TIGIT+ NK cells in combination with specific gut microbiota features predict response to checkpoint inhibitor therapy in melanoma patients.

BACKGROUND: Composition of the intestinal microbiota has been correlated to therapeutic efficacy of immune checkpoint inhibitors (ICI) in various cancer entities including melanoma. Prediction of the outcome of such therapy, however, is still unavailable. This prospective, non-interventional study was conducted in order to achieve an integrated assessment of the connection between a specific intestinal microbiota profile and antitumor immune response to immune checkpoint inhibitor therapy (anti-PD-1 and/or anti-CTLA-4) in melanoma patients. METHODS: We assessed blood and stool samples of 29 cutaneous melanoma patients who received immune checkpoint inhibitor therapy. For functional and phenotypical immune analysis, 12-color flow cytometry and FluoroSpot assays were conducted. Gut microbiome was analyzed with shotgun metagenomics sequencing. To combine clinical, microbiome and immune variables, we applied the Random Forest algorithm. RESULTS: A total of 29 patients was analyzed in this study, among whom 51.7% (n = 15) reached a durable clinical benefit. The Immune receptor TIGIT is significantly upregulated in T cells (p = 0.0139) and CD56high NK cells (p = 0.0037) of responders. Several bacterial taxa were associated with response (e.g. Ruminococcus torques) or failure (e.g. Barnesiella intestinihominis) to immune therapy. A combination of two microbiome features (Barnesiella intestinihominis and the Enterobacteriaceae family) and one immune feature (TIGIT+ CD56high NK cells) was able to predict response to ICI already at baseline (AUC = 0.85; 95% CI: 0.841-0.853). CONCLUSIONS: Our results reconfirm a link between intestinal microbiota and response to ICI therapy in melanoma patients and furthermore point to TIGIT as a promising target for future immunotherapies.

Torque teno virus-DNA load as individual cytomegalovirus risk assessment parameter upon allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Immune recovery following allogeneic hematopoietic stem cell transplantation (allo-HSCT) decisively influences the occurrence of opportunistic infections, one of the leading causes of death among this group of patients. Yet, today, there are no laboratory parameters mirroring immune function sufficiently. Torque teno virus (TTV) has already proven itself as a functional immune marker in other settings. AIMS: In this analysis, we investigated whether monitoring of TTV-DNA load in whole blood is able to provide additional information on the capacity of the immune system to control cytomegalovirus (CMV) replication in allo-HSCT recipients. METHODS: Whole blood samples from 59 patients were collected upon allo-HSCT (between Day -7 and +10), on Day +14, +21, +28, +56, +90, and +365 post-transplant. TTV-DNA loads and other relevant clinical information were correlated with the risk of CMV infections or reactivations, defined by evidence of viral replication in blood. RESULTS: CMV serostatus of the recipient and a TTV load below 1000 copies/mL upon allo-HSCT were significantly associated with an increased incidence of CMV infection or reactivation. CONCLUSIONS: Quantification of TTV load in the early phase of allo-HSCT procedure could provide additional information in order to identify patients at risk for CMV infection or reactivation.

The gut microbiome in intravenous immunoglobulin-treated chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: The gut microbiome is involved in autoimmunity. Data on its composition in chronic inflammatory demyelinating polyneuropathy (CIDP), the most common chronic autoimmune disorder of peripheral nerves, are currently lacking. METHODS: In this monocentric exploratory pilot study, stool samples were prospectively collected from 16 CIDP patients (mean age 58 ± 10 years, 25% female) before and 1 week after administration of intravenous immunoglobulin (IVIg). Gut microbiota were analyzed via bacterial 16S rRNA gene sequencing and compared to 15 age-matched healthy subjects (mean age 59 ± 15 years, 66% female). RESULTS: The gut microbiota of CIDP patients showed an increased alpha-diversity (p = 0.005) and enrichment of Firmicutes, such as Blautia (p = 0.0004), Eubacterium hallii (p = 0.0004), or Ruminococcus torques (p = 0.03), and of Actinobacteriota (p = 0.03) compared to healthy subjects. IVIg administration did not alter the gut microbiome composition in CIDP in this short-term observation (p = 0.95). CONCLUSIONS: The gut microbiome in IVIg-treated CIDP shows distinct features, with increased bacterial diversity and enrichment of short-chain fatty acid producing Firmicutes. IVIg had no short-term impact on the gut microbiome in CIDP patients. As the main limitation of this exploratory pilot study was small cohort size, future studies also including therapy-naïve patients are warranted to verify our findings and to explore the impact of long-term IVIg treatment on the gut microbiome in CIDP.

Combined analysis of gut microbiota, diet and PNPLA3 polymorphism in biopsy-proven non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a global health burden. Risk factors for disease severity include older age, increased body mass index (BMI), diabetes, genetic variants, dietary factors and gut microbiota alterations. However, the interdependence of these factors and their individual impact on disease severity remain unknown. METHODS: In this cross-sectional study, we performed 16S gene sequencing using fecal samples, collected dietary intake, PNPLA3 gene variants and clinical and liver histology parameters in a well-described cohort of 180 NAFLD patients. Principal component analyses were used for dimensionality reduction of dietary and microbiota data. Simple and multiple stepwise ordinal regression analyses were performed. RESULTS: Complete data were available for 57 NAFLD patients. In the simple regression analysis, features associated with the metabolic syndrome had the highest importance regarding liver disease severity. In the multiple regression analysis, BMI was the most important factor associated with the fibrosis stage (OR per kg/m2 : 1.23, 95% CI 1.10-1.37, P < .001). The PNPLA3 risk allele had the strongest association with the histological grade of steatosis (OR 5.32, 95% CI 1.56-18.11, P = .007), followed by specific dietary patterns. Low abundances of Faecalibacterium, Bacteroides and Prevotella and high abundances of Gemmiger were associated with the degree of inflammation, ballooning and stages of fibrosis, even after taking other cofactors into account. CONCLUSIONS: BMI had the strongest association with histological fibrosis, but PNPLA3 gene variants, gut bacterial features and dietary factors were all associated with different histology features, which underscore the multifactorial pathogenesis of NAFLD.

Fecal microbiota transfer for refractory intestinal graft-versus-host disease - Experience from two German tertiary centers.

RATIONALE: Steroid refractory graft-vs-host disease (sr-GvHD) represents a challenging complication after allogeneic hematopoietic cell transplantation (allo-HCT). Intestinal microbiota (IM) diversity and dysbiosis were identified as influencing factors for the development of acute GvHD. Fecal microbiota transfer (FMT) is hypothesized to restore IM dysbiosis, but there is limited knowledge about the significance of FMT in the treatment of sr-GvHD. OBJECTIVES: We studied the effects of FMT on sr-GvHD in allo-HCT patients from two German tertiary clinical centers (n = 11 patients; period: March 2017 until July 2019). To assess safety and clinical efficacy, we analyzed clinical data pre- and post-FMT (day -14 to +30 relative to FMT). Moreover, IM were analyzed in donor samples and in a subset of patients pre- and post-FMT by 16S rRNA sequencing. RESULTS: Post-FMT, we observed no intervention-associated, systemic inflammatory responses and only minor side effects (5/11 patients: abdominal pain and transformation of peristalsis-each 3/11 and vomiting-1/11). Stool frequencies and volumes were significantly reduced [pre- vs post-FMT (d14): P < .05, respectively] as well as clear attenuation regarding both grading and staging of sr-GvHD was present upon FMT. Moreover, IM analyses revealed an increase of alpha diversity as well as a compositional shifts toward the donor post-FMT. CONCLUSIONS: In our study, we observed positive effects on sr-GVHD after FMT without the occurrence of major adverse events. Although these findings are in line with published data on beneficial effects of FMT in sr-GvHD, further randomized clinical studies are urgently needed to better define the clinical validity including mode of action.

Phenotyping non-alcoholic fatty liver disease by the gut microbiota: Ready for prime time?

BACKGROUND AND AIM: Several studies observed alterations in the gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD). However, analyzed patient populations and methods strongly differ among these studies. The aim of this study was to prove the reproducibility of published results and to provide a detailed overview of all findings in our NAFLD cohort using next generation sequencing methods. METHODS: The individual taxonomic microbiota composition of fecal samples from 90 NAFLD patients and 21 healthy controls was analyzed using 16S rRNA gene sequencing. Study participants were grouped according to their disease stage and compared regarding their gut microbiota composition. Studies were identified from PubMed listed publications, and the results were compared with the findings in our cohort. RESULTS: Results from 13 identified studies were compared with our data. A decreased abundance of the Bacteroidetes and Ruminococcaceae as well as an increased abundance of Lactobacillaceae and Veillonellaceae and Dorea were the most frequently reported changes among NAFLD patients in 4/13, 5/13, 4/13, 2/13, and 3/13 studies, respectively. Even though these alterations in the gut microbiota composition were also observed in our patient cohort, the majority of published differences could not be reproduced, neither in our own nor in other NAFLD cohort studies. CONCLUSION: Despite repeatedly reproduced abundance patterns of specific bacteria, the heterogeneous study results did not reveal a consistent disease specific gut microbiota signature. Further prospective studies with homogenous patient cohorts and standardized methods are necessary to phenotype NAFLD by the gut microbiota.

Changes in the cervical microbiota of cervical cancer patients after primary radio-chemotherapy.

OBJECTIVE: Several recent studies have identified a potential interaction between the vaginal microbiota and gynecological cancers, but little is known about the cervical microbiota and its changes during cancer treatment. Therefore, the aim of the study was to evaluate the quantitative and qualitative changes of cervical microbiota in patients undergoing concurrent chemotherapy and radiation treatment for locally advanced cervical cancer. METHODS: Cervical cytobrush samples of 15 cervical patients undergoing chemoradiation treatment were collected 1 day before starting external beam radiation therapy and on the day of the last fraction of brachytherapy. After DNA extraction, 16S rRNA amplicon sequencing of the V3-V4 region was performed on the MiSeq platform, followed by data processing and statistical analyses concerning the alpha and beta diversity of 16 samples (7 samples were excluded because of incomplete sample sets). RESULTS: The amount of amplicon yield after polymerase chain reaction analysis in post-radiation samples was significantly lower compared with the baseline samples (pre 31.49±24.07 ng/µl; post 1.33±1.94 ng/µl; p=0.007). A comparison of pre-treatment and post-treatment samples did not show significant differences regarding beta diversity (weighted UniFrac). There was no significant difference in alpha diversity, which is used to characterize species diversity within a particular community and takes into account both number and abundance (Shannon Diversity Index pre-treatment samples: 2.167±0.7504 (95% CI 1.54 to 2.79); post-treatment samples: 1.97±0.43 (95% CI 1.61 to 2.33); p=0.38). Interindividual differences in patients could partly explain some variation of the samples (permutational multivariate analysis of variance). CONCLUSION: There was a strong reduction in cervical bacterial loads after chemoradiation. Neither alpha nor beta diversity varied significantly when baseline samples were compared with post-treatment samples.

Controlling intestinal colonization of high-risk haematology patients with ESBL-producing Enterobacteriaceae: a randomized, placebo-controlled, multicentre, Phase II trial (CLEAR).

OBJECTIVES: We assessed the efficacy and safety of an oral antimicrobial regimen for short- and long-term intestinal eradication of ESBL-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EC/KP) in immunocompromised patients. METHODS: We performed a randomized (2:1), double-blind multicentre Phase II study in four haematology-oncology departments. Patients colonized with ESBL-EC/KP received a 7 day antimicrobial regimen of oral colistin (2 × 106 IU 4×/day), gentamicin (80 mg 4×/day) and fosfomycin (three administrations of 3 g every 72 h), or placebo. Faecal, throat and urine specimens were collected on day 0, 6 ± 2, 11 ± 2, 28 ± 4 and 42 ± 4 after treatment initiation, and the quantitative burden of ESBL-EC/KP, resistance genes and changes in intestinal microbiota were analysed. Clinicaltrials.gov: NCT01931592. RESULTS: As the manufacture of colistin powder was suspended worldwide, the study was terminated prematurely. Overall, 29 (18 verum/11 placebo) out of 47 patients were enrolled. The short-term intestinal eradication was marginal at day 6 (verum group 15/18, 83.3% versus placebo 2/11, 18.2%; relative risk 4.58, 95% CI 1.29-16.33; Fisher's exact test P = 0.001) and not evident at later timepoints. Quantitative analysis showed a significant decrease of intestinal ESBL-EC/KP burden on day 6. Sustained intestinal eradication (day 28 + 42) was not achieved (verum, 38.9% versus placebo, 27.3%; P = 0.299). In the verum group, mcr-1 genes were detected in two faecal samples collected after treatment. Microbiome analysis showed a significant decrease in alpha diversity and a shift in beta diversity. CONCLUSIONS: In this prematurely terminated study of a 7 day oral antimicrobial eradication regimen, short-term ESBL-EC/KP suppression was marginal, while an altered intestinal microbiota composition was clearly apparent.

