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

• Published in: Ticks and tick-borne diseases Vol. 7; no. 3; pp. 457 - 461
• Main Authors: Segura, Ferran, Pons, Immaculada, Sanfeliu, Isabel, Nogueras, María-Mercedes
• Format: Journal Article
• Language: English
• Published: Netherlands 01.04.2016
• Subjects: Amphotericin B - pharmacology; Anti-Bacterial Agents - pharmacology; Antigens, Bacterial - analysis; Bacterial Typing Techniques; Blood Culture; Boutonneuse Fever - blood; Boutonneuse Fever - diagnosis; Boutonneuse Fever - microbiology; Centrifugation; DNA, Bacterial - analysis; Fluorescent Antibody Technique, Indirect; Gentamicins - pharmacology; Humans; Real-Time Polymerase Chain Reaction; Rickettsia - drug effects; Rickettsia - genetics; Rickettsia - immunology; Rickettsia - isolation & purification; Rickettsia conorii - drug effects; Rickettsia conorii - genetics; Rickettsia conorii - immunology; Rickettsia conorii - isolation & purification; Vancomycin - pharmacology; Rickettsia massiliae-Bar29; Real-time PCR; Shell-vial assay; Rickettsia conorii; Mediterranean spotted fever
• ISSN: 1877-959X; 1877-9603
• DOI: 10.1016/j.ttbdis.2016.01.008

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.