Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization.
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.
Department of Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany. sven.poppert@medizin.uni-ulm.de
Sven
S
Poppert
Andreas
A
Essig
Barbara
B
Stoehr
Adelinde
A
Steingruber
Beate
B
Wirths
Stefan
S
Juretschko
Udo
U
Reischl
Nele
N
Wellinghausen
D002555:Cerebrospinal Fluid / Q000382:microbiology*; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification; D006095:Gram-Positive Cocci / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016920:Meningitis, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D008853:Microscopy; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial