Development of a national EUCAST challenge panel for antimicrobial susceptibility testing.

A challenge panel of bacterial strains useful for clinical laboratories to validate their European Committee on Antimicrobial Susceptibility Testing (EUCAST) antimicrobial susceptibility test (AST) system was established. A total of 117 strains, obtained from Belgian Reference Centres (n = 57) and from routine clinical samples (n = 60) was selected based on resistance pattern. These strains were analysed in seven different laboratories by three different automated AST systems (Vitek (n = 2), Phoenix (n = 2) and Microscan (n = 2)) and by disc diffusion from five different manufacturers (Rosco (n = 2), Becton-Dickinson (n = 2), Biomérieux (n = 1), Bio-rad (n = 1) and i2a (n = 1)). To select the challenge panel, selection criteria were set for categorical agreement between the different systems and the number of very major errors, major errors and minor errors. Very major and major errors for at least two antibiotics were observed in 43% of all strains, leading to the exclusion of these strains from the selected panel. In only 10% of all tested strains was there 100% categorical agreement for all antibiotics. Finally, 28 strains (14 Gram-positive and 14 Gram-negative) covering a wide spectrum of resistance mechanisms were selected. Pilot-testing of this challenge panel in 20 laboratories mainly confirmed the results of the validation study. Only six strains withheld for the pilot study could not be used as challenge strain due to an overall (very) major error rate of >5% for a particular antibiotic (n = 5) or for two antibiotics (n = 1). To conclude, this challenge panel should facilitate the implementation and use of EUCAST breakpoints in laboratories.

[Acute respiratory failure due to Mycoplasma pneumonia]. / Détresse respiratoire aiguë due à Mycoplasma pneumoniae.

INTRODUCTION: Respiratory infections due to Mycoplasma pneumoniae are typically mild and subacute. We report the case of a 40-year-old man hospitalized for acute respiratory distress in the context of an acute infection with Mycoplasma pneumoniae. Radiological and pulmonary function test were consistent with an acute infectious bronchiolitis. CASE REPORT: The patient presented with isolated respiratory failure with profound hypoxemia requiring oxygen delivered at high concentration by face mask. The CT appearance of the lesions corresponded to a spread of bilateral micro-connected pulmonary nodules (a "tree-in-bud" pattern) associated with obstructive ventilatory disorder. The only pathogen identified by PCR on BAL and serology was Mycoplasma pneumoniae. The evolution was favorable with antibiotic therapy combined with corticosteroids. CONCLUSION: Mycoplasma pneumoniae may be responsible for severe respiratory illness in the form of bronchiolitis. In the setting of severe acute community pneumoniae antibiotic treatment which is also effective against Mycoplasma pneumonia should be considered. In this case, corticosteroids may be an effective adjunct by their action on the small airways.

[Non-invasive ventilation era: is there still a place for long-term tracheostomy?]. / Place de la trachéotomie au long cours dans l'insuffisance respiratoire chronique sévère à l'ère de la ventilation non invasive.

INTRODUCTION: At a time when non-invasive ventilation (NIV) is commonly used in acute as well as chronic respiratory failure, it is important to consider the current place, if any, of long-term tracheostomy. BACKGROUND: Except in emergency situations where tracheostomy is mandatory to ensure safe access to the airway, long-term ventilation with tracheostomy (LTVT) is generally considered in the case of inability to wean from NIV after an episode of acute respiratory failure requiring endotracheal ventilation or because of the development of bulbar signs (swallowing, phonation) in advanced neuromuscular disease. It is also appropriate when ventilatory dependence on NIV exceeds 20 hours per day. Historical retrospective studies confirmed the feasibility of LTVT, but this has to be seen in perspective with the results obtained 20 years later with NIV. VIEWPOINT AND CONCLUSION: Even if the indications for LTVT have diminished considerably since the emergence of NIV, tracheostomy remains mandatory in some situations of respiratory distress and it should be considered as a potential resource, possibly temporary in some cases in the light of recent work on the possibility of decanulation after LTVT.

Seroprevalence of celiac disease in Belgian children and adolescents.

BACKGROUND: Celiac Disease (CD) has a high prevalence worldwide. Because the disease may be underdiagnosed in Belgium, we wanted to evaluate the seroprevalence of CD in children and adolescents. PATIENTS AND METHODS: Serum samples from 1159 apparently healthy children and adolescents aged between 1-19 years, were prospectively collected in 2006 by 15 Belgian diagnostic laboratories. In September 2009, all samples were analyzed for human tissue transglutaminase IgA antibodies (IgA tTG) and total IgA levels by, respectively a commercial ELISA and immunonephelometry. Sera, found positive, were assessed by immunofluorescence for the presence of IgA and IgG antiendomysium antibodies (IgA/IgG EMA). In patients with IgA deficiency IgG antibodies against deamidated gliadin peptides (IgG DGP) and IgG antiendomysium antibodies (IgG EMA) were determined. RESULTS: Ten of the 1159 individuals (0.86%) tested positive for IgA tTG. A further 0.86% showed borderline IgA tTG results. In almost two percent (198%) of the analyzed samples total IgA levels below the lower limit of normal were observed. Four out of eight positive IgA tTG samples tested positive for IgA EMA. All samples with borderline IgA tTG results were negative for IgA EMA. Twenty-six percent (6/23) of the IgA deficient samples showed positive IgG DGP antibodies, but none of those tested positive for IgG EMA. CONCLUSION: The seroprevalence of IgA tTG in this non-IgA deficient population (n = 1136) in Belgium is 1:114. The combined seroprevalence of IgA tTG and IgA EMA in that same population is 1:284. The seroprevalence based on positive IgA tTG or DPG IgG in, respectively, non-IgA deficient and IgA deficient subjects is 1:72 (n = 1159). These seroprevalences are similar to those found in neighbouring countries.

[Mature mediastinal teratoma and sarcoid-like granulomatosis]. / Tératome mature médiastinal et granulomatose sarcoid-like.

INTRODUCTION: Sarcoidosis is a systemic granulomatous disorder. However, when epithelioid cell granulomata are found in association with malignancy, they could correspond to a sarcoid reaction. CASE REPORT: We report the case of a young woman with mediastinal lymphadenopathy containing non-caseating granulomata. She also had a mass in the anterior mediastinum which proved, on surgical resection, to be an intrathymic mature teratoma. CONCLUSION: An association between a sarcoid-like reaction and testicular germ cell tumours has been described in literature. Our observation of a sarcoid reaction associated with a teratoma could be due to a hypersensitivity reaction to antigen released by the tumour.

Benzoate treatment and the glycine index in nonketotic hyperglycinaemia.

High-dose benzoate treatment aimed at reducing plasma glycine levels to normal reduces seizures and increases wakefulness in patients with nonketotic hyperglycinaemia (NKH). Since benzoate metabolism is dependent on the available glycine pool, and since the glycine pool is variably affected by the deficiency in the glycine cleavage enzyme system, we examined the importance of interpatient variability in benzoate requirement. To correct for the dietary glycine contribution, the glycine index was introduced as the molar requirement of benzoate dose necessary to normalize plasma glycine levels and subtracting from that the dietary glycine intake, both corrected for weight. The glycine index varied between 3.62 and 4.87 mmol/kg per day in five patients with a poor neurodevelopmental outcome and between 0.92 and 1.90 mmol/kg per day in four patients with a better neurodevelopmental outcome, and was 2.54 mmol/kg per day in a single patient with an intermediate outcome. The glycine index was stable over time within each patient. Exceeding the balance by either increasing food glycine intake or decreasing the benzoate dose resulted in increased glycine levels. Exceeding the glycine tolerance by increasing benzoate resulted in elevated and toxic levels of benzoate. The glycine index is a stable, individually specific parameter in patients with NKH. It has clinical consequences for the dose of benzoate required and the role of dietary management. Through its correlation with neurodevelopmental outcome, the glycine index points to potential genetic factors that could contribute to the psychomotor retardation in NKH.

The neuronal migration defect in mice with Zellweger syndrome (Pex5 knockout) is not caused by the inactivity of peroxisomal beta-oxidation.

The purpose of this study was to investigate whether deficient peroxisomal beta-oxidation is causally involved in the neuronal migration defect observed in Pex5 knockout mice. These mice are models for Zellweger syndrome, a peroxisome biogenesis disorder. Neocortical development was evaluated in mice carrying a partial or complete defect of peroxisomal beta-oxidation at the level of the second enzyme of the pathway, namely, the hydratase-dehydrogenase multifunctional/bifunctional enzymes MFP1/L-PBE and MFP2/D-PBE. In contrast to patients with multifunctional protein 2 deficiency who present with neocortical dysgenesis, impairment of neuronal migration was not observed in the single MFP2 or in the double MFP1/MFP2 knockout mice. At birth, the double knockout pups displayed variable growth retardation and about one half of them were severely hypotonic, whereas the single MFP2 knockout animals were all normal in the perinatal period. These results indicate that in the mouse, defective peroxisomal beta-oxidation does not cause neuronal migration defects by itself. This does not exclude that the inactivity of this metabolic pathway contributes to the brain pathology in mice and patients with complete absence of functional peroxisomes.

Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse).

Zellweger syndrome (cerebro-hepato-renal syndrome) is the most severe form of the peroxisomal biogenesis disorders leading to early death of the affected children. To study the pathogenetic mechanisms causing organ dysfunctions in Zellweger syndrome, we have recently developed a knockout-mouse model by disrupting the PEX5 gene, encoding the targeting receptor for most peroxisomal matrix proteins (M Baes, P Gressens, E Baumgart, P Carmeliet, M Casteels, M Fransen, P Evrard, D Fahimi, PE Declercq, D Collen, PP van Veldhoven, GP Mannaerts: A mouse model for Zellweger syndrome. Nat Genet 1997, 17:49-57). In this study, we present evidence that the absence of functional peroxisomes, causing a general defect in peroxisomal metabolism, leads to proliferation of pleomorphic mitochondria with severe alterations of the mitochondrial ultrastructure, changes in the expression and activities of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial compartment in various organs and specific cell types (eg, liver, proximal tubules of the kidney, adrenal cortex, heart, skeletal and smooth muscle cells, neutrophils). The changes of mitochondrial respiratory chain enzymes are accompanied by a marked increase of mitochondrial manganese-superoxide dismutase, as revealed by in situ hybridization and immunocytochemistry, suggesting increased production of reactive oxygen species in altered mitochondria. This increased oxidative stress induced probably by defective peroxisomal antioxidant mechanisms combined with accumulation of lipid intermediates of peroxisomal beta-oxidation system could contribute significantly to the pathogenesis of multiple organ dysfunctions in Zellweger syndrome.

Oxidative catabolism of alpha-tocopherol in rat liver microsomes.

The goal of this study was to clarify the mechanism responsible for the catabolism of alpha-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 microM of alpha-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that alpha-tocopherol is omega-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).

Isoprenoid biosynthesis is not compromised in a Zellweger syndrome mouse model.

Because several studies indicated that peroxisomes are important for the biosynthesis of isoprenoids, we wanted to investigate whether a reduced availability of isoprenoids could be one of the pathogenic factors contributing to the severe phenotype of the Pex5(-/-) mouse, a model for Zellweger syndrome. Total cholesterol was determined in plasma, brain and liver of newborn mice. In none of these tissues a significant difference was observed between Pex5(-/-) and wild type or heterozygous mice. The hepatic ubiquinone content was found to be even higher in Pex5(-/-) mice as compared to wild type or heterozygous littermates. To investigate whether the Pex5(-/-) fetuses are able to synthesise their own isoprenoids, fibroblasts derived from these mice were incubated with radiolabeled mevalonolactone as a substrate for isoprenoid synthesis. No significant difference was observed between the cholesterol production rates of Pex5(-/-) and normal fibroblasts. Our results show that there is no deficiency of isoprenoids in newborn Pex5(-/-) mice, excluding the possibility that a lack of these compounds is a determinant factor in the development of the disease state before birth.

Prenatal and postnatal development of peroxisomal lipid-metabolizing pathways in the mouse.

The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: alpha-oxidation, beta-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5(-/-) knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C(26:0) was observed in comparison with control littermates, indicating that ether phospholipid synthesis and beta-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the beta-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the alpha-oxidation pathway suggested that alpha-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in alpha-oxidation and beta-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal beta-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.

Do sphingoid bases interact with the peroxisome proliferator activated receptor alpha (PPAR-alpha)?

In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-alpha, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-gamma-ATP, the binding of purified 32P-PPAR-alpha to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-alpha did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-alpha to sphingoid bases might be of interest given the role of PPAR-alpha and sphingolipids in various cellular processes.

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids.

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.

Docosahexaenoic acid deficit is not a major pathogenic factor in peroxisome-deficient mice.

Docosahexaenoic acid (DHA), a major component of membrane phospholipids in brain and retina, is profoundly reduced in patients with peroxisome biogenesis disorders (Zellweger syndrome). Supplementing newborn patients with DHA resulted in improved muscular tone and visual functions. The purpose of this study was to investigate (a) whether DHA levels were also reduced in newborn PEX5 knockout mice, the mouse model of Zellweger syndrome that we recently generated; (b) whether these levels could be normalized by supplying DHA; and (c) whether this results in longer survival. The DHA concentration in brain of newborn PEX5-/- mice was reduced by 40% as compared with levels in normal littermates; in liver, no differences were noticed. The daily administration of 10 mg of DHA-ethyl ester (EE) to pregnant heterozygous mothers during the last 8 days of gestation resulted in a normalization of brain DHA levels in Zellweger pups. However, no clinical improvement was observed in these pups, and the neuronal migration defect was unaltered. These data suggest that the accretion of DHA in the brain at the end of embryonic development is not only supported by the maternal supply but also depends on synthesis in the fetal brain. Furthermore, the DHA deficit does not seem to be a major pathogenic factor in the newborn Zellweger mice.

Channelling of intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine in mammalian cells.

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T12 fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus alpha-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to <1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of alpha-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, alpha-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.

Effects of 2',3'-dideoxycytidine and 2',3'-dideoxycytidine 5'-triphosphate on phospholipid metabolism in permeabilized rat hepatocytes.

Both 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) inhibit the synthesis of the major phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in permeabilized rat hepatocytes. For PC, this appears to be based on competitive inhibition of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase; EC 2.7.8.2). The study was based on short-term incubations (6-12 min) of the nucleoside/nucleotide analogs with alpha-toxin permeabilized rat hepatocytes. At a concentration of 1 mM, ddC and ddCTP decreased the incorporation of radiolabelled glycerol-3-phosphate into PC by approximately 50% as compared with control. This was accompanied by a significant increase in diacylglycerol labelling. In the presence of 1 mM CDP-ethanolamine and increasing concentrations of ddC(TP) (0.01-1 mM), the incorporation of radiolabelled glycerol-3-phosphate into PE was decreased to approximately 60% of the control value. When both PC and PE synthesis were operative, the inhibition by ddC(TP) was restricted to PC synthesis. ddC and ddCTP were found to have inhibition constants (K(i)) of 496 microM and 452 microM, respectively, for the inhibition of PC synthesis from CDP-choline. Although the inhibitory concentrations of the nucleoside analog and its triphosphate ester are much higher than the in vivo plasma concentrations, the possibility is raised that the peripheral neuropathy, seen as a dose-dependent adverse effect of ddC treatment in acquired immunodeficiency syndrome therapy is, at least partly, caused by a perturbation of the phospholipid constitution of neuronal membranes.

A mouse model for Zellweger syndrome.

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Metabolism of dolichol, dolichoic acid and nordolichoic acid in cultured cells.

The uptake and metabolism of [1-(14)C]-labelled dolichol, dolichoic acid and nordolichoic acid were investigated in MDCK and HepG2 cells. Each of the three isoprenoids, bound to human serum albumin, was taken up effectively. None of the compounds was broken down in HepG2 cells, although these converted dolichol into fatty acid esters. In MDCK cells dolichoic acid gave rise to the formation of [14C]CO2 and radiolabelled formic acid, indicating that dolichoic acid can be broken down by alpha-oxidation. Dolichoic acid was also converted to a mixture of polar compounds, possibly polyols. MDCK cells generated radiolabelled CO2 from nordolichoic acid, presumably through beta-oxidation, although we could not find any labelled propionic acid. No oxidative breakdown of dolichol was found, apparently due to the lack of or very low conversion to dolichoic acid.

Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells.

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.