title: Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene.
abstract: Listeria monocytogenes is 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 quantifying L. monocytogenes DNA in the CSF. The designed primers specifically amplify the L. monocytogenes hly gene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for the hly gene (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 the L. monocytogenes DNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases in which L. monocytogenes was 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 by L. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment.
authors.affiliation: Département de Microbiologie, Hópital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Université Paris Descartes, Paris, France. alemonnier@ch-versailles.fr
authors.forename: Alban
authors.initials: A
authors.lastname: Le Monnier
authors.forename: Eric
authors.initials: E
authors.lastname: Abachin
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Beretti
authors.forename: Patrick
authors.initials: P
authors.lastname: Berche
authors.forename: Samer
authors.initials: S
authors.lastname: Kayal
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001427:Bacterial Toxins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D002555:Cerebrospinal Fluid / Q000382:microbiology; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008584:Meningitis, Listeria / Q000175:diagnosis* / Q000382:microbiology; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes