Biochips for Direct Detection and Identification of Bacteria in Blood Culture-Like Conditions.

Bloodstream bacterial infections are life-threatening conditions necessitating prompt medical care. Rapid pathogen identification is essential for early setting of the best anti-infectious therapy. However, the bacterial load in blood samples from patients with bacteremia is too low and under the limit of detection of most methods for direct identification of bacteria. Therefore, a preliminary step enabling the bacterial multiplication is required. To do so, blood cultures still remain the gold standard before bacteremia diagnosis. Bacterial identification is then usually obtained within 24 to 48 hours -at least- after blood sampling. In the present work, the fast and direct identification of bacteria present in blood cultures is completed in less than 12 hours, during bacterial growth, using an antibody microarray coupled to a Surface Plasmon Resonance imager (SPRi). Less than one bacterium (Salmonella enterica serovar Enteritidis) per milliliter of blood sample is successfully detected and identified in blood volumes similar to blood tests collected in clinics (i.e. several milliliters). This proof of concept demonstrates the workability of our method for human samples, despite the highly complex intrinsic nature of unprocessed blood. Our label-free method then opens new perspectives for direct and faster bacterial identification in a larger range of clinical samples.

Assessment of linezolid prescriptions in three French hospitals.

The use of linezolid to treat gram-positive cocci infections is increasing in France. Linezolid is approved in pneumonia and complicated skin and soft tissue infections. Overuse and misuse of linezolid can favor the emergence and spreading of linezolid-resistant strains. We aimed to assess the appropriateness of linezolid use in French hospitals. This is a multicenter, retrospective study conducted in three tertiary care hospitals. Appropriateness of linezolid indications and adequacy (composite score concerning dosage, route of administration and blood monitoring) were assessed. Over a three-month period, all prescriptions of linezolid were extracted and analyzed by two independent infectious disease experts. Among the 81 initial prescriptions that were evaluated, indication was appropriate in 48% of cases. Among those, 51% complied with international guidelines. Fifty-seven percent of the prescriptions were adequate regarding dosage, route of administration and blood monitoring. Overall, 23% of prescriptions combined both appropriateness and adequacy. The most frequent reasons for inappropriateness were the possibility of choosing narrower-spectrum antibiotics and the empirical use of linezolid in severe sepsis or septic shock. Initial treatment was the most frequently appropriate in bone and joint infection cases (p = 0.001). Our study shows that even if modalities of use were mostly correct, appropriateness of linezolid indications is low. Educational programs are mandatory to improve practices, as well as clinical studies to better assess the efficacy and safety of linezolid in clinical situations other than pneumonia or complicated skin and soft tissue infections.

Novel portable platform for molecular detection of toxigenic Clostridium difficile in faeces: a diagnostic accuracy study.

BACKGROUND: A novel portable platform for nucleic acid amplification enables rapid detection of diarrhoea causing toxigenic Clostridium difficile directly from faeces, even in resource-limited settings. We evaluated the accuracy and precision of the new commercial molecular test system. METHODS: One thousand one hundred and sixty faecal samples from patients suspected of having Clostridium difficile infection (CDI) were analysed using the Orion GenRead C. difficile test system (Orion Diagnostica Oy, Espoo, Finland) and comparative methods in three teaching hospital laboratories in Finland and France. The precision of the Orion GenRead C. difficile test system was evaluated in a reproducibility study with a set of blind-coded samples. The test system is based on a new isothermal amplification technology (Strand Invasion Based Amplification, SIBA®) and detection of the tcdB gene of C. difficile. We calculated the sensitivity, specificity, and the overall agreement according to Clinical and Laboratory Standards Institute recommendations. FINDINGS: The overall agreement of the Orion GenRead C. difficile test when compared to the comparative methods in routine use in the participating laboratories was between 96.7% and 98.8%. In the reproducibility study; the total percent agreement between three laboratories was 99.8%. INTERPRETATION: The identification of toxigenic C. difficile from faeces with the light-weight portable Orion GenRead test system was highly sensitive and specific, and the results were reproducible in the participating laboratories. This platform could enable fast and accurate molecular pathogen detection even in resource-limited or point-of-care settings.

Impact of systemic antifungal therapy on the detection of Candida species in blood cultures in clinical cases of candidemia.

The diagnosis and follow-up of candidemia still rely on blood cultures (BCs). In vitro studies show that antifungals can significantly modify the result of blood culture not containing adsorbing agents. We aimed to evaluate, under clinical conditions, the impact on BC yeast detection of systemic antifungal therapy (SAT). Patients (n = 125) experiencing candidemia at Grenoble University Hospital (France) were included in a 4-year retrospective study. The Plus Aerobic/F (Aerobic) and Plus Anaerobic/F (Anaerobic) bottles, which both contain adsorbing resins and the non-resin selective Mycosis IC/F (Mycosis) bottles, were compared using multivariate hierarchical models adjusted for clinical characteristics. The positivity rate (PR) is decreased in patients with SAT (p < 0.01), abdominal surgery (p = 0.01), and hemodialysis (p = 0.02). In all bottles, SAT reduces PR by a factor of 0.16 (95 % CI: [0.08; 0.32]) and increases the time to positivity (TTP) by a factor of 1.76 ([1.30; 2.40]; p < 0.01). In the presence of SAT, TTP is higher in non-resin bottles (Mycosis) than in resin bottles (RR = 1.76, [1.30; 2.40]); however, the TTP in nonresin and resin bottles remains comparable. Although discordant results are observed with and without SAT (37 and 58 % respectively), we showed that the presence of SAT decreases significantly the agreement rate by a factor of 0.29 (CI: [0.12; 0.68]). The combination of Anaerobic and Mycosis bottles allowed a 100 % positivity rate for C. glabrata. SAT significantly affects BC results. Because they provide additional and complementary results, this study supports the concomitant use of resin and selective bottles, especially in patients receiving SAT.

Francisella tularensis aortitis.

Francisella tularensis, the agent of tularemia, is a Gram-negative coccobacillus primarily pathogen for animals and occasionally for humans. The clinical manifestations of tularemia include pneumonia, ulceroglandular, oropharyngeal, or typhoidal disease. Rare manifestations are also described, but to our knowledge, we describe here the first case of F. tularensis aortitis in a human. Diagnosis was confirmed by the presence of F. tularensis in blood culture, by the presence of F. tularensis DNA in the aortic biopsy and by specific IgG and IgM responses against the bacteria. The outcome was favorable after surgery and specific antimicrobial therapy.

Correlation between clinical data and antibiotic resistance in coagulase-negative Staphylococcus species isolated from 68 patients with acute post-cataract endophthalmitis.

Coagulase-negative staphylococci (CNS) cause the majority of post-cataract endophthalmitis, which can lead to anatomical and/or functional loss of the eye. This study reports the antibiotic susceptibilities of CNS isolates associated with acute post-cataract endophthalmitis cases and correlates antibiotic resistance with severity and outcome of infection in these patients. Clinical data (initial ocular examination, final prognosis, antibiotic treatment) and the antibiotic susceptibilities of the isolated CNS strains were obtained from 68 patients with post-surgical endophthalmitis recruited during a 7-year period by the FRench Institutional ENDophthalmitis Study (FRIENDS) group. The CNS strains displayed 100% susceptibility to vancomycin, 70% to fluoroquinolones, 83% to fosfomycin, 46% to imipenem and 18% to piperacillin. The most effective antibiotic combinations were fosfomycin plus a fluoroquinolone and imipenem plus a fluoroquinolone, which were considered adequate in 80% and 58% of patients, respectively. Methicillin resistance was significantly associated with older age (p 0.001), diabetes mellitus (p 0.004), absence of fundus visibility (p 0.06), and poor visual prognosis (p 0.03). Resistance to fluoroquinolones was significantly associated with absence of fundus visibility (p 0.05) and diabetes mellitus (p 0.02). This large prospective study demonstrates that methicillin resistance and, to a lesser extent, fluoroquinolone resistance in CNS strains causing postoperative endophthalmitis are both prevalent in France and associated with a poorer visual prognosis. These results emphasize the need for an effective surveillance of this antibiotic resistance and the development of new diagnostic tools for rapid detection for early optimization of antibiotic therapy in endophthalmitis patients.

FRIENDS Group: clinical and microbiological characteristics of post-filtering surgery endophthalmitis.

PURPOSE: To study the clinical and microbiological characteristics as well as the prognostic factors for post-filtering surgery endophthalmitis. METHODS: Twenty-three eyes were included in the study in four tertiary centres between 2004 and 2010. The clinical and microbiological data were collected prospectively (minimum follow-up, 6 months). Microbiological diagnosis was based on conventional cultures and panbacterial PCR (16SrDNA amplification and sequencing). RESULTS: The onset of endophthalmitis was early (<6 weeks) in 22 % of the cases and delayed in 78 %. Elevated intraocular pressure and hypopyon were more frequent in delayed than in early presentations (p = 0.04). By combining the results of culture and panbacterial PCR, a bacterial species could be identified in 73.9 % of the cases, including 56.5 % of commensal species of the digestive tract such as Moraxella spp., oropharyngeal streptococci and Enterococcus faecalis. Good final visual acuity (VA ≥ 20/40) was correlated with initial VA greater than light perception (p = 0.05). Poor final VA (≤20/400) was correlated with a higher virulence of the infecting bacterial species (p = 0.006), and was noted in all patients with early-onset endophthalmitis. CONCLUSION: Acute early- or delayed-onset post-filtering surgery endophthalmitis is frequently caused by bacteria of the digestive tract (e.g., Streptococcus and Enterococcus spp.). The combination of conventional cultures and panbacterial PCR allowed us to identify the causative microorganism in three-quarters of the cases, i.e., 21 % more cases than through culture alone. Despite adequate antibiotic and surgical treatment, the anatomical and visual prognosis remains poor.

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

Clostridium sordellii brain abscess diagnosed by 16S rRNA gene sequencing.

Clostridium sordellii is usually associated with skin and soft tissue infections. We describe the first case to our knowledge of a Clostridium sordellii-associated brain abscess, diagnosed by 16S rRNA gene sequencing, expanding the microbiological spectrum of brain abscesses, with emphasis on the role of 16S rRNA gene PCR in their etiologic diagnosis.

[Efflux pumps: their role in Staphylococcus aureus antibiotic resistance]. / Les phénomènes de résistance aux antibiotiques liés aux pompes à efflux : exemple de Staphylococcus aureus.

Efflux pumps located in the bacterial membranes are responsible for low level resistance to antibiotics, considered not to be relevant in the clinic and thus often neglected. However, these pumps contribute to the emergence of high level antibiotic resistance mechanisms, which are responsible for severe complications during the treatment of infectious diseases. Therefore it is necessary to take into account these pumps while developing novel antibacterial agents. Among these new research strategies, the development of efflux pump inhibitors seems to be an attractive approach to restore the activity of some "classical" antibiotics and to limit the emergence of multiresistant strains associated with hospital-acquired infections. In this review, we focalise on Staphylococcus aureus efflux pumps and their potential inhibitors.

[New microbiologic diagnostic techniques in infective endocarditis]. / Nouvelles techniques de diagnostic microbiologique dans l'endocardite infectieuse.

The new techniques used for identifying causal germs in infective endocarditis are reviewed. Techniques such as polymerase chain reaction are now used to identify previously unrecognised germs. Choosing the appropriate microbiologic technique requires close collaboration between clinicians and microbiologists.

Acute postoperative endophthalmitis caused by Staphylococcus lugdunensis.

Acute postoperative endophthalmitis caused by Staphylococcus lugdunensis is infrequently reported in clinical studies. Five cases of acute postcataract surgery endophthalmitis caused by S. lugdunensis were taken from a multicenter prospective study conducted in four university-affiliated hospitals in France (2004 to 2005). These cases were characterized by severe ocular inflammation occurring with a mean delay of 7.6 days after cataract surgery, severe visual loss (hand motions or less in three cases), and dense infiltration of the vitreous. Each of these patients was initially treated by using a standard protocol with intravitreal (vancomycin and ceftazidime), systemic, and topical antibiotics. Given the severity of the endophthalmitis, even though bacteria were sensitive to intravitreal antibiotics, pars plana vitrectomy was needed in four cases. The final visual prognosis was complicated by severe retinal detachment in three cases. The microbiological diagnosis was reached by using conventional cultures with specific biochemical tests and eubacterial PCR amplification followed by direct sequencing.