Diagnosis of Listeria monocytogenes Meningoencephalitis by Real-Time PCR for thehlyGene

Abstract

ABSTRACTListeria monocytogenesis a bacterial pathogen that can invade the central nervous system (CNS), causing meningoencephalitis and brain abscesses. The diagnosis of CNS listeriosis, based on the isolation of the bacteria in the cerebrospinal fluid (CSF), can be difficult because of previous antibiotic treatment and a low number of bacteria in the CSF. To improve the sensitivity of microbiological diagnosis, we have developed a real-time PCR assay for detecting and quantifyingL. monocytogenesDNA in the CSF. The designed primers specifically amplify theL. monocytogeneshlygene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for thehlygene (PCR-hly) provides reproducible quantitative results over a wide dynamic range of concentrations and was highly sensitive while detecting a single gene copy/ml. By assaying a large panel of bacterial species, including species secreting pore-forming cytolysin, we determined the specificity of the PCR-hly, which exclusively detects theL. monocytogenesDNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases in whichL. monocytogeneswas isolated by culture. Positive PCR-hly of the CSF was also obtained for five additional, clinically confirmed cases of CNS listeriosis for which bacterial cultures were negative presumably due to previous treatment with antibiotics. As a complement to classical bacteriological CSF culture, our designed real-time PCR-hly assay proved to be valuable by enhancing the rapidity and the accuracy of the diagnosis of CNS infection byL. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment.