe (Spn) (A) in combination with the FITC-labeled eubacterial probe (EUB 338) (B). As a further control, the DNA stain DAPI was implemented, which stained bacteria as well as the nucleus of a granulocyte (C). The overlay (D) demonstrates that the bacteria fluoresce in all channels, resulting in a white color, while the weak autofluorescence of the erythrocytes in the FITC and Cy3 channel results in a brown color.

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References

    1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. - PMC - PubMed
    1. Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919-1925. - PMC - PubMed
    1. Balganesh, M., M. K. Lalitha, and R. Nathaniel. 2000. Rapid diagnosis of acute pyogenic meningitis by a combined PCR dot-blot assay. Mol. Cell. Probes 14:61-69. - PubMed
    1. Fuchs, B. M., G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann. 1998. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64:4973-4982. - PMC - PubMed
    1. Greisen, K., M. Loeffelholz, A. Purohit, and D. Leong. 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32:335-351. - PMC - PubMed

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