BackgroundDuring the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.ResultsComparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence.ConclusionThe assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

Background and purposePolymerase chain reaction (PCR) methods enable detection and species identification of many pathogens. We assessed the efficacy of a new PCR and microarray-based platform for detection of bacteria in prosthetic joint infections (PJIs).MethodsThis prospective study involved 61 suspected PJIs in hip and knee prostheses and 20 negative controls. 142 samples were analyzed by Prove-it Bone and Joint assay. The laboratory staff conducting the Prove-it analysis were not aware of the results of microbiological culture and clinical findings. The results of the analysis were compared with diagnosis of PJIs defined according to the Musculoskeletal Infection Society (MSIS) criteria and with the results of microbiological culture.Results38 of 61 suspected PJIs met the definition of PJI according to the MSIS criteria. Of the 38 patients, the PCR detected bacteria in 31 whereas bacterial culture was positive in 28 patients. 15 of the PJI patients were undergoing antimicrobial treatment as the samples for analysis were obtained. When antimicrobial treatment had lasted 4 days or more, PCR detected bacteria in 6 of the 9 patients, but positive cultures were noted in only 2 of the 9 patients. All PCR results for the controls were negative. Of the 61 suspected PJIs, there were false-positive PCR results in 6 cases.InterpretationThe Prove-it assay was helpful in PJI diagnostics during ongoing antimicrobial treatment. Without preceding treatment with antimicrobials, PCR and microarray-based assay did not appear to give any additional information over culture.

Background The morbidity and mortality from severe sepsis depends largely on how quickly and comprehensively evidencebased therapies are administered. As such, a huge opportunity exists. However, optimal care requires not only factual knowledge, but also numerous practical strategies including the ability to recognize a disease, to identify impending crises, to communicate effectively, to run a team, to work under stress and to simultaneously coordinate multiple tasks. Medical simulation offers a way to practice these essential crisis management skills, and without any risk to patients. Methods Following a didactic lecture on the key components of the Surviving Sepsis Campaign Guidelines, we trained 20 emergency medicine residents on a portable Laerdal Patient Simulator. Pre-programmed sepsis scenarios were developed following a needs assessment and modified Delphi technique. To maximize realism, this was performed in the acute care area of the Emergency Department and included a pre-briefed respiratory therapist and nurse. We videotaped resident performance and provided nonpunitive feedback, focusing on the comprehensiveness of therapy (for example, whether broad-spectrum antibiotics were given) and crisis resource management strategies (for example, whether help was asked for and tasks were appropriately allocated). Results Evaluation using a five-point Likert scale demonstrated that participants found this very useful (4.5/5), that lessons were complementary and supplementary to those learned from lectures (4.5/5) and that medical simulation was realistic (4/5). In addition, despite prior sepsis lectures, comparison of pre-tests and posttests showed that more emergency medicine residents would: administer broad-spectrum antibiotics as soon as possible following hypotension (14/20 pre-test, compared with 16/20 posttest), administer low-dose corticosteroids for those with refractory shock (10/20 pre-test, compared with 13/20 post-test), and would favour norepinephrine as a vasopressor (8/20 pre-test, compared with 12/20 post-test). Participants specifically valued the chance to observe and practice crisis resource management skills, which they felt had not been previously addressed (19/20). Conclusion Medical simulation appears to be an effective way to change both knowledge and behaviours in the treatment of severe sepsis. Many education and licensing boards also expect trainees to become not only content experts, but also effective communicators, collaborators, resource managers and advocates. These laudable goals are difficult to capture with traditional lectures but are comparably easy using medical simulation. We hope others will consider medical simulation as a complementary teaching and quality-assurance strategy in the fight against sepsis. P2 Protective potential of 2-chloroadenosine in Klebsiella pneumoniae B5055 induced sepsis in BALB/c mice

Introduction Urokinase receptor (uPAR, CD87), a glycosylphosphatidylinositol-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in Southeast Asia. We here investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. Methods Using a translational approach we conducted a patient study in patients with culture-confirmed sepsis caused by B. pseudomallei, in vitro experiments using wild-type (WT) and uPAR knockout (KO) cells, and mouse studies using WT and uPAR KO mice inoculated with B. pseudomallei. Results uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with WT mice, corresponding with increased pulmonary and hepatic inflammation. uPAR KO mice demonstrated significantly reduced neutrophil migration towards the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPARdeficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. Conclusions These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils towards the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei. P2 A comparison of acute lung inflammation in Klebsiella pneumoniae B5055-induced pneumonia and sepsis in BALB/c mice