Profiling of volatile organic compounds produced by clinical Aspergillus isolates using gas chromatography-mass spectrometry.

Volatile organic compounds (VOCs) in exhaled breath may identify the presence of invasive pulmonary aspergillosis. We aimed to detect VOC profiles emitted by in vitro cultured, clinical Aspergillus isolates using gas chromatography-mass spectrometry (GC-MS). Three clinical Aspergillus isolates and a reference strain were cultured while conidiation was prevented. Headspace samples were analyzed using a standardized method. Breath samples of patients from which the cultures were obtained were checked for the presence of the VOCs found in vitro. Each Aspergillus isolate produced a distinct VOC profile. These profiles could not be confirmed in exhaled breath in vivo.

Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies.

Rapid diagnostic testing of methicillin-resistant Staphylococcus aureus carriage at different anatomical sites: costs and benefits of less extensive screening regimens.

Multiple body site screening and pre-emptive isolation of patients at risk for methicillin-resistant Staphylococcus aureus (MRSA) carriage are considered essential for control of nosocomial spread. The relative importance of extranasal screening when using rapid diagnostic testing (RDT) is unknown. Using data from a multicentre study evaluating BD GeneOhm™ MRSA PCR (IDI), Xpert MRSA (GeneXpert) and chromogenic agar, added to conventional cultures, we determined cost-effectiveness assuming isolation measures would have been based on RDT results of different hypothetical screening regimes. Costs per isolation day avoided were calculated for regimes with single or less extensive multiple site RDT, regimes without conventional back-up cultures and when PCR would have been performed with pooling of swabs. Among 1764 patients at risk, MRSA prevalence was 3.3% (n = 59). In all scenarios the negative predictive value is above 98.4%. With back-up cultures of all sites as a reference, the costs per isolation day avoided were €15.19, €30.83 and €45.37 with 'nares only' screening using chromogenic agar, IDI and GeneXpert, respectively, as compared with €19.95, €95.77 and €125.43 per isolation day avoided when all body sites had been screened. Without back-up cultures costs per isolation day avoided using chromogenic agar would range from €9.24 to €76.18 when costs per false-negative RDT range from €5000 up to €50 000; costs for molecular screening methods would be higher in all scenarios evaluated. In conclusion, in a low endemic setting chromogenic agar screening added to multiple site conventional cultures is the most cost-effective MRSA screening strategy.

[Treatment of recurrent Clostridium difficile-associated diarrhoea with a suspension of donor faeces]. / Behandeling van recidiverende Clostridium dificile-geassocieerde diarree met een suspensie van donorfeces.

OBJECTIVE: To study the effect of treating recurrent Clostridium difficile-associated diarrhoea (CDAD) with a suspension of donor faeces. DESIGN: Uncontrolled interventional study. METHOD: Patients that, despite adequate antibiotic therapy, had developed at least 2 recurrences ofCDAD, including at least one recurrence that had been treated with a vancomycin tapering regimen, were included in the study. Relatives or volunteers served as faeces donor. All donors were previously examined for the presence of HIV, hepatitis B- and C-virus, and acute infection with cytomegalovirus or Epstein-Barr virus. The donor faeces were examined for the presence of C. difficile, Yersinia, Campylobacter, Shigella, Salmonella, and parasites. Before the infusion of donor faeces, the patients were treated for 4 days with vancomycin 500 mg q.i.d., followed by colon lavage. The suspension of 150 g of donor faeces dissolved in 300-400 ml of NaCl was infused into the jejunum via a duodenal catheter or into the caecum via colonoscopy. RESULTS: 7 CDAD patients were included and treated, including 2 with the hypervirulent C. difficile-strain PCR ribotype 027, toxinotype III. In 5 patients, the defaecation frequency returned to normal almost immediately after treatment and the cultures and toxin tests for C. difficile were repeatedly negative. In the remaining 2 patients, the treatment was successful after a repeated infusion of faeces from a different donor. CONCLUSION: Treatment with donor faeces seems promising for patients who develop repeated recurrences despite adequate therapy and could be valuable in the future during (local) epidemics of the PCR ribotype 027 strain. A randomised nationwide study (FECAL trial) has been started in order to determine the efficacy of this treatment.

Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology.

A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.

[Life-threatening infections with a new strain of Clostridium difficile]. / Levensbedreigende infecties met een nieuwe variant van Clostridium difficile.

Three men, aged 39, 73, and 66 years, respectively, developed an infection with a new strain ofClostridium difficile, ribotype 027.C.difficile-associated diarrhoea (CDAD) occurred in two patients after multiple abdominal surgery and in the third patient one week after autologous haematopoietic cell transplantation. Within a few days, despite antibiotic therapy, all three patients developed severe (pseudomembranous) colitis with sepsis for which admission to the Intensive Care Unit was required. Two patients underwent (sub)total colectomy and received an intensive course of oral and/or rectal vancomycin. In all patients who develop diarrhoea in hospital, especially during or after treatment with antibiotics or chemotherapeutic agents, an infection with C. difficile ribotype 027 should be suspected. Recent outbreaks of this hypervirulent strain of C. difficile have been reported in Canada, the United States, United Kingdom, and The Netherlands. Demonstration of C. difficile toxin in faeces confirms the clinical suspicion of CDAD and ribotyping of the strain may reveal whether the 027 strain is present. For treatment of these 027 infections, vancomycin is preferred to metronidazole. After a severe course of colitis or in case of recurrence a 'tapering and pulse' course ofvancomycin can be prescribed; alternatively, treatment with bovine antibody-enriched whey may be considered. The introduction of this hypervirulent strain has led to reinforcement of the hygienic measures in accordance with the recommendations of the Dutch Working Party on Infection Prevention and a policy to deter the use of fluoroquinolones.

Chemokines produced by mesothelial cells: huGRO-alpha, IP-10, MCP-1 and RANTES.

Recently we showed the in vivo relevance of chemokines in cases of bacterial peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. Mesothelial cells, the most numerous cells in the peritoneal cavity, are hypothesized to function as a main source of chemokine production. We investigated the time- and dose-dependent expression patterns of four chemokines by mesothelial cells at the mRNA and protein level in response to stimulation with physiological doses of proinflammatory mediators that are present at the site of bacterial inflammation. Besides the chemokines huGRO-alpha (attractant for neutrophils), MCP-1 and RANTES (monocyte attractants), the expression and production of IP-10 was analysed. Mesothelial cells were cultured and stimulated with either IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma or combinations of these. The time- and dose-dependent mRNA expression of the chemokines was determined by Northern blot analysis and the protein production by ELISA. It was concluded that mesothelial cells could indeed be triggered by the mentioned stimuli to induce mRNA and protein production (huGRO-alpha and IP-10) or to augment constitutive protein production (MCP-1). However, RANTES mRNA and protein production could only be induced in some cases and only in small amounts. The chemokine response of mesothelial cells was regulated differentially, depending on the stimulus and the chemokine measured. In distinct cases, combination of the stimuli led to synergy in mRNA expression and protein production. The presented in vitro data support our hypothesis that mesothelial cells in vivo are the main source of relevant chemokines in response to proinflammatory mediators, suggesting an important role for mesothelial cells in host defence.

Production of IL-1 beta and TNF-alpha by peritoneal macrophages depends on the bacterial species and the inoculum.

Peritoneal macrophages (PMs) are very potent producers of proinflammatory stimuli, such as interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha. After contact with invading micro-organisms, PMs produce different cytokines, both pro- and anti-inflammatory. Therefore, they are crucial in the regulation of inflammatory events. The aim of this study was to investigate whether the production of IL-1 beta and TNF-alpha is dependent on the bacterial species used. PMs were harvested from spent peritoneal dialysis effluent and subsequently stimulated with five strains of bacteria in two different concentrations. After 24 hours of stimulation, supernatants were harvested and analyzed for both IL-1 beta and TNF-alpha content. IL-1 beta was measured with a commercial ELISA, and TNF-alpha was determined with a bioassay. Both the IL-1 beta and the TNF-alpha production were species-dependent. One strain of Staphylococcus aureus and one strain of Staphylococcus epidermidis induced a markedly higher response in both IL-1 beta and TNF-alpha than the other species. This response was also dose-dependent, and this holds true for all species. In conclusion, the IL-1 beta and TNF-alpha response by PMs is both species- and dose-dependent.

Identification of the major chemokines that regulate cell influxes in peritoneal dialysis patients.

To investigate which members of the recently discovered family of chemotactic cytokines (chemokines) are important in leukocyte recruitment to a bacterial inflammation site, four different chemokines in the effluent of peritoneal dialysis patients suffering from acute bacterial peritonitis were measured. The presence of two neutrophil-attracting chemokines, interleukin-8 and human melanoma growth-stimulating activity (huGRO alpha), and two monocyte-attracting members of the chemokine superfamily, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES), was investigated in patient effluents just before, during, and after a peritonitis episode. This was studied in seven peritonitis effluents of five patients by using chemokine-specific enzyme-linked immunosorbent assays. Cell populations in the dialysates were differentiated on cytocentrifuge preparations. The contribution of the detected chemokines to neutrophilic and monocytic cell influxes in the inflamed peritoneal cavity was analyzed by correlating concentrations of chemokines to the relevant cell numbers present in the dialysates of these patients. The detection of the neutrophil-attracting chemokine interleukin-8 during peritonitis was in accordance with other studies. Moreover, a second neutrophil chemoattractant, huGRO alpha, was identified in vivo. Both were elevated during inflammation (P < 0.02) and contributed significantly to the neutrophilic cell influx (P < 0.05). One of the monocyte-attracting chemokines, RANTES, could not be detected in any of the effluents, whereas the other, MCP-1, was significantly elevated during peritonitis (P < 0.02). In contrast to the other chemokines measured, MCP-1 concentration was relatively high in steady-state peritoneal dialysates. An absolute correlation between dialysate MCP-1 concentration and the number of macrophages in these effluents was absent. However, in a 48-well chemotaxis assay, monocyte migration toward peritonitis, as well as steady-state patient dialysates, could be blocked with antibodies to MCP-1. It was concluded, therefore, that MCP-1 is the most important monocyte chemoattractant in peritoneal dialysis steady-state and peritonitis patients; whereas, besides interleukin-8, huGRO alpha was identified as a major neutrophil-attracting chemokine in the peritonitis situation.

Ingestion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli by human peritoneal mesothelial cells.

In the present study we examined whether mesothelial cells can ingest and digest bacteria. The results showed that all strains were ingested. Ingested staphylococci proliferated abundantly, and only a few were digested. Escherichia coli, however, was digested during the first 8 h, whereafter the mesothelial cells disintegrated and proliferation of bacteria could be observed. The clinical implications of these findings are discussed.

Renal function influences interleukin-8 background production by cultured human mesothelial cells.

Previous studies have demonstrated that mesothelial cells (MC) are important in the local host defense system of the peritoneal cavity. Most studies on the function of MC are performed on MC derived from material of patients with normal renal function (NRF). The aim of the present study was to examine differences in interleukin (IL)-8 expression by MC from patients with NRF and from patients with end-stage renal disease (ESRD). Therefore, MC were isolated from the omentum and pleural exudate of patients with NRF, from spent effluent of stable peritoneal dialysis (PD) patients, and from omentum obtained during catheter implantation prior to PD treatment. MC were stimulated with increasing doses of IL-1 beta or tumor necrosis factor-alpha for 24 hours, after which the supernatant was analyzed for IL-8 content. The IL-8 background level of MC isolated from patients with NRF was significantly lower than the IL-8 background level of MC derived from patients with ESRD. Although IL-8 production appeared to be higher in the ESRD MC, this difference was not significant after stimulation. While the overall immunity is depressed in uremia, MC are activated. The relatively high background of IL-8 might lead to an insensitivity of neutrophils by blocking the receptors and explain their impaired chemotaxis in uremia.

Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs.

Interleukin-8 production by human mesothelial cells after direct stimulation with staphylococci.

Mesothelial cells (MC) are able to produce interleukin-8 (IL-8) after stimulation with IL-1 beta or tumor necrosis factor alpha. The aim of our study was to investigate whether MC are able to produce IL-8 after direct stimulation with clinically relevant bacteria. We observed a significant IL-8 response by the MC which were directly stimulated with viable staphylococci.

Cancer antigen 125: a bulk marker for the mesothelial mass in stable peritoneal dialysis patients.

Mesothelial cells that line the peritoneal cavity are capable of producing several proinflammatory cytokines such as interleukin-6 and interleukin-8. Since they are the most numerous cell in the peritoneal cavity when the lining mesothelial cells are included, they may play a major role in the local antibacterial defence mechanism. Cancer antigen (CA)125 is expressed by mesothelial cells (as by other coelomic epithelium-derived cells) and might therefore be considered a marker of the mesothelium. The aim of this study was to determine whether CA125 is a bulk or an activation stage mesothelial cell marker. A positive correlation was found between the mesothelial cell number and the CA125 concentration in dialysate of stable PD patients (P = 0.03). CA125 release by mesothelial cell cultures during confluence showed that the release per cell was constant in time. Stimulation of mesothelial cells in a confluential phase with IL1 beta, TNF alpha, IFN gamma and TGF beta did not result in an increase in CA125 release. Cell lysis showed that CA125 is also present intracellularly. This implies that release of intracellular CA125 can be a disturbing factor in interpreting the CA125 concentration of dialysate in situations where mesothelial cell death may occur, such as in peritonitis. It can be concluded, that our data show that dialysate CA125 is a bulk marker for the mesothelial cell mass in stable PD patients and can thus provide data on the state of the peritoneal membrane in the follow-up of the individual patient.

Analysis of the peritoneal cellular immune system during CAPD shortly before a clinical peritonitis.

We analysed the peritoneal cellular immune system 24-48 h before the onset of a clinical peritonitis. Peritoneal cells were obtained from the overnight dialysis effluents 1 or 2 days (day-1 and day-2) preceding the day of peritonitis, the last overnight effluent before the peritonitis effluent (day P), and the first peritonitis effluent. Nine peritonitis episodes of six patients were studied. The number of Fc receptor positive cells, the chemotactic activity, and immunophenotype of the peritoneal cell population at day-2 and day-1 were similar to the postperitonitis control effluent. However, immunophagocytosis and phagocytosis capacity of the peritoneal macrophages was decreased in five of six episodes at day-2 and -1 compared to control peritoneal macrophages. The overnight effluents of day P revealed a moderate influx of neutrophilic granulocytes and an increase of bacterial killing capacity and chemotactic activity. Activation of the peritoneal T cells at day P was shown by the increase in MHC class II positive T cells and an increase in the CD4/CD8 ratio. Bacterial cell cultures of the effluents were positive in three episodes 24-48 h before peritonitis, and of all overnight effluents at day P. These results indicate that malfunctioning of phagocytosis by peritoneal macrophages may contribute to the development of a CAPD peritonitis.

Microwave treatment of xenogeneic cartilage transplants.

Human rib cartilage was irradiated with microwaves according to six different methods and transplanted into rabbits. Untreated rib cartilage preserved in Cialit served as a control. After 12 and 40 wk of implantation, the microscopic appearance of these xenogeneic cartilage transplants was given a score in comparison with the transplants preserved in Cialit. The microwave treatment of the cartilage appeared to improve the results. The Cialit-preserved transplants showed progressive resorption by macrophages with central necrosis and fragmentation, which was not present in the microwave-treated grafts. Microwaves seem to stabilize the cartilage matrix and enhance the diffusion of fixatives. Irradiation in ethanol as an immersion fluid appeared to be the best method. The results of transplantation can benefit from the use of microwaves in the preservation of the cartilage. It is argued that, in addition, microwave irradiation might be used for inactivation of human immunodeficiency virus in human cartilage used for transplantation.

[Immunology of cholesteatoma]. / L'immunologie du cholestéatome.

The presence of Langerhans cells and T cells in human cholesteatoma is demonstrated by immunohistochemical and submicroscopic analyses. Various monoclonal antibodies directed against Langerhans cells and T lymphocytes reveal the presence of these cell populations in the cholesteatoma matrices but not in the normal tympanic membrane. Their quantitative and spatial distribution in and around the cholesteatoma matrix might be of importance in the pathophysiology of human cholesteatoma.

Spatial distribution of Langerhans' cells and T-lymphocyte subpopulations in human tympanic membrane and aural cholesteatoma.

Immunohistochemical analysis of human cholesteatoma matrices revealed the presence of Langerhans' cells and T-lymphocytes. Through cell-to-cell interaction, Langerhans' cells probably play a key role in skin-related disorders, including cholesteatomas. They probably originate from a mobile cell population of monocyte origin and migrate into and out of the body's lining. Their custodial function is often carried out in close relation with T-lymphocytes. Monoclonal antibodies against Langerhans' cells and T-lymphocyte membrane receptors reveal the presence of these cell populations in cholesteatoma matrices but not in the tympanic membrane. Langerhans' cells and T-cell "traffic" through cholesteatomas are discussed in relation to the pathogenesis, natural course and recurrence of cholesteatomas. Through immunopathologic evaluation the clinical aggressiveness of a cholesteatoma may become predictable. It may even have consequences for the future handling of cholesteatomas.

Immunobiology of Langerhans' cells migrating into aural cholesteatomas.

Immunohistochemical and submicroscopic analyses of human cholesteatoma matrices reveal the presence of Langerhans' cells and T lymphocytes. Through cell-to-cell interaction, Langerhans' cells probably play a key role in skin-related disorders, including cholesteatomas. They originate from a mobile cell population of monocyte origin and migrate into and out of the body's lining. Their custodial function is always carried out in close relation with T lymphocytes. Various monoclonal antibodies directed against Langerhans' cell and T lymphocyte membrane receptors reveal the presence of these cell populations in cholesteatoma matrices but not in the tympanic membrane. Langerhans' cell and T cell traffic through cholesteatomas are discussed in relation to the pathogenesis, natural course, and recurrence rate of cholesteatomas. Through immunopathologic evaluation the clinical aggressiveness of a cholesteatoma will probably become predictable. Medical manipulation of Langerhans' cell and T cell functions- as an adjuvant to surgery - may have consequences for the future handling of cholesteatomas.