We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-betalactamase-producing (ESBL ؉ ) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL ؉ bacteria detected in samples obtained from patients in our intensive care unit.Extended-spectrum beta-lactamases (ESBLs) are enzymes in gram-negative bacilli that confer resistance to the majority of ␤-lactam antibiotics up to the extended-spectrum cephalosporins. Their worldwide dissemination concerns clinicians, because infections with ESBL-producing (ESBL ϩ ) microorganisms are often not adequately covered with empirically started antibiotics. The proper choices of antibiotic therapy and infection control measures depend upon early and accurate ESBL detection; it is therefore pivotal to have a rapid and sensitive laboratory assay (4).The sensitivity of methicillin (meticillin)-resistant Staphylococcus aureus detection by culture is increased 9 to 25% by overnight enrichment of culture swabs in broth before inoculation on solid medium (2, 6). To the best of our knowledge, the effect of preenrichment on the sensitivity of detection of ESBL ϩ bacteria has not yet been determined. We have evaluated the effect of overnight enrichment in broth by culturing fecal samples that were spiked with genotypically characterized ESBL ϩ strains to see if normal flora of a fecal sample would interfere with detection of low numbers of ESBL-positive strains. The enrichment broth was also evaluated with clinical samples obtained from adult patients in two intensive care units (ICUs) of our hospital.For the spiking experiments, we used the Klebsiella pneumoniae K6 ATCC 700603 strain, which produces an SHV-18 ESBL (5), and two clinical isolates of Escherichia coli with a CTX-M-type ESBL. Bacterial suspensions of these strains with an optical density at a 0.5 McFarland standard were serially diluted in phosphate-buffered saline (PBS); nine 10-fold dilutions were made. To quantify the viable bacteria in each dilution step, a MacConkey agar was inoculated with 100 l of a suspension and incubated overnight at 37°C; the number of grown colonies was counted the following day. Spiked samples were made by adding 100 l of each dilution in PBS to 900 l of a fecal suspension that was obtained by suspending 6 grams of fresh feces from healthy volunteers in 60 ml of antibioticfree trypticase soy broth (TSB) with 0.5% sodium chloride (Becton Dickinson, Breda, The Netherlands). A fecal suspension without the addition of an ESBL ϩ strain was used as a negative control. Aliquots of 100 l of the spiked samples were subcultured in 900 l of TSB and onto beta-lactamase screening (BLSE) agar (Aes Chemunex, Bruz cedex, France). The BLSE agar is a commercially available double plate containing Drigalski medium supplemented with 1.5 g per ml cefotaxime and MacConkey agar with 2 g per ml ceftazidime. Gramnegative bacteria that are resistant to cephalosporins (including AmpC producers) can grow on this sele...

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptideloaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.

Favipiravir is a novel antiviral drug approved for influenza treatment in Japan. Little is known about favipiravir pharmacokinetics in critically ill patients. Here, we report a patient with influenza treated with favipiravir and undergoing continuous venovenous haemofiltration (CVVH) on the Intensive Care Unit of a tertiary hospital in the Netherlands. Pharmacokinetic analyses showed increased clearance and decreased plasma levels compared to healthy volunteers. CVVH has no clinically relevant contribution to total clearance. Despite susceptibility to favipiravir, the influenza virus was not cleared. A multi-disciplinary approach is needed to ensure optimal favipiravir treatment in critically ill patients.