title: Shell-vial culture, coupled with real-time PCR, applied to Rickettsia conorii and Rickettsia massiliae-Bar29 detection, improving the diagnosis of the Mediterranean spotted fever.
abstract: Rickettsia conorii and Rickettsia massiliae-Bar29 are related to Mediterranean spotted fever (MSF). They are intracellular microorganisms. The Shell-vial culture assay (SV) improved Rickettsia culture but it still has some limitations: blood usually contains low amount of microorganisms and the samples that contain the highest amount of them are non-sterile. The objectives of this study were to optimize SV culture conditions and monitoring methods and to establish antibiotic concentrations useful for non-sterile samples. 12 SVs were inoculated with each microorganism, incubated at different temperatures and monitored by classical methods and real-time PCR. R. conorii was detected by all methods at all temperatures since 7th day of incubation. R. massiliae-Bar29 was firstly observed at 28°C. Real-time PCR allowed to detected it 2-7 days earlier (depend on temperature) than classical methods. Antibiotics concentration needed for the isolation of these Rickettsia species from non-sterile samples was determined inoculating SV with R. conorii, R. massiliae-Bar29, biopsy or tick, incubating them with different dilutions of antibiotics and monitoring them weekly. To sum up, if a MSF diagnosis is suspected, SV should be incubated at both 28°C and 32°C for 1-3 weeks and monitored by a sensitive real-time PCR. If the sample is non-sterile the panel of antibiotics tested can be added.
authors.affiliation: Corporació Sanitària Parc Taulí - Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, Parc Taulí 1, Sabadell, Barcelona, 08208, Spain. Electronic address: fsegura@tauli.cat.
authors.forename: Ferran
authors.initials: F
authors.lastname: Segura
authors.affiliation: Corporació Sanitària Parc Taulí - Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, Parc Taulí 1, Sabadell, Barcelona, 08208, Spain. Electronic address: ipons@tauli.cat.
authors.forename: Immaculada
authors.initials: I
authors.lastname: Pons
authors.affiliation: Corporació Sanitària Parc Taulí - Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, Parc Taulí 1, Sabadell, Barcelona, 08208, Spain. Electronic address: isanfeliu@tauli.cat.
authors.forename: Isabel
authors.initials: I
authors.lastname: Sanfeliu
authors.affiliation: Corporació Sanitària Parc Taulí - Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, Parc Taulí 1, Sabadell, Barcelona, 08208, Spain. Electronic address: mnogueras@tauli.cat.
authors.forename: María-Mercedes
authors.initials: MM
authors.lastname: Nogueras
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000032:analysis*; D015373:Bacterial Typing Techniques; D000071997:Blood Culture; D001907:Boutonneuse Fever / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D002498:Centrifugation; D004269:DNA, Bacterial / Q000032:analysis*; D019084:Fluorescent Antibody Technique, Indirect; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D012281:Rickettsia / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D020114:Rickettsia conorii / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D005839:Gentamicins; D014640:Vancomycin; D000666:Amphotericin B
keywords: Mediterranean spotted fever; Real-time PCR; Rickettsia conorii; Rickettsia massiliae-Bar29; Shell-vial assay