Development of a database for the rapid and accurate routine identification of Achromobacter species by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

OBJECTIVES: Achromobacter spp. are emerging pathogens in respiratory samples from cystic fibrosis patients. The current reference methods (nrdA-sequencing or multilocus sequence typing) can identify 18 species which are often misidentified by conventional techniques as A. xylosoxidans. A few studies have suggested that matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) provides accurate identification of the genus but not of species. The aims of this study were (a) to generate a database for MALDI-TOF/MS Bruker including the 18 species, (b) to evaluate the suitability of the database for routine laboratory identification, and (c) to compare its performance with that of the currently available Bruker default database. METHODS: A total of 205 isolates belonging to the 18 species identified by nrdA sequencing were used to build a local database. Main spectra profiles (MSPs) were created according to Bruker's recommendations for each isolate with the Biotyper software. Performance of the default Bruker database and ours for routine use were compared by testing 167 strains (including 38 isolates used from MSP creation) belonging to the 18 species identified by nrdA sequencing directly from colonies cultivated on various media. RESULTS: Our new database accurately identified 99.4% (166/167) of the isolates from the 18 species (score ≥2.0) versus only 50.9% (85/167) with the Bruker database. In the Bruker database 17.3% of the isolates (29/167) were incorrectly identified as another species despite a score of ≥2.0. CONCLUSIONS: The use of MALDI-TOF/MS in combination with a database developed with samples from 18 Achromobacter species provides rapid and accurate identification. This tool could be used to help future clinical studies.

16S rRNA PCR on clinical specimens: Impact on diagnosis and therapeutic management.

OBJECTIVE: 16S rRNA PCR (16S PCR) performed on clinical samples contributes to bacterial identification in cases of negative culture due to an antibiotic therapy. Sensitivity of the 16S PCR is low (19-42%). Little data is available on its impact on the management of patients. We aimed to evaluate the contribution of 16S PCR to diagnosis and therapeutic management at the university hospital of Dijon, France. PATIENTS AND METHODS: 16S PCR was performed on the clinical specimens of 132 patients. Clinical settings, laboratory results, and data on antibiotic therapy were collected, as well as conclusions drawn from the 16S PCR result by physicians. Each case was analyzed to determine if the 16S PCR was helpful. The relevance of the 16S PCR was also assessed. RESULTS: The 16S PCR yield was 27.3%, ranging from 14.3% to 64.3% depending on the type of specimen. 16S PCR had a positive impact on diagnosis in 28.8% of cases. Five negative 16S PCR results were considered helpful as they contributed to ruling out bacterial infection. 16S PCR led to treatment changes in six patients (4.5%): three narrower spectrums, two treatment adaptations, and one discontinuation. The 16S PCR was considered "non-relevant" in 35 cases (26.5%). None of these 35 PCRs contributed to the patient's management. CONCLUSION: Physicians should be aware of performances of 16S PCR. Dialogue between physicians and bacteriologists is essential for appropriate selection of indications and correct interpretation of results.

Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

Inventory of Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study.

The objective of this study was to perform an inventory of the extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB, aac(6')-Ib-cr, and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

Distribution of innate efflux-mediated aminoglycoside resistance among different Achromobacter species.

Achromobacter spp. are emerging respiratory pathogens in cystic fibrosis patients. Since 2013 the genus Achromobacter includes 15 species for which innate antibiotic resistance is unknown. Previously the AxyXY-OprZ efflux system has been described to confer aminoglycoside (AG) resistance in A. xylosoxidans. Nevertheless, some Achromobacter spp. strains are susceptible to AG. This study including 49 Achromobacter isolates reveals that AG resistance is correlated with different Achromobacter spp. It is noteworthy that the axyXY-oprZ operon is detected only in AG-resistant species, including the most frequently encountered in cystic fibrosis patients: A. xylosoxidans, A. ruhlandii, A. dolens and A. insuavis.

Effects of a primary rehabilitation programme on arterial vascular adaptations in an individual with paraplegia.

OBJECTIVE: Evaluation of the effects of 6 weeks of wheelchair endurance training on arterial stiffness in an individual with paraplegia. METHODS: A 22-year-old male patient with complete (ASIA A) paraplegia (T11) was tested before and after training (30 minutes three times per week). Physical performance and cardiorespiratory response were evaluated during a maximal progressive test. Heart rate (HR), blood pressure, stroke volume and arterial carotid-wrist and carotid-ankle pulse wave velocity (PWV) were measured at rest. RESULTS: Maximal responses registered (maximal tolerated power, VO2 peak) during the exercise test were increased after training. At rest, HR as PWV decreased, whereas cardiac output and blood pressure remained constant. CONCLUSION: Continuous exposure of the subject to a repeated high intensity exercise bout for 6 weeks elevated fitness level. Such a regular practice might also constitute a major way to trigger vascular remodelling beyond to the trained body part.

Towards the molecular elucidation of congenital myasthenic syndromes: identification of mutations in MuSK.

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and characterized by a dysfunction of the neurotransmission. They are heterogeneous at the pathophysiological level and can be classified in three categories according to their origin: presynaptic, synaptic or postsynaptic. The strategy for the diagnosis and characterization of CMS relies on the clinic, EMG, muscle biopsy, identification of mutations in genes known to be responsible for CMS and the demonstration that the gene mutations are the cause of the disease by using experimental approaches. As an example of such strategy, we report briefly here the characterization of the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed marked pre- and postsynaptic structural abnormalities of the neuromuscular junction as well as a severe decrease in acetylcholine receptor epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The results obtained strongly suggested that the missense mutation, in the presence of a null mutation on the other allele, was responsible for the severe synaptic changes observed in the patient and, hence, is causing the disease. However the molecular origin of a large number of CMS is still unknown. There are hundreds of molecules known to be present at the NMJ and mutations in the genes coding for these synaptic molecules are likely to be responsible for a neuromuscular block.

Virological and immunological responses to HAART in asymptomatic therapy-naive HIV-1-infected subjects according to CD4 cell count.

OBJECTIVE: When to start highly active antiretroviral therapy (HAART) in asymptomatic chronically HIV-1-infected subjects with CD4 cell counts of 300 x 10(6)-500 x 10(6)/l is debated extensively. Retrospective analyses of virological and immunological responses following HAART have been evaluated in both blood and lymph nodes according to pre-treatment levels of CD4 cells either above or below 500 x 10(6)/l. DESIGN: Open-label, observational, non-randomized, prospective study. SETTING: Outpatients attending the Centre of Clinical Investigation in Infectious Diseases, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland. PARTICIPANTS: Fifty-four HIV-1-infected antiretroviral-naive subjects with CD4 cell count > or = 250 x 10(6)/l and plasma viraemia > or = 5000 copies/ml who had been treated with HAART for at least 48 weeks. Controls were 49 HIV-negative subjects. INTERVENTIONS: All patients received abacavir, nelfinavir, saquinavir soft gel capsules, and amprenavir in varying combinations for 72 weeks. MAIN OUTCOME MEASURES: The extent of immune reconstitution following HAART in 43 and 11 subjects with either more or fewer than 500 x 10(6) CD4 cells/l at baseline was evaluated in blood and lymph node, and compared with immunological measures observed in 49 HIV-negative controls. RESULTS: After 48 weeks of therapy, plasma viraemia was suppressed effectively in both groups of patients. Normalization of both CD4 cell count in blood, divided equally between memory and naive cells, and percentage of CD4 cells in lymph nodes occurred in the two groups. Consistently, the net increase over baseline in CD4 cell count and in memory and naive CD4 subsets was greater in patients with fewer than 500 x 10(6) CD4 cells/l at baseline. Recovery of HIV-specific responses was similar in the two groups. CONCLUSIONS: This study suggests that virological and immunological responses are comparable in asymptomatic therapy-naive HIV-1-infected subjects with CD4 cell counts above or below 500 x 10(6)/l.

Immunological and virological responses in HIV-1-infected adults at early stage of established infection treated with highly active antiretroviral therapy.

OBJECTIVE: To evaluate the immunological and virological responses to highly active antiretroviral therapy (HAART) in blood and lymphoid compartments of HIV-1-infected patients at an early stage of infection. DESIGN: An open-label, observational, non-randomized, prospective trial of outpatients attending the Centre of Clinical Investigation in Infectious Diseases, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland. SUBJECTS: Forty-one antiretroviral-naive HIV-1-infected adults with 400 CD4 T cells/microl or greater and 5000 plasma HIV-1-RNA copies/ml or greater were enrolled, and 32 finished the study. Forty-nine HIV-negative individuals were included as controls. All subjects gave written informed consent. INTERVENTIONS: All patients received abacavir 300 mg by mouth every 12 h and amprenavir 1200 mg by mouth every 12 h for 72 weeks. MAIN OUTCOME MEASURES: The extent of immune reconstitution in blood and lymph nodes after 72 weeks of HAART was evaluated, and compared with immunological measures of 49 HIV-negative subjects. RESULTS: Virus replication was effectively suppressed (-3.5 log10 at week 72). Substantial increments of CD4 T cell count in blood and percentage in lymph nodes were observed over time, and these measures were comparable to HIV-negative subjects by week 24 in blood and by week 48 in lymph nodes. The increase was equally distributed between naive and memory CD4 T cells. Recovery of HIV-specific CD4 responses occurred in 40% of patients. CONCLUSION: The initiation of HAART at an early stage of established HIV infection induces systemic quantitative normalization of CD4 T cells, a partial recovery of HIV-specific CD4 cell responses, and effective and durable suppression of virus replication.

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy.

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4(+) T lymphocytes, i.e., CD4(+)Ki67(+) T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8(+)Ki67(+) T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4(+)Ki67(+) T lymphocytes increased over time and was associated with an increase of both naive and memory CD4(+) T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4(+) T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Predicting the duration of antiviral treatment needed to suppress plasma HIV-1 RNA.

Effective therapeutic interventions and clinical care of adults infected with HIV-1 require an understanding of factors that influence time of response to antiretroviral therapy. We have studied a cohort of 118 HIV-1-infected subjects naive to antiretroviral therapy and have correlated the time of response to treatment with a series of virological and immunological measures, including levels of viral load in blood and lymph node, percent of CD4 T cells in lymph nodes, and CD4 T-cell count in blood at study entry. Suppression of viremia below the limit of detection, 50 HIV-1 RNA copies/mL of plasma, served as a benchmark for a successful virological response. We employed these correlations to predict the length of treatment required to attain a virological response in each patient. Baseline plasma viremia emerged as the factor most tightly correlated with the duration of treatment required, allowing us to estimate the required time as a function of this one measure.

Moderate intake of n-3 fatty acids for 2 months has no detrimental effect on glucose metabolism and could ameliorate the lipid profile in type 2 diabetic men. Results of a controlled study.

OBJECTIVE: To evaluate the effect of a moderate dose of fish oil on glycemic control and in vivo insulin action in type 2 diabetic men with elevated plasma triacylglycerols and to determine the effect of the same treatment on gene expression of GLUT4, lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL) in the abdominal adipose tissue. RESEARCH DESIGN AND METHODS: A total of 12 type 2 diabetic men were randomly allocated to 2 months of 6 g daily of either fish oil or sunflower oil, separated by a 2-month washout interval, in a double-blind crossover design. RESULTS: For glucose metabolism, 2 months of fish oil supplementation compared with sunflower oil led to similar fasting plasma insulin, glucose, and HbA1c. Basal hepatic glucose production did not increase after fish oil. There was no difference in insulin suppression of hepatic glucose production nor in insulin stimulation of whole-body glucose disposal measured by the euglycemic-hyperinsulinemic clamp. Fish oil did not ameliorate the low mRNA level of GLUT4 in adipose tissue of these patients. For lipid profile, fish oil lowered plasma triacylglycerol more than sunflower oil (P < 0.05) and tended to increase the amount of mRNA of both LPL and HSL in adipose tissue. CONCLUSIONS: A moderate dose of fish oil did not lead to deleterious effects on glycemic control or whole-body insulin sensitivity in type 2 diabetic men, with preserved triacylglycerol-lowering capacities.

[Neurophysiopathology of spinal lesions, evaluation of the 80s. Post-injury assessment and treatment of the neurogenic bladder in the acute period]. / La neurophysiopathologie de la lésion médullaire, bilan des années 80. Le bilan post-traumatique et le traitement de la neurovessie à la période aiguë.

This introductory presentation deals with the recent pathophysiological aspects of the post-traumatic spinal cord lesion and the population concerned. The neurophysiology of the bladder and its sphincters is summarized so as to try and explain some disturbances occurring during the acute phase following a spinal cord injury; their clinical and urological aspects and the methods of bladder drainage.

The locked-in syndrome: a review and presentation of two chronic cases.

The locked-in syndrome (LIS) is a state of an upper motor neurone quadriplegia involving the cranial nerve pairs with usually a lateral gaze palsy, paralytic mutism, full consciousness and awareness by the patient of his environment. A historical presentation of the LIS is given as well as a short description of the clinicoanatomic lesion causing LIS. The usual cause is vascular and corresponds to a pontine infarction due to an obstruction of the basilar artery but other lesions in the brainstem can also be the cause. Non-vascular aetiologies, especially traumatic, are reviewed. The use of electroencephalography (EEG), brain auditory evoked potentials (BAEP) and somesthesic evoked potentials (SEP) are discussed as well as the use in the acute stage of computed tomography (CT), angiography, and magnetic resonance imagery (MRI). The last method may show well delineated ischaemic lesions some time after the event. The communication disability is probably the most difficult to overcome. Two cases of LIS are presented.