Is TNF-α a prognostic factor in patients with sepsis?

Objective: To determine tumor necrosis factor-α (TNF-α) levels in a prospective study in 58 hospitalized patients in a department of internal medicine (63 episodes, 29 in immunocompromised patients) during a 7-month period.Methods: Patients fulfilling the following criteria were included: clinical evidence of acute infection, temperature >38.2°C, tachycardia >90 beats/min, tachypnea >20 breaths/min. Samples were taken from day 1 up to day 13 after an infection was diagnosed, and TNF-α was determined by enzyme immunoassay.Results: In 29 episodes (46.0%) the infection was microbiologically documented. The median of the TNF-α levels in the Gram-negative episodes was significantly higher than that in the Gram-positive episodes (p = 0.002). Thirteen of 63 episodes (20.6%) had a fatal outcome. With respect to all measured values, the non-survivors had a significantly higher median of TNF-α levels than the survivors (p = 0.0001). There was, however, great interpatient and intrapatient variability in TNF-α levels; thus, no unequivocal correlation between TNF-α and outcome could be documented.Conclusions: Our data indicate that the influence of the infecting organism on TNF-α kinetics is less pronounced than that of the underlying disease

The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles

Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18-24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL of vancomycin and 8 μg mL of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation

Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco’s modified Eagle’s medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory