[Carbapenemase producing Enterobacteriaceae in the Netherlands: unnoticed spread to several regions]. / Carbapenemase-producerende enterobacteriën in Nederland.

- Carbapenemase producing Enterobacteriaceae (CPE), including Klebsiella pneumoniae and Escherichia coli, are only sporadically seen in the Netherlands and then mainly in patients who have been transferred from foreign hospitals.- CPE are resistant to virtually all beta-lactam antibiotics, including carbapenems, e.g., meropenem and imipenem. Several genes, e.g., OXA-48, KPC and NDM-1, code for carbapenemase enzymes that deactivate carbapenems.- Control of CPE focuses on timely identification of patients who are infected or are carriers and the application of preventive measures to prevent spread.- Genotypic analysis of CPE isolates submitted to the national CPE surveillance revealed close relationships between 8 NDM-1 positive K. pneumoniae isolates of patients from different parts of the Netherlands and isolates obtained through contact tracing during a known hospital outbreak.- Based on retrospective epidemiological investigation, no shared exposure could be found.- These findings indicate unnoticed spread of CPE in the Netherlands.

The aetiology of community-acquired pneumonia and implications for patient management.

PURPOSE: Understanding which pathogens are associated with clinical manifestation of community-acquired pneumonia (CAP) is important to optimise treatment. We performed a study on the aetiology of CAP and assessed possible implications for patient management in the Netherlands. METHODS: Patients with CAP attending the emergency department of a general hospital were invited to participate in the study. We used an extensive combination of microbiological techniques to determine recent infection with respiratory pathogens. Furthermore, we collected data on clinical parameters and potential risk factors. RESULTS: From November 2007 through January 2010, 339 patients were included. Single bacterial infection was found in 39% of these patients, single viral infection in 12%, and mixed bacterial-viral infection in 11%. Streptococcus pneumoniae was the most frequently identified pathogen (22%; n=74). Infection with atypical bacteria was detected in 69 (20%) of the patients. CONCLUSION: Initial empirical antibiotics should be effective against S. pneumoniae, the most common pathogen identified in CAP patients. The large proportion of patients with infection with atypical bacteria points to the need for improved diagnostic algorithms including atypical bacteria, especially since these atypical bacteria are not covered by the first-choice antibiotic treatment according to the recently revised Dutch guidelines on the management of CAP.

Dynamics of antiviral-resistant influenza viruses in the Netherlands, 2005-2008.

In the Netherlands, influenza specific antivirals are used for the therapy of influenza in nursing homes and hospitals, for prophylaxis in high risk groups and neuraminidase inhibitors are stockpiled as part of pandemic preparedness plans. To monitor the antiviral susceptibility profile, human influenza virus isolates derived from the Dutch influenza surveillance in 2005-2006 (n=87), 2006-2007 (n=58) and 2007-2008 (n=128) were analyzed with phenotypic assays and sequencing. For adamantanes, a high proportion (>74%) of A(H3N2) viruses had the S31N mutation in M2 protein, while variation in the HA(1) region of adamantane-sensitive viruses suggested that adamantane-sensitive variants were reseeded into the Dutch population and re-emerged as drug-sensitive due to M-segment reassortment. For neuraminidase inhibitors oseltamivir and zanamivir, 98% of types A and B influenza viruses prior to 2007-2008 were sensitive for both, whereas 24% of the A(H1N1) viruses obtained in 2007-2008 were oseltamivir-resistant while retaining sensitivity to zanamivir and adamantanes. Furthermore, oseltamivir-resistant A(H1N1) or adamantane-resistant A(H3N2) virus infections were not associated with differences in clinical symptoms compared to infections with sensitive variants. Our data show the dynamic nature of emergence of drug-resistant influenza viruses, stressing the need for surveillance of resistance trends as part of influenza monitoring programs.

Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model.

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.

Chitosan for mucosal vaccination.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.

Chitosan and its derivatives in mucosal drug and vaccine delivery.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.

In vivo uptake of chitosan microparticles by murine Peyer's patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry.

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.

Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches.

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this study chitosan microparticles were prepared and characterized with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyer's patches was studied using confocal laser scanning microscopy (CLSM). Chitosan microparticles were made according to a precipitation/coacervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3+/-0.7 microm in size and positively charged (20+/-1 mV). Since only microparticles smaller than 10 microm can be taken up by M-cells of Peyer's patches, these microparticles are suitable to serve as vaccination systems. CLSM visualization studies showed that the model antigen ovalbumin was entrapped within the chitosan microparticles and not only associated to their outer surface. These results were verified using field emission scanning electron microscopy, which demonstrated the porous structure of the chitosan microparticles, thus facilitating the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in loading capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) remained entrapped in the microparticles. Because the prepared chitosan microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be taken up by the epithelium of the murine Peyer's patches. Since uptake by Peyer's patches is an essential step in oral vaccination, these results show that the presently developed porous chitosan microparticles are a very promising vaccine delivery system.