Delayed Diagnosis of a Diffuse Invasive Gastrointestinal Aspergillosis in an Immunocompetent Patient.

Invasive aspergillosis represents a clinical picture frequently associated with host's immunosuppression which usually involves a high morbidity and mortality. In general, the most frequent fungal entry is the lungs with secondary hematogenous dissemination, but there are other hypotheses like a gastrointestinal portal of entry. There are some rare publications of cases with invasive aspergillosis in immunocompetent patients. We present the case of an immunocompetent patient without any risk factors except for age, ICU stay, and surgical intervention, who developed a septic shock by invasive gastrointestinal aspergillosis as primary infection. Due to the unusualness of the case, despite all the measures taken, the results were obtained postmortem. We want to emphasize the need not to underestimate the possibility for an invasive aspergillosis in an immunocompetent patient. Not only pulmonary but also gastrointestinal aspergillosis should be taken into account in the differential diagnosis to avoid a delay of treatment.

Accuracy of Sensititre YeastOne echinocandins epidemiological cut-off values for identification of FKS mutant Candida albicans and Candida glabrata: a ten year national survey of the Fungal Infection Network of Switzerland (FUNGINOS).

OBJECTIVES: Echinocandins represent the first-line treatment of candidaemia. Acquired echinocandin resistance is mainly observed among Candida albicans and Candida glabrata and is associated with FKS hotspot mutations. The commercial Sensititre YeastOne™ (SYO) kit is widely used for antifungal susceptibility testing, but interpretive clinical breakpoints are not well defined. We determined echinocandins epidemiological cut-off values (ECV) for C. albicans/glabrata tested by SYO and assessed their ability to identify FKS mutants in a national survey of candidaemia. METHODS: Bloodstream isolates of C. albicans and C. glabrata were collected in 25 Swiss hospitals from 2004 to 2013 and tested by SYO. FKS hotspot sequencing was performed for isolates with an MIC≥ECV for any echinocandin. RESULTS: In all, 1277 C. albicans and 347 C. glabrata were included. ECV 97.5% of caspofungin, anidulafungin and micafungin were 0.12, 0.06 and 0.03 µg/mL for C. albicans, and 0.25, 0.12 and 0.03 µg/mL for C. glabrata, respectively. FKS hotspot sequencing was performed for 70 isolates. No mutation was found in the 52 'limit wild-type' isolates (MIC=ECV for at least one echinocandin). Among the 18 'non-wild-type' isolates (MIC>ECV for at least one echinocandin), FKS mutations were recovered in the only two isolates with MIC>ECV for all three echinocandins, but not in those exhibiting a 'non-wild-type' phenotype for only one or two echinocandins. CONCLUSION: This 10-year nationwide survey showed that the rate of echinocandin resistance among C. albicans and C. glabrata remains low in Switzerland despite increased echinocandin use. SYO-ECV could discriminate FKS mutants from wild-type isolates tested by SYO in this population.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland.

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Quantitative PCR to diagnose Pneumocystis pneumonia in immunocompromised non-HIV patients.

The utility of quantitative Pneumocystis jirovecii PCR in clinical routine for diagnosing Pneumocystis pneumonia (PCP) in immunocompromised non-HIV patients is unknown. We analysed bronchoalveolar lavage fluid with real-time quantitative P. jirovecii PCR in 71 cases with definitive PCP defined by positive immunofluorescence (IF) tests and in 171 randomly selected patients with acute lung disease. In those patients, possible PCP cases were identified by using a novel standardised PCP probability algorithm and chart review. PCR performance was compared with IF testing, clinical judgment and the PCP probability algorithm. Quantitative P. jirovecii PCR values >1,450 pathogens·mL(-1) had a positive predictive value of 98.0% (95% CI 89.6-100.0%) for diagnosing definitive PCP. PCR values of between 1 and 1,450 pathogens·mL(-1) were associated with both colonisation and infection; thus, a cut-off between the two conditions could not be identified and diagnosis of PCP in this setting relied on IF and clinical assessment. Clinical PCP could be ruled out in 99.3% of 153 patients with negative PCR results. Quantitative PCR is useful for diagnosing PCP and is complementary to IF. PCR values of >1,450 pathogens·mL(-1) allow reliable diagnosis, whereas negative PCR results virtually exclude PCP. Intermediate values require additional clinical assessment and IF testing. On the basis of our data and for economic and logistical limitations, we propose a clinical algorithm in which IF remains the preferred first test in most cases, followed by PCR in those patients with a negative IF and strong clinical suspicion for PCP.

Systematic improvement of a chemically-defined protein-free medium for hybridoma growth and monoclonal antibody production.

A chemically-defined protein-free medium (FMX-Turbodoma) has been improved for the production of monoclonal IgA antibodies by hybridoma cells, using a systematic method. Cell growth rate, IgA production, activity and molecular weight pattern have been used as optimization criteria. Of potentially important supplements, glucose, glutamine, Pluronic acid F-68 as well as several amino acids had significant beneficial effects. A determination of amino acids profiles via HPLC analysis allowed the formulation of a balanced medium. Unbalanced supplementations of amino acids were found undesirable because of the toxicity of some amino acids at high concentration. Compared with the basal medium, the maximum viable cell and final IgA concentrations in the final version of the protein-free medium were increased by 130% and 700%, respectively, whereas the IgA molecular weight pattern and in vitro activity were not affected. The IgA production was even higher than in a serum-containing medium (RPMI 1640 + 10% FCS) and the price of the protein-free medium is about 20% of this serum-containing medium. This makes such a protein-free medium very convenient for laboratory and large-scale production.

The skin as a target organ for the investigation of antiallergic drugs: comparison between cetirizine and terfenadine.

Two double-blind clinical pharmacology studies were performed in atopics in order to compare the inhibitory effects of cetirizine (CTZ) 2 HCl and terfenadine (TER) on histamine and antigen-induced skin reactions. In the first study, the subjects took single intakes of CTZ 10 mg and TER 60 mg. In the second study, they took CTZ 10 mg once a day and TER 60 mg b.i.d. for 3 weeks. CTZ was more effective than TER in inhibiting histamine skin reactivity. CTZ and TER were equally effective in inhibiting antigen-induced reactions. There was no tachyphylaxis, either for CTZ or for TER.

[Hyposensitization in pollinosis. Results of a 3-year controlled study with 2 depot-allergoid grass pollen extracts: aluminum hydroxide-adsorbed allergoid and tyrosine-adsorbed allergoid]. / Zur Hyposensibilisierung der Pollinose. Ergebnisse einer kontrollierten Studie über drei Jahre mit zwei Depotallergoid-Graspollenextrakten: Aluminiumhydroxid-adsorbiertes Allergoid (AGD) und Tyrosin-adsorbiertes Allergoid (TA).

For controlled hyposensitization treatment over a period of three years 36 patients with confirmed grass pollen sensitization had been selected in 1986 and randomly distributed to receive preseasonal injection therapy: 23 patients were treated with an average of seven AGD (aluminium-adsorbed allergoid) injections, and 13 patients had received six TA (tyrosine-adsorbed allergoid) injections. Evaluation of the trial data collected during three years of preseasonal treatment showed the following results of tolerance and efficacy: Systemic side-reactions registered during therapy were only mild and transient and occurred in the average after 3% of the AGD injections and after 10% of the TA injections. Local reactions over 5 cm diameter were registered after 7% in the AGD group and after 9% in the TA group. Before therapy there was no significant difference (p greater than 0.05) between the groups; after three years of therapy the AGD injections had resulted in a mean net rise of specific IgG of 220% (significant, p = 0.001); during the same time, TA injections had resulted in a final net increase of 10% (not significant, p greater than 0.05). Both treatment forms did not lead to any statistically relevant changes of specific IgE values. After three years of hyposensitization treatment, patients of both groups had improved; but an advantage was documented for patients treated with AGD on the basis of scores for objective assessment as well as for registered symptom and medication scores.

[Angioedema caused by enalapril (Reniten)]. / Enalapril-(Reniten-) assoziierte Angioödeme.

Two cases of enalapril(Reniten)-induced angioedema are described. In both patients the time lag between the first manifestation of angio-edema and diagnosis was more than one year, during which several bouts of edema occurred. One patient developed life-threatening swelling of the tongue and the larynx followed by asystole and apnea. The second patient had recurrent edema of the tongue and dyspnea. In general, enalapril-induced edema is not thought to be based on immunological mechanisms. However, in both patients we found elevated titres of antinuclear antibodies, which were reversible upon cessation of enalapril medication. The possible pathomechanisms are discussed.

[Anaphylactic reaction to latex, an immediate-type allergen of increasing significance]. / Anaphylaktische Reaktion auf Latex, ein Soforttypallergen von zunehmender Bedeutung.

We report on two patients with immediate-type allergy to latex, an allergen that is receiving increasing attention. Both patients presented with anaphylactic symptoms after gynecological examination. In addition, one patient reacted with an anaphylactic shock immediately after insertion of a latex intestinal tube for a Holzknecht test. Of all the cases of immediate-type allergy to latex published so far, 45% had localized urticaria, 43% mucocutaneous reaction, and 10% an anaphylactic shock; 74% of the patients were atopic.

The preparation and characterisation of native photoreceptor units from the thylakoids of Rhodopseudomonas viridis.

The photosynthetic membranes of Rhodopseudomonas viridis consist of a regular array of structural units. Each unit is composed of a central core (thought to contain the reaction centre complex) surrounded by a subdivided ring of protein (of likely antennae function). These individual units can be dissociated from the membrances using a variety of detergent treatments. The absorption spectrum, used as a criterion of a native state, is retained. All of the seven major polypeptides, the four reaction centre polypeptides (cytochrome, H, M and L chain) as well as the three light-harvesting polypeptides (B1015-alpha, beta and xi) are shown to be present. Electron microscopy of the units shows a similar structure to the units within the membrane. surface-specific iodination of both membranes and units labels predominantly polypeptides H, B1015-alpha, and xi. M and L are weakly labelled. In addition, B1015-beta is labelled in the isolated units. This, with other evidence, supports an allocation of light-harvesting polypeptides to the outer ring. Further solubilisation of these units separates the reaction centre (as a native complex containing all four polypeptides) from the light-harvesting polypeptides. The light-harvesting polypeptides are obtained in a form containing all three polypeptides and bound pigment, however the peak at 1015 nm corresponding to native bacteriochlorophyll b is lost.

The structure of the photoreceptor unit of Rhodopseudomonas viridis.

The thylakoid membrane of Rhodopseudomonas viridis contains extensive, regular arrays of photoreceptor complexes arranged on a hexagonal lattice with a repeat distance of 130 A. Single membrane sheets were obtained by mild treatment of the thylakoid fraction with the detergent Triton X-100. Heavy metal shadowing and electron microscopy of isolated thylakoids indicated a strong asymmetry of the membrane, showing a smooth plasmic and a rough exoplasmic side. Fourier processing of rotary-shadowed specimens showed the different surface relief on both sides of the membrane. Structural units on both sides were roughly circular and showed 6-fold symmetry at a resolution close to 20 A. The structural unit was characterised by a central core that seemed to extend through the membrane, protruding on the exoplasmic side. The core was surrounded by a ring showing 12 subunits on the plasmic side. Rotary-shadowed as well as negatively-stained membranes indicated a handedness of the structure. Treatment of thylakoid vesicles with higher detergent concentrations yielded a fraction of particles showing the same features as Fourier maps of the structural units. The isolated particles therefore appeared to represent structurally intact units of photosynthesis.

Localisation of Rhodopseudomonas viridis reaction centre and light harvesting proteins using ferritin-antibody labelling.

Rhodopseudomonas viridis thylakoid membrane polypeptides were characterised by SDS gels, 2 D gels and surface-specific iodination. Four polypeptides with apparent molecular weights of 38 000, 33 000, 27 000, and 24 000 (reaction centre) and three low molecular weight polypeptides 11 000, 8000 and 6000 (probably light harvesting polypeptides) were identified. Antibodies were produced against the polypeptides eluted from SDS gels and tested for specificity by an immunoblotting assay. The antibodies were bound to the membranes and viewed by electron microscopy using a modification of the ferritin labelling technique. It is suggested that antigenic determinants for the 38 000, 33 000, and 27 000 reaction centre polypeptides and the 11 000 and 8000 low molecular weight polypeptides are present on the cytoplasmic membrane surface. The 33 000, 27 000, 11 000 and 6000 polypeptides appear to have surface-located residues which can be iodinated. The photosynthetic membrane of Rps. viridis appears to be a highly asymmetrical membrane.

On the linkage of exoplasmatic freeze-fracture particles to phycobilisomes.

The thylakoids of the thermophilic cyanobacterium Mastigocladus laminosus were examined by freeze-fracture analysis. The expolasmatic (EF)-freeze-fracture particles are organized in rows, separated by 45 nm or more with a 12-nm center-tocenter spacing of neighboring particles. Phycobilisomes, associated to the outer thylakoid surfaces show a similar spacing pattern. Fractures exposing simultaneously phycobilisomes and EF-freeze-fracture particles on the same thylakoid show a direct alignment of both systems. Consequently the phycobilisomes are concluded to be associated peripherally on top of the EF-freeze-fracture particles in a 1:1 assembly pattern. The periodicity of the EF-freeze-fracture particles determines the arrangement of the phycobilisomes in the rows. The planar phycobilisome model of Mörschel et al. (1977) easily allows a successive arrangement of the phycobilisomes in a row, whereas with the staggered model developed by Bryant et al. (1979), only a cogged arrangement of neighboring phycobilisomes is possible.

Isolation and localization of plasma membrane-bound invertase in yeast (Saccharomyces cerevisiae).

Latex spheres of 60 nm diameter (synthesized by aqueous emulsion copolymerization of methacrylate derivatives according to [22]) were coated with bovine serum albumin (BSA) and concanavalin A. By virtue of their size and their high density (1.32-1.35 g/ml) they are well suited as scanning electron microscopy markers and as affinity density perturbation reagents. Yeast protoplasts could be labeled with these spheres and the amount of binding depended upon incubation time and temperature. Isolated and solubilized yeast plasma membranes were incubated with these spheres and by density gradient centrifugation the membrane glycoproteins could be separated from the other proteins by the method of affinity density perturbation. Since the yeast plasma membrane glycoproteins exhibit invertase activity [1, 19] the activity of the different fractions was either detected on gels by staining for invertase activity or measured in vitro and quantified; a 6 to 7fold purification of the enzyme was achieved. Protoplasts labeled with antibodies directed against these glycoproteins exhibited a distribution of ferritin marker molecules that was very similar to that of the intramembranous particles. Antibodies against extracellular invertase cross reacted with the plasma membrane of glycoproteins and showed the same distribution of markers as the antibodies against the glycoproteins. It can therefore be concluded that the yeast plasma membrane glycoproteins exhibit invertase activity and that they are associated with the intramembranous particles.

Specific labeling of glycoproteins in yeast plasma membrane with concanavalin A.

The intramembranous particles of yeast Saccharomyces cereisiae plasma membrane form paracrystalline arrays or are randomly distributed as seen by freeze-fracture electron microscopy. Protoplasts with randomly distributed particles and with paracrystalline arrays were isolated and subsequently labeled with 3H-Con A, Con A and ferritin-Con A. The distribution of the Con A or the ferritin-Con A molecules on deep-etched exoplasmic surfaces strongly resembled the distribution of the intramembranous particles. The influence upon labeling of buffer ionic strength was investigated. Binding assays with 3H-Con A and freeze-etch electron microscopy demonstrated that the amount of non-specifically bound lectin molecules decreases by increasing buffer ionic strength. Only partial removal of Con A molecules was achieved by adding various concentrations of the specific sugar Methyl-alpha-D-Mannoside (alpha MM) to labeled protoplasts. By means of analytical ultracentrifugation it was found that alpha MM also promotes the formation of Con A dimers. fixed protoplasts were treated with detergents and 2-chloroethanol at various concentrations and subsequently labeled with 3H-Con A or ferritin-Con A. The amount of Con A bound to extracted cells did not decrease but ultrastructural changes of the deep-etched surfaces were observed. From our data it can be concluded that only the glycoproteins are labeled with Con A and they seem to be associated with the intramembranous particles [15]. Each intramembranous particle seems to bind 36 to 44 Con A molecules and therefore the glycoproteins seem to possess very long sugar chains. This further supports the hypothesis that the intramembranous particles are associated with the membrane-bound invertase.

Isolation and characterization of paracrystalline arrays of the plasma membrane of baker's yeast Saccharomyces cerevisiae.

Yeast plasma membranes were isolated from homogenized cells and analyzed by SDS-PAGE. Two glycoproteins of 160 000 and 240 000 molecular weight were found, both of which exhibited invertase activity (EC 3.2.1.26). By density gradient centrifugation a heavy membrane fraction which consisted of the glycoproteins and two hydrophobic proteins was isolated. Antibody labeling of protoplasts revealed a good correlation between the distribution of binding sites of the antibodies against the heavy fraction and the distribution of the intramembranous particles. The cytoplasmic surface of the yeast plasma membrane was visualized by freeze drying and subsequent platinum/carbon shadowing of membrane vesicles adsorbed to cationized glass and squirted with a hypotonic buffer stream. In contrast to the smooth exoplasmic surface the cytoplasmic surface showed paracrystalline arrays of particles which resembled in size, number and lattice constant the intramembranous particles. Removal of the adsorbed paracrystalline arrays and subsequent SDS-PAGE revealed the same protein pattern as the heavy membrane fraction. It can therefore be concluded that the glycoproteins which show invertase activity and the two hydrophobic proteins are the major components of the paracrystalline arrays. It is proposed that the glucose level of the nutrient medium influences the appearance and disappearance of the paracrystalline arrays, which consist mainly of invertase, because synthesis of invertase is inhibited by glucose levels higher than 1%.

Immunochemical analysis of the plasma membrane from baker's yeast Saccharomyces cerevisiae.

Yeast plasma membranes have been isolated from homogenized yeast cells, identified as pure plasma membrane vesicles which were used as antigens. By crossed immunoelectrophoresis with anti-membrane immunoglobulins, 17 discrete antigens have been detected in Triton X-100 extracts from plasma membranes. Three different immunoabsorption experiments were performed with : a) isolated membranes exposing the cytoplasmic surfaces (PS) and the external surfaces (ES), b) yeast protoplasts exposing only antigenic determinants on the ES, c) lysed protoplasts which had been saturated on the ES with antibodies prior to lysis. These absorption experiments demonstrated that seven of the antigens are expressed on the ES while eight immunogens expose antigenic determinants on the PS. Four of the principal immunoprecipitates are not affected by absorption with surface antigens whereas two of the antigens indicate transmembrane characteristics. Of these 17 immunoprecipitates four were shown by zymograms to possess enzymatic activities: ATPase (EC 3.6.1.3) and NADH-dehydrogenase (EC 1.8.99.3) (three separate components). Three of these enzymes are expressed on the PS, and one NADH-dehydrogenase exposes determinants on the ES of the protoplasts. The presence of antigens on the PS of the plasma membrane could also be demonstrated on micrographs by the indirect ferritin-antibody labeling technique followed by freeze-etching and shadowing of the membranes.