A metagenomics method for the quantitative detection of bacterial pathogens causing hospital-associated and ventilator-associated pneumonia.

IMPORTANCE: The management of ventilator-associated pneumonia and hospital-acquired pneumonia requires rapid and accurate quantitative detection of the infecting pathogen. To this end, we propose a metagenomic sequencing assay that includes the use of an internal sample processing control for the quantitative detection of 20 relevant bacterial species from bronchoalveolar lavage samples.

Oral and intestinal dysbiosis in Parkinson's disease.

The suspicion of an origin of Parkinson's disease (PD) at the periphery of the body and the involvement of environmental risk factors in the pathogenesis of PD have directed the attention of the scientific community towards the microbiota. The microbiota represents all the microorganisms residing both in and on a host. It plays an essential role in the physiological functioning of the host. In this article, we review the dysbiosis repeatedly demonstrated in PD and how it influences PD symptoms. Dysbiosis is associated with both motor and non-motor PD symptoms. In animal models, dysbiosis only promotes symptoms in individuals genetically susceptible to Parkinson's disease, suggesting that dysbiosis is a risk factor but not a cause of Parkinson's disease. We also review how dysbiosis contributes to the pathophysiology of PD. Dysbiosis induces numerous and complex metabolic changes, resulting in increased intestinal permeability, local and systemic inflammation, production of bacterial amyloid proteins that promote α-synuclein aggregation, as well as a decrease in short-chain fatty acid-producing bacteria that have anti-inflammatory and neuroprotective potential. In addition, we review how dysbiosis decreases the efficacy of dopaminergic treatments. We then discuss the interest of dysbiosis analysis as a biomarker of Parkinson's disease. Finally, we give an overview of how interventions modulating the gut microbiota such as dietary interventions, pro-biotics, intestinal decontamination and fecal microbiota transplantation could influence the course of PD.

Acute myeloid leukaemia in patients we judge as being older and/or unfit.

The definition of older age in AML is arbitrary. In the context of the clinical studies, it starts with age ≥60 or ≥65 years and in recent years ≥70 or 75, depending on the selection of the studied population. In clinical practice, with older age, we often mean that the patient is unfit for intensive chemotherapy. Higher age overlaps with categories such as worse performance status, unfitness, comorbidities, poor-risk cytogenetics, adverse mutation patterns, age-related clonal haematopoiesis and specific disease ontogeny. Intensive induction therapy can result in prolonged overall survival, at least in a subset of elderly patients aged up to 75 years despite the reluctance of some physicians and patients to use treatment regimens perceived as toxic. Venetoclax and azacitidine combination is the new standard of comparison for persons unfit for intensive therapy. New oral hypomethylating agent CC-486 as maintenance therapy led to a prolonged overall survival in a randomized trial of patients ≥55 years of age who were in first complete remission, but not eligible for allogeneic stem cell transplantation. Any therapy is better than no therapy, but a substantial proportion of older patients still receive only palliative care. Making a decision for AML diagnosed in older age should be individualized and shared through the dialog with the patient and relatives or cohabitants, considering medical issues and social factors including personal goals. Although we are witnesses of the advances in basic research and therapy, we are still a very long way from curing older patients with AML.

Sustained benefit of cognitive behaviour therapy for health anxiety in medical patients (CHAMP) over 8 years: a randomised-controlled trial.

BACKGROUND: Health anxiety is an under-recognised but a frequent cause of distress. It is particularly common in general hospitals. METHODS: We carried out an 8-year follow-up of medical out-patients with health anxiety (hypochondriasis) enrolled in a randomised-controlled trial in five general hospitals in London, Middlesex and Nottinghamshire. Randomisation was to a mean of six sessions of cognitive behaviour therapy adapted for health anxiety (CBT-HA) or to standard care in the clinics. The primary outcome was a change in score on the Short Health Anxiety Inventory, with generalised anxiety and depression as secondary outcomes. Of 444 patients aged 16-75 years seen in cardiology, endocrinology, gastroenterology, neurology and respiratory medicine clinics, 306 (68.9%) were followed-up 8 years after randomisation, including 36 who had died. The study is registered with controlled-trials.com, ISRCTN14565822. RESULTS: There was a significant difference in the HAI score in favour of CBT-HA over standard care after 8 years [1.83, 95% confidence interval (CI) 0.25-3.40, p = 0.023], between group differences in generalised anxiety were less (0.54, 95% CI -0.29 to 1.36), p = 0.20, ns), but those for depression were greater at 8 years (1.22, 95% CI 0.42-2.01, p < 0.003) in CBT-HA than in standard care, most in standard care satisfying the criteria for clinical depression. Those seen by nurse therapists and in cardiology and gastrointestinal clinics achieved the greatest gains with CBT-HA, with greater improvement in both symptoms and social function. CONCLUSIONS: CBT-HA is a highly long-term effective treatment for pathological health anxiety with long-term benefits. Standard care for health anxiety in medical clinics promotes depression. Nurse therapists are effective practitioners.

From genotype to antibiotic susceptibility phenotype in the order Enterobacterales: a clinical perspective.

OBJECTIVES: Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in the order Enterobacterales. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacterales species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. METHODS: We sequenced (Illumina MiSeq, 2×250 bp) 187 clinical isolates from species possessing (n = 98) or not (n = 89) an intrinsic AmpC-type cephalosporinase. Phenotypic antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. Piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and ciprofloxacin were considered for prediction. RESULTS: A total of 1496 isolate/antibiotic combinations (187 isolates × 8 antibiotics) were considered. In 230 cases (15.4%), no attempt of prediction was made because it could not be supported by current knowledge. Among the 1266 attempts, 1220 (96.4%) were correct (963 for predicting susceptibility and 257 for predicting resistance), 24 (1.9%) were major errors (MEs) and 22 (1.7%) were very major errors (VMEs). Concordance were similar between non-AmpC and AmpC-producing Enterobacterales (754/784 (96.2%) vs 466/482 (96.7%), chi-square test p 0.15), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (19/784 (2.4%) vs 3/466 (0.6%), chi-square test p 0.02). The majority of VMEs were putatively due to the overexpression of chromosomal genes. CONCLUSIONS: In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacterales species. However, more knowledge about the mechanisms underlying the derepression of AmpC are needed.

In vivo selection of a multidrug-resistant Aeromonas salmonicida during medicinal leech therapy.

We report the selection in a 15-year-old boy of a multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing Aeromonas salmonicida after medicinal leech therapy that required an antibiotic prophylaxis based on piperacillin/tazobactam and cotrimoxazole. Whole genome sequencing of the strain indeed revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual ß-lactamase SCO-1.

Unexpected persistence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in the faecal microbiota of hospitalised patients treated with imipenem.

Imipenem is active against extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) but favours the intestinal emergence of resistance. The effects of imipenem on intestinal microbiota have been studied using culture-based techniques. In this study, the effects were investigated in patients using culture and metagenomic techniques. Seventeen hospitalised adults receiving imipenem were included in a multicentre study (NCT01703299, http://www.clinicaltrials.gov). Most patients had a history of antibiotic use and/or hospitalisation. Stools were collected before, during and after imipenem treatment. Bacterial and fungal colonisation was assessed by culture, and microbiota changes were assessed using metagenomics. Unexpectedly, high colonisation rates by imipenem-susceptible ESBL-E before treatment (70.6%) remained stable over time, suggesting that imipenem intestinal concentrations were very low. Carriage rates of carbapenem-resistant Gram-negative bacilli (0-25.0%) were also stable over time, whereas those of yeasts (64.7% before treatment) peaked at 76.5% during treatment and decreased thereafter. However, these trends were not statistically significant. Yeasts included highly diverse colonising Candida spp. Metagenomics showed no global effect of imipenem on the bacterial taxonomic profiles at the sequencing depth used but demonstrated specific changes in the microbiota not detected with culture, attributed to factors other than imipenem, including sampling site or treatment with other antibiotics. In conclusion, culture and metagenomics were highly complementary in characterising the faecal microbiota of patients. The changes observed during imipenem treatment were unexpectedly limited, possibly because the microbiota was already disturbed by previous antibiotic exposure or hospitalisation.

The farming environment protects mice from allergen-induced skin contact hypersensitivity.

BACKGROUND: Being born and raised in a farm provides a long-lasting protection for allergies. The microbial environment provided by farm animals is crucial to induce this protective effect, although underlying immune mechanisms remain elusive. OBJECTIVE: To establish a mouse model of global exposure to the farming environment and to study immunologic changes linked to protection of allergy. METHODS: Mice colonies were bred in parallel in a farm cowshed and the university animal facility (AF). Mice from both locations were subjected to a skin contact allergy model. Peripheral blood cells and cell cytokine production were assessed in both populations. In addition, the gut microbiome at various ages was characterized. RESULTS: Mice born in the farm were less prone to develop allergy than mice bred in the AF. Mice transfers between the AF and the farm showed a better protection when mice were moved to the farm early in life. As compared to AF-bred mice, farm mice displayed early immune activation with higher CD4+ T cell population, in particular CD4+ CD25+ FoxP3- (activated cells). The cytokine profile of mice from the farm was skewed towards an IL-17 and IL-22 secreting cell profile accompanied by increased IL-10 secretion. These differences were mostly seen within a specific age window between birth and 8 weeks of age. Microbiome analysis showed differences between 4 and 20 weeks old mice and between farm and AF mice with an increased number of Murine mastadenovirus B in young farm mice exclusively. CONCLUSION: The farming environment provides a strong, allergy protective IL-22 stimulus and generates activated CD4+ T cells. Exposure to the farm environment early in their life may also provide a better protection for contact skin allergy. Whether a viral trigger might decisively influence protection for allergies remains to be determined.

Collateral damage from oral ciprofloxacin versus nitrofurantoin in outpatients with urinary tract infections: a culture-free analysis of gut microbiota.

Recent treatment guidelines for uncomplicated urinary tract infections (UTIs) discourage fluoroquinolone prescription because of collateral damage to commensal microbiota, but the ecologic impact of alternative agents has not been evaluated by culture-free techniques. We prospectively collected faecal samples at three time points from ambulatory patients with UTIs treated with ciprofloxacin or nitrofurantoin, patients not requiring antibiotics and household contacts of ciprofloxacin-treated patients. We described changes in gut microbiota using a culture-independent approach based on pyrosequencing of the V3-V4 region of the bacterial 16S rRNA gene. All groups were similar at baseline. Ciprofloxacin had a significant global impact on the gut microbiota whereas nitrofurantoin did not. The end of ciprofloxacin treatment correlated with a reduced proportion of Bifidobacterium (Actinobacteria), Alistipes (Bacteroidetes) and four genera from the phylum Firmicutes (Faecalibacterium, Oscillospira, Ruminococcus and Dialister) and an increased relative abundance of Bacteroides (Bacteroidetes) and the Firmicutes genera Blautia, Eubacterium and Roseburia. Substantial recovery had occurred 4 weeks later. Nitrofurantoin treatment correlated with a reduced relative proportion of the genus Clostridium and an increased proportion of the genus Faecalibacterium. This study supports use of nitrofurantoin over fluoroquinolones for treatment of uncomplicated UTIs to minimize perturbation of intestinal microbiota.

Incidence and prognostic significance of karyotypic subgroups in older patients with acute myeloid leukemia: the Swedish population-based experience.

The Swedish population-based acute myeloid leukemia registry contains data from 3251 patients (excluding acute promyelocytic leukemia) diagnosed between 1997 and 2006. Informative cytogenetic data from 1893 patients were retrospectively added, including 1054 patients aged between 60 and 79 years. Clonal abnormalities were found in 57% of the informative karyotypes. Karyotypic patterns differed by age: t(8;21), inv(16) and t(11q23) were more common in younger patients, whereas loss of 5q, 7q and 17p, monosomal karyotype (MK) and complex karyotypes were more common in older patients. Loss of 5q, 7q and 17p often occurred together within MK. Patients with 5 chromosome abnormalities had worse overall survival than those with fewer abnormalities or normal karyotype in all age groups. Loss of 5q, 7q and/or 17p had, in contrast to MK, a further negative impact on survival. Multivariable Cox regression analyses on risk factors in patients <80 years with cytogenetic abnormalities and intensive treatment revealed that age and performance status had the most significant impact on survival (both P<0.001), followed by sex (P=0.0135) and a karyotype including -7/del(7q) (P=0.048).

Effects of amoxicillin treatment on the salivary microbiota in children with acute otitis media.

Amoxicillin is a first-line antibiotic treatment for acute otitis media in children and one of the most commonly used antibiotics for human bacterial infections. We investigated changes in salivary bacterial communities among children treated with amoxicillin for acute otitis media (n = 18), using a culture-independent approach based on pyrosequencing of the V3 region of the bacterial 16S rRNA gene. The control group consisted of children with acute otitis media who were not given antibiotics (n = 15). One species-level phylotype assigned to the genus Streptococcus was identified across all (n = 99) saliva samples. Two additional species-level phylotypes from the genera Gemella and Granulicatella were shared by all (n = 45) samples of control subjects. Amoxicillin treatment resulted in reduced species richness and diversity, and a significant shift in the relative abundance of 35 taxa at different ranks from phylum to species-level phylotype. At the phylum level, prevalence of TM7 and Actinobacteria decreased at the end of treatment, whereas Proteobacteria had a higher relative abundance post-treatment. Multivariate analysis showed that samples from the same control subject taken over time intervals tended to cluster together. Among antibiotic-treated subjects, samples taken before and at the end of amoxicillin treatment formed two relatively well-separated clusters both of which greatly overlapped with samples taken about 3 weeks post-treatment. Our results point to a substantial but incomplete recovery of the salivary bacterial community from the antibiotic about 3 weeks after the end of treatment.

Allogeneic haematopoietic stem-cell transplantation with reduced intensity conditioning for advanced stage Hodgkin's lymphoma in Sweden: high incidence of post transplant lymphoproliferative disorder.

Allogeneic transplantation after reduced intensity conditioning (allo-RIC) is a treatment option for patients with Hodgkin's lymphoma (HL) relapsing after autologous transplantation. In all, 23 adult patients with HL underwent allo-RIC in Sweden between 2000 and 2007. The median number of previous treatment lines was five and 20 patients (87%) were previously autografted. TRM at 100 days and at 1 year was 13 and 22% respectively. Acute GVHD grades II-IV developed in 7 out of 23 patients (30%) and chronic GVHD in 10 out of 20 patients at risk (50%). The OS and EFS at three years was 59 and 27%, respectively. Four patients (17%) developed post transplant lymphoproliferative disease (PTLD) after a median time of 55 days (range 38-95); two of these patients later died. The study confirmed that allo-RIC is feasible, but associated with a substantial relapse rate: only 20% of the patients were still alive 7 years after the transplant. A finding of high incidence of PTLD needs to be confirmed in a larger trial that includes patients with non-HL and CLL.

Epidemiological characteristics of the metabolic syndrome in apparently healthy persons.

The risk factors that define the metabolic syndrome lead to an accelerated development of atherosclerosis, cardiovascular diseases in apparently healthy persons. The goal of the research is determining the prevalence of the metabolic syndrome as well as the participation of the risk factors that define this condition in apparently healthy persons. The metabolic syndrome is defined according to the National Cholesterol Education Program Expert Panel. The prevalence of the metabolic syndrome in the examined population, aged 53 +/- 8, is 62 (39.7%). In the group of subjects that have the metabolic syndrome,the largest number of subjects had 3 risk factors and only 5 subjects had 5 risk factors. With the increase of the number of components of the metabolic syndrome, all the observed parameters of the metabolic syndrome also increase, while the level of HDL decreases. In all the examined groups of subjects, the low level of HDL-H is the most present metabolic risk factor, while in the groups of subjects that have the metabolic syndrome the hypertension is the second most present factor, and the waist circumference is the least present factor. According to our research, the prevalence of the metabolic syndrome in apparently healthy persons that have a sedentary life style is 39.7%. In all the subjects the low level of HDL-h is the most present, and in the subjects with the metabolic syndrome compared to subjects that do not have this syndrome the frequency of all the components of the metabolic syndrome is statistically considerably higher.