Pharmacokinetic Modeling of Voriconazole To Develop an Alternative Dosing Regimen in Children.

The pharmacokinetic variability of voriconazole (VCZ) in immunocompromised children is high, and adequate exposure, particularly in the first days of therapy, is uncertain. A population pharmacokinetic model was developed to explore VCZ exposure in plasma after alternative dosing regimens. Concentration data were obtained from a pediatric phase II study. Nonlinear mixed effects modeling was used to develop the model. Monte Carlo simulations were performed to test an array of three-times-daily (TID) intravenous dosing regimens in children 2 to 12 years of age. A two-compartment model with first-order absorption, nonlinear Michaelis-Menten elimination, and allometric scaling best described the data (maximal kinetic velocity for nonlinear Michaelis-Menten clearance [Vmax] = 51.5 mg/h/70 kg, central volume of distribution [V1] = 228 liters/70 kg, intercompartmental clearance [Q] = 21.9 liters/h/70 kg, peripheral volume of distribution [V2] = 1,430 liters/70 kg, bioavailability [F] = 59.4%, Km = fixed value of 1.15 mg/liter, absorption rate constant = fixed value of 1.19 h-1). Interindividual variabilities for Vmax, V1, Q, and F were 63.6%, 45.4%, 67%, and 1.34% on a logit scale, respectively, and residual variability was 37.8% (proportional error) and 0.0049 mg/liter (additive error). Monte Carlo simulations of a regimen of 9 mg/kg of body weight TID simulated for 24, 48, and 72 h followed by 8 mg/kg two times daily (BID) resulted in improved early target attainment relative to that with the currently recommended BID dosing regimen but no increased rate of accumulation thereafter. Pharmacokinetic modeling suggests that intravenous TID dosing at 9 mg/kg per dose for up to 3 days may result in a substantially higher percentage of children 2 to 12 years of age with adequate exposure to VCZ early during treatment. Before implementation of this regimen in patients, however, validation of exposure, safety, and tolerability in a carefully designed clinical trial would be needed.

Fecal microbiota transplantation in a kidney transplant recipient with recurrent urinary tract infection.

PURPOSE: We report on a kidney transplant recipient treated with fecal microbiota transplantation (FMT) for recurrent urinary tract infections. METHODS: FMT was administered via frozen capsulized microbiota. Before and after FMT, urinary, fecal and vaginal microbiota compositions were analyzed. RESULTS: The patient remained without symptoms after FMT. CONCLUSIONS: Underlying mechanisms of action need to be addressed in depth by future research.

High Intracellular Concentrations of Posaconazole Do Not Impact on Functional Capacities of Human Polymorphonuclear Neutrophils and Monocyte-Derived Macrophages In Vitro.

Posaconazole is a commonly used antifungal for the prophylaxis and treatment of invasive fungal infections. We previously demonstrated that the intracellular concentration of posaconazole in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) was greatly increased compared to the plasma concentration. As these professional phagocytes are crucial to combat fungal infections, we set out to investigate if and how, beneficial or deleterious, this high loading of intracellular posaconazole impacts the functional capacities of these cells. Here, we show that high intracellular concentrations of posaconazole do not significantly impact PMN and monocyte-derived macrophage function in vitro In particular, killing capacity and cytoskeletal features of PMN, such as migration, are not affected, indicating that these cells serve as vehicles for posaconazole to the site of infection. Moreover, since posaconazole as such slowed the germination of Aspergillus fumigatus conidia, infected neutrophils released less reactive oxygen species (ROS). Based on these findings, we propose that the delivery of posaconazole by neutrophils to the site of Aspergillus species infection warrants control of the pathogen and preservation of tissue integrity at the same time.

Antibiotics-Induced Intestinal Immunomodulation Attenuates Experimental Autoimmune Neuritis (EAN).

BACKGROUND: The composition of gut microbiota plays a pivotal role in priming the immune system and thus impacts autoimmune diseases. Data on the effects of gut bacteria eradication via systemic antibiotics on immune neuropathies are currently lacking. This study therefore assessed the effects of antibiotics-induced gut microbiota alterations on the severity of experimental autoimmune neuritis (EAN), a rat model of Guillain-Barré Syndrome (GBS). Myelin P0 peptide 180-199 (P0 180-199)-induced EAN severity was compared between adult Lewis rats (12 weeks old) that received drinking water with or without antibiotics (colistin, metronidazole, vancomycin) and healthy rats, beginning antibiotics treatment immediately after immunization (day 0), and continuing treatment for 14 consecutive days. Neuropathy severity was assessed via a modified clinical score, and then related to gut microbiota alterations observed after fecal 16S rRNA gene sequencing at baseline and after EAN induction. Effectors of gut mucosal and endoneurial immunity were assessed via immunostaining. EAN rats showed increased gut mucosal permeability alongside increased mucosal CD8+ T cells compared to healthy controls. Antibiotics treatment alleviated clinical EAN severity and reduced endoneurial T cell infiltration, decreased gut mucosal CD8+ T cells and increased gut bacteria that may be associated with anti-inflammatory mechanisms, like Lactobacillus or Parasutterella. Our findings point out a relation between gut mucosal immunity and the pathogenesis of EAN, and indicate that antibiotics-induced intestinal immunomodulation might be a therapeutic approach to alleviate autoimmunity in immune neuropathies. Further studies are warranted to evaluate the clinical transferability of these findings to patients with GBS.

Population pharmacokinetics of escalating doses of caspofungin in a phase II study of patients with invasive aspergillosis.

Caspofungin (CAS) is approved for second-line management of proven or probable invasive aspergillosis at a dose of 50 mg once daily (QD). Preclinical and limited clinical data support the concept of the dose-dependent antifungal efficacy of CAS with preservation of its favorable safety profile. Little is known, however, about the pharmacokinetics (PKs) of higher doses of CAS in patients. In a formal multicenter phase II dose-escalation study, CAS was administered as a 2-h infusion at doses ranging from 70 to 200 mg QD. CAS PK sampling (n = 468 samples) was performed on day 1 and at peak and trough time points on days 4, 7, 14, and 28 (70 mg, n = 9 patients; 100 mg, n = 8 patients; 150 mg, n = 9 patients; 200 mg, n = 20 patients; total, n = 46 patients). Drug concentrations in plasma were measured by liquid chromatography tandem mass spectroscopy. Population pharmacokinetic analysis (PopPK) was performed using NONMEM (version 7) software. Model evaluation was performed using bootstrap analysis, prediction-corrected visual predictive check (pcVPC), as well as standardized visual predictive check (SVPC). The four investigated dose levels showed no difference in log-transformed dose-normalized trough levels of CAS (analysis of variance). CAS concentration data fitted best to a two-compartment model with a proportional-error model, interindividual variability (IIV) fitted best on clearance (CL), central and peripheral volume of distribution (V(1) and V(2), respectively) covariance fitted best on CL and V(1), interoccasion variability (IOV) fitted best on CL, and body weight fitted best as a covariate on CL and V(1) (CL, 0.411 liters/h ± 29% IIV; IOV on CL, 16%; V(1), 5.785 liters ± 29% IIV; intercompartmental clearance, 0.843 liters/h; V2, 6.53 liters ± 67% IIV). None of the other examined covariates (dose level, gender, age, serum bilirubin concentration, creatinine clearance) improved the model further. Bootstrap results showed the robustness of the final PopPK model. pcVPC and SVPC showed the predictability of the model and further confirmed the linear PKs of CAS over the dosage range of 70 to 200 mg QD. On the basis of the final model, geometric mean simulated peak plasma levels at steady state ranged from 13.8 to 39.4 mg/liter (geometric coefficient of variation, 31%), geometric mean trough levels ranged from 4.2 to 12.0 mg/liter (49%), and geometric mean areas under the concentration-time curves ranged from 170 to 487 mg · h/liter (34%) for the dosage range of 70 to 200 mg QD. CAS showed linear PKs across the investigated dosage range of 70 to 200 mg QD. Drug exposure in the present study population was comparable to that in other populations. (This study has been registered with the European Union Drug Regulating Authorities Clinical Trials website under registration no. 2006-001936-30 and at ClinicalTrials.gov under registration no. NCT00404092.).

Mucormycosis treated with posaconazole: review of 96 case reports.

Mucormycosis is an emerging invasive fungal infection, primarily affecting immunocompromised patients. The disease is difficult to diagnose and mortality reaches 40% even if treated adequately. Depending on site of infection and risk factors, surgical debridement in combination with systemically active antifungal drugs are the mainstay treatment strategies. Lipid-based amphotericin B is the treatment of choice for first-line therapy while posaconazole may be a promising alternative. We performed a PubMed search on reports of patients with mucormycosis treated with posaconazole. From 2003 to 2011, 96 cases have been published. Diagnosis was based on histology alone in 2 (2.1%) and microbiological evidence in 67 (69.8%), while no data on the diagnostic approach was reported in 27 (28.1%) patients. The most frequent pathogens were Rhizopus spp. (31.2%), followed by Mucor spp. (14.6%). The site of infection was predominantly rhino-orbital (38.5%, of which 43% also had central nervous system [CNS] involvement), followed by disseminated disease (22.1%). A complete response was achieved in 62 (64.6%), partial response in 7 (7.3%) patients, and stable disease in 1 (1%). Overall mortality was 24% (lacking data for three patients). In published case reports on posaconazole treatment for mucormycosis, the drug was frequently and successfully used in combination or as second line therapy.

Longitudinal variability in the urinary microbiota of healthy premenopausal women and the relation to neighboring microbial communities: A pilot study.

BACKGROUND: The understanding of longitudinal changes in the urinary microbiota of healthy women and its relation to intestinal microbiota is limited. METHODS: From a cohort of 15 premenopausal women without known urogenital disease or current symptoms, we collected catheter urine (CU), vaginal and periurethral swabs, and fecal samples on four visits over six months. Additionally, ten participants provided CU and midstream urine (MU) to assess comparability. Urine was subjected to expanded culture. 16S rRNA gene sequencing was performed on all urine, fecal, and selected vaginal and periurethral samples. Sequence reads were processed (DADA2 pipeline) and analyzed using QIIME 2 and R. RESULTS: Relative abundances of urinary microbiota were variable over 6-18 months. The degree of intraindividual variability of urinary microbiota was higher than that found in fecal samples. Still, nearly half of the observed beta diversity of all urine samples could be attributed to differences between volunteers (R2 = 0.48, p = 0.001). After stratification by volunteer, time since last sexual intercourse was shown to be a factor significantly contributing to beta diversity (R2 = 0.14, p = 0.001). We observed a close relatedness of urogenital microbial habitats and a clear distinction from intestinal microbiota in the overall betadiversity analysis. Microbiota compositions derived from MU differed only slightly from CU compositions. Within this analysis of low-biomass samples, we identified contaminating sequences potentially stemming from sequencing reagents. CONCLUSIONS: Results from our longitudinal cohort study confirmed the presence of a rather variable individual urinary microbiota in premenopausal women. These findings from catheter urine complement previous observations on temporal dynamics in voided urine. The higher intraindividual variability of urinary microbiota as compared to fecal microbiota will be a challenge for future studies investigating associations with urogenital diseases and aiming at identifying pathogenic microbiota signatures.

Dietary omega-6/omega-3 ratio is not associated with gut microbiota composition and disease severity in patients with nonalcoholic fatty liver disease.

In this cross-sectional study, we hypothesized that a high dietary ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids could be associated with an altered gut bacterial composition and with the disease severity in patients with nonalcoholic fatty liver disease (NAFLD). A total of 101 NAFLD patients were included in the study, of which 63 underwent a liver biopsy. All 101 patients completed a 14-day food and activity record. Ebispro 2016 professional software was used to calculate individual macronutrients and micronutrients consumed. Patients were grouped into 3 quantiles (Q) according to a low (Q1: <6.1, n = 34), moderate (Q2: 6.1-7.8, n = 33), or high (Q3: >7.8, n = 34) dietary n-6/n-3 ratio. Stool samples were analyzed using 16S rRNA gene sequencing. Spearman correlation coefficients and principal coordinate analysis were used to detect differences in the bacterial composition of the gut microbiota. The median dietary n-6/n-3 ratio of all patients was 6.7 (range, 3.1-14.9). No significant associations between the dietary n-6/n-3 ratio and the gut microbiota composition or disease severity were observed. However, the abundance of specific bacteria such as Catenibacterium or Lactobacillus ruminis were found to be positively correlated and the abundance of Clostridium were negatively correlated with dietary n-6 fatty acid intake. The results indicate that a high dietary n-6/n-3 ratio is probably not a highly relevant factor in the pathogenesis of human NAFLD. Further studies are needed to clarify the importance of interactions between gut bacterial taxa and n-6 fatty acids in the pathophysiology of NAFLD.

Analysis of Human Gut Microbiota Composition Associated to the Presence of Commensal and Pathogen Microorganisms in Côte d'Ivoire.

BACKGROUND: The human gut microbiota is a microbial ecosystem contributing to the maintenance of host health with functions related to immune and metabolic aspects. Relations between microbiota and enteric pathogens in sub-Saharan Africa are scarcely investigated. The present study explored gut microbiota composition associated to the presence of common enteric pathogens and commensal microorganisms, e.g., Blastocystis and Entamoeba species, in children and adults from semi-urban and non-urban localities in Côte d'Ivoire. METHODS: Seventy-six stool samples were analyzed for microbiota composition by 16S rRDNA sequencing. The presence of adeno-, entero-, parechoviruses, bacterial and protozoal pathogens, Blastocystis, and commensal Entamoeba species, was analyzed by different molecular assays. RESULTS: Twelve individuals resulted negative for any tested microorganisms, 64 subjects were positive for one or more microorganisms. Adenovirus, enterovirus, enterotoxigenic Escherichia coli (ETEC), and Blastocystis were frequently detected. CONCLUSIONS: The bacterial composition driven by Prevotellaceae and Ruminococcaceae confirmed the biotype related to the traditional dietary and cooking practices in low-income countries. Clear separation in UniFrac distance in subjects co-harboring Entamoeba hartmanni and Blastocystis was evidenced. Alpha diversity variation in negative control group versus only Blastocystis positive suggested its possible regulatory contribution on intestinal microbiota. Pathogenic bacteria and virus did not affect the positive outcome of co-harbored Blastocystis.

A quality improvement study on the reduction of central venous catheter-associated bloodstream infections by use of self-disinfecting venous access caps (STERILE).

BACKGROUND: Contamination of the catheter hub is an important source of central line-associated bloodstream infections (CLABSI); catheter hub caps incorporating a 70% isopropyl alcohol aim are designed to reduce contamination and hence CLABSI rates. Supporting data in high-risk hematological and oncological patients on the clinical effectiveness of this approach are sparse. METHODS: We conducted a before-after single center study accompanying the introduction of such caps at our department. Retrospective data from the year prior to the introduction were compared to 1 year of prospective data. RESULTS: The control and antiseptic barrier cap (ABC) groups consisted of 309 and 289 patients presenting a CLABSI rate of 15.28 and 10.38 per 1,000 catheter days (P= .042), respectively. However, after multivariate analysis, ABCs were not identified as a statistically significant independent protective factor for the occurrence of CLABSI (hazard ratio 0.69, P= .120). There was no significant difference between the groups with respect to time to CLABSI (P= .681), nor the proportion of catheters removed due to suspicion of infection (P= .076). CONCLUSIONS: The introduction of ABCs in this high-risk population did not significantly alter CLABSI rates.

Intracellular concