Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR) assay, using <it>ecfX </it>as the specific target gene, for the rapid and accurate identification of <it>P. aeruginosa </it>from positive blood cultures (BCs).</p> <p>Methods</p> <p>Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification).</p> <p>Results</p> <p>Thirty-three strains of <it>P. aeruginosa</it>, 53 strains of Enterobactericaeae, nine strains of <it>Stenotrophomonas maltophilia </it>and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing <it>P. aeruginosa</it>. All <it>P. aeruginosa </it>clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%.</p> <p>Conclusions</p> <p>This reliable technique may offer a rapid (<1.5 h) tool that would help clinicians to initiate an appropriate treatment earlier. Further investigations are needed to assess the clinical benefit of this novel strategy as compared to phenotypic methods.</p

Loss of seryl-tRNA synthetase (SARS1) causes complex spastic paraplegia and cellular senescence

Genetic research; Nervous system diseases; PediatricsInvestigación genética; Enfermedades del sistema nervioso; PediatríaRecerca genètica; Malalties del sistema nerviós; PediatriaBackground Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 (SARS1) were reported. Methods We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient’s fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant’s pathogenicity. Results A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels). Conclusion We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence.This study was supported by grants from the Undiagnosed Rare Disease programme URD-Cat (SLT002/16/00174) from the Generalitat de Catalunya, the Center for Biomedical Research on Rare Diseases (CIBERER) (ACCI19-759), the ASL-HSP France and the Hesperia Foundation to AP. This study was also funded by the Instituto de Salud Carlos III and ‘Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, una manera de hacer Europa’ (FIS PI20/00758) to CC and ‘La Marató de TV3’ Foundation (202006-30) to CC and AP. This study was also funded by Instituto de Salud Carlos III (Sara Borrell programme, CD19/00221) to EV, the Spanish Ministerio de Economia, Industria y Competividad (Juan de la Cierva programme FJCI-2016-28811) to EV, and the Center for Biomedical Research on Rare Diseases (CIBERER) to MR and NL. This study was supported by the French National Programme Investissement d’Avenir administered by the ‘Agence National de la Recherche’ (ANR), ‘MitoCross’ Laboratory of Excellence (Labex), funded as ANR-10-IDEX-0002-02, the University of Strasbourg and CNRS (to HB, BS). The CERCA Programme/Generalitat de Catalunya provided institutional support. AP is member of the Undiagnosed Disease Network International (UDNI)

A novel mutation in the GFAP gene expands the phenotype of Alexander disease

Background: Alexander disease, an autosomal dominant leukodystrophy, is caused by missense mutations in GFAP. Although mostly diagnosed in children, associated with severe leukoencephalopathy, milder adult forms also exist. Methods: A family affected by adult-onset spastic paraplegia underwent neurological examination and cerebral MRI. Two patients were sequenced by WES. A candidate variant was functionally tested in an astrocytoma cell line. Results: The novel variant in GFAP N-terminal head domain (p.Gly18Val) cosegregated in multiple relatives (LOD score: 2.7). All patients, even those with the mildest forms, showed characteristic signal changes or atrophy in the brainstem and spinal cord MRIs, and abnormal MRS. In vitro, this variant did not cause significant protein aggregation, in contrast to most Alexander disease mutations characterized so far. However, cell area analysis showed larger size, a feature previously described in patients and mouse models. Conclusion: We suggest that this variant causes variable expressivity and an attenuated phenotype of Alexander Disease type II, probably associated with alternative pathogenic mechanisms, i.e. astrocyte enlargement. GFAP analysis should be considered in adult-onset neurologic presentations with pyramidal and bulbar symptoms, in particular when characteristic findings, such as the tadpole sign, are present in MRI. WES is a powerful tool to diagnose atypical cases

Tauroursodeoxycholic bile acid arrests axonal degeneration by inhibiting the unfolded protein response in X-linked adrenoleukodystrophy

Altres ajuts: We are indebted to the NIH NeuroBioBank for supplying the case material used for the human studies. This study was supported by grants from the European Leukodystrophy Association [ELA2012-033C1], the Center for Biomedical Research on Rare Diseases (CIBERER) to N.L. and M.R. Locomotor experiments were performed by the SEFALer unit F5 led by A.P. which belongs to the CIBERER structure.The online version of this article (doi:10.1007/s00401-016-1655-9) contains supplementary material, which is available to authorized users

Kinetic DTI of the cervical spine: diffusivity changes in healthy subjects

Introduction The study aims to assess the influence of neck extension on water diffusivity within the cervical spinal cord. Methods IRB approved the study in 22 healthy volunteers. All subjects underwent anatomical MR and diffusion tensor imaging (DTI) at 1.5 T. The cervical cord was imaged in neutral (standard) position and extension. Segmental vertebral rotations were analyzed on sagittal T2-weighted images using the SpineView® software. Spinal cord diffusivity was measured in cross-sectional regions of interests at multiple levels (C1–C5). Results As a result of non-adapted coil geometry for spinal extension, 10 subjects had to be excluded. Image quality of the remaining 12 subjects was good without any deteriorating artifacts. Quantitative measurements of vertebral rotation angles and diffusion parameters showed good intra-rater reliability (ICC= 0.84–0.99). DTI during neck extension revealed significantly decreased fractional anisotropy (FA) and increased radial diffusivity (RD) at the C3 level and increased apparent diffusion coefficients (ADC) at the C3 and C4 levels (p < 0.01 Bonferroni corrected). The C3/C4 level corresponded to the maximal absolute change in segmental vertebral rotation between the two positions. The increase in RD correlated positively with the degree of global extension, i.e., the summed vertebral rotation angle between C1 and C5 (R= 0.77, p= 0.006). Conclusion Our preliminary results suggest that DTI can quantify changes in water diffusivity during cervical spine extension. The maximal differences in segmental vertebral rotation corresponded to the levels with significant changes in diffusivity (C3/C4). Consequently, kinetic DTI measurements may open new perspectives in the assessment of neural tissue under biomechanical constraint

Value of 18-F-FDG PET/CT and CT in the Diagnosis of Indeterminate Adrenal Masses

The purpose of this paper was to study the value of 18-FDG PET/CT and reassess the value of CT for the characterization of indeterminate adrenal masses. 66 patients with 67 indeterminate adrenal masses were included in our study. CT/MRI images and 18F-FDG PET/CT data were evaluated blindly for tumor morphology, enhancement features, apparent diffusion coefficient values, maximum standardized uptake values, and adrenal-to-liver maxSUV ratio. The study population comprised pathologically confirmed 16 adenomas, 19 metastases, and 32 adrenocortical carcinomas. Macroscopic fat was observed in 62.5% of the atypical adenomas at CT but not in malignant masses. On 18F-FDG PET/CT, SUVmax and adrenal-to-liver maxSUV ratio were significantly lower in adenomas than in malignant tumors. An SUVmax value of less than 3.7 or an adrenal-to-liver maxSUV ratio of less than 1.29 is highly predictive of benignity

Oxidative stress and mitochondrial dynamics malfunction are linked in Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) is a fatal hypomyelinating disorder characterized by early impairment of motor development, nystagmus, choreoathetotic movements, ataxia and progressive spasticity. PMD is caused by variations in the proteolipid protein gene PLP1, which encodes the two major myelin proteins of the central nervous system, PLP and its spliced isoform DM20, in oligodendrocytes. Large duplications including the entire PLP1 gene are the most frequent causative mutation leading to the classical form of PMD. The Plp1 overexpressing mouse model (PLP-tg66/66 ) develops a phenotype very similar to human PMD, with early and severe motor dysfunction and a dramatic decrease in lifespan. The sequence of cellular events that cause neurodegeneration and ultimately death is poorly understood. In this work, we analyzed patient-derived fibroblasts and spinal cords of the PLP-tg66/66 mouse model, and identified redox imbalance, with altered antioxidant defense and oxidative damage to several enzymes involved in ATP production, such as glycolytic enzymes, creatine kinase and mitochondrial proteins from the Krebs cycle and oxidative phosphorylation. We also evidenced malfunction of the mitochondria compartment with increased ROS production and depolarization in PMD patient's fibroblasts, which was prevented by the antioxidant N-acetyl-cysteine. Finally, we uncovered an impairment of mitochondrial dynamics in patient's fibroblasts which may help explain the ultrastructural abnormalities of mitochondria morphology detected in spinal cords from PLP-tg66/66 mice. Altogether, these results underscore the link between redox and metabolic homeostasis in myelin diseases, provide insight into the pathophysiology of PMD, and may bear implications for tailored pharmacological intervention

RINT1 deficiency disrupts lipid metabolism and underlies a complex hereditary spastic paraplegia

The Rad50 interacting protein 1 (Rint1) is a key player in vesicular trafficking between the ER and Golgi apparatus. Biallelic variants in RINT1 cause infantile-onset episodic acute liver failure (ALF). Here, we describe 3 individuals from 2 unrelated families with novel biallelic RINT1loss-of-function variants who presented with early onset spastic paraplegia, ataxia, optic nerve hypoplasia, and dysmorphic features, broadening the previously described phenotype. Our functional and lipidomic analyses provided evidence that pathogenic RINT1 variants induce defective lipid-droplet biogenesis and profound lipid abnormalities in fibroblasts and plasma that impact both neutral lipid and phospholipid metabolism, including decreased triglycerides and diglycerides, phosphatidylcholine/phosphatidylserine ratios, and inhibited Lands cycle. Further, RINT1 mutations induced intracellular ROS production and reduced ATP synthesis, affecting mitochondria with membrane depolarization, aberrant cristae ultrastructure, and increased fission. Altogether, our results highlighted the pivotal role of RINT1 in lipid metabolism and mitochondria function, with a profound effect in central nervous system development

Sphingolipid desaturase DEGS1 is essential for mitochondria-associated membrane integrity

Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental disorders to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase Delta 4-dihydroceramide desaturase 1 (DEGS1) acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18) (OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane-resident (MAM-resident) enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multiomics, and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: (a) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; (b) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; (c) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; and (d) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAM function