Molecular Microbiology in Osteo-arthritis Infection

Overview

Osteoarticular infections are painful and disabling diseases that require antimicrobial treatment adapted to the microorganisms implicated. Microbiological cultures are currently regarded as the reference for identification of pathogenic bacteria. However, the sensitivity of these cultures is very variable and depends both on the context in which clinical samples are taken, and on the pathogen involved. The rate of detection varies according to infection type: from 50 to 70% for infectious spondylodiscitis, 65 to 95% for prosthetic joint infections, 50% for gonococcal arthritis and 90% for non-gonococcal arthritis. The aim of the study is to evaluate the diagnostic performances of microbiological cultures and molecular methods in case of osteoarticular infections. The gold standard will be established by an expert group of osteoarticular infection (composed by a bacteriologist, a radiologist, a surgeon, an anatomy-pathologist and a rheumatologist), which established the final diagnosis of infected or not infected patients.

Background: Osteoarticular infections are painful and disabling diseases that require antimicrobial treatment adapted to the microorganisms implicated. Microbiological cultures are currently regarded as the reference for identification of pathogenic bacteria and it seems that the microbiological cultures are sufficient in most cases to establish the diagnosis. However, the sensitivity of these cultures is very variable and depends both on the context in which clinical samples are taken, and on the pathogen involved. In addition, previous experimental data show that molecular methods could produce a diagnosis missing by conventional cultures. Main objective: The aim of the study is to evaluate the diagnostic performances of microbiological cultures and molecular method by considering for the definition of cases an accumulation of clinical, radiological and biological arguments usually used to establish the diagnosis of bone and joint infections. The discordance of the two methods will be secondary classified by an expert group of osteoarticular infection (composed by a bacteriologist, a radiologist, a surgeon, a anatomy-pathologist and a rheumatologist).Tested hypothesis:Interest of molecular methods for the diagnosis of osteoarticular infections.Secondary objective:Quantification of the delay of the results obtained by the two methods Study design: · One centre specialized in osteoarticular infections· Observational prospective study· Two methods of diagnosis: cultures and molecular methods· An expert group Included patients and number of patients: Total of 284 patients composed by 194 cases and 90 controlsIncluded patients (n=194) will all have a suspected diagnosis of osteoarticular infections included discitis, septic arthritis and prosthetic joint infection defined by inclusions criteria.Results of the included patients will be compared to those obtained from the group of control patients (n=90).Protocol of duration: Study duration is 6 months for each patientThe protocol duration is planned for 2 yearsProcedures:All samples will be treated with the same procedure - Samples (synovial fluids, biopsies, ..) will be recovered either during surgery, by puncture aspiration at the bedside, or by guided ultrasonography, all under sterile conditions (mask, sterile gloves, cautious skin preparation and towelling) and transported to the Bacteriology department in sterile tubes without additional substrate in less than 12 hours. - Bacteriological procedures will be performed according to the guidelines applied for osteoarticular infection · Liquid samples: erythrocyte and leukocyte counts, polymorphonuclear percentage · Solid specimens: crushed using a mechanic apparatus · Gram and auramine staining· Cultures at 37°C on Columbia sheep blood agar (aerobically and anaerobically for 5 days), chocolate agar supplemented with IsoVitaleX (with 5% CO2 for 5 days), brain heart infusion broth (anaerobically for 15 days with subcultures done after 10 days on the same agar media, even if aspect was not cloudy).· Mycobacterial culture : Lowenstein-Jensen and COLETSOS media, MGIT incubated for 3 months.- Molecular procedures· DNA manipulations pre- and post-PCR: in separate rooms with separate pipetting devices to prevent contamination of samples with foreign DNA. · Equipment used in pre-PCR steps: DNA-free and UV-irradiated. · DNA extraction based on Boom's method: QIAGEN kit and EZ1 BioRobot · Broad-range 16S rRNA gene amplification · Amplicons: sequenced in both directions on an ABIPRISM 3100 analyser (Applied Bio systems, COURTABOEUF, France). · Cloning experiments for several sequences in one sample· Analysis of sequence: GenBank using the BLASTN program through the website of the National Centre of Biotechnology Information.· Internal control PCR: b-actin gene· Real time PCR: for the detection of mycobacteria and gonococcal arthritis- Potential implications: We could have chosen, like the majority of the studies published to date, to compare our molecular method with another method of diagnosis (culture, histology). However, there is not currently gold standard and the diagnosis of osteoarticular infections generally rests on an accumulation of arguments. The rule of the expert group is to determine, with less possible error, if each patient included will be "infected". This methodology is close to daily clinical reality and trusted in physician who was treating the patient. If the study shows an advantage of the molecular biology versus classical methods in our groups it would then be justified to discuss the diagnosis diagram of osteoarticular infections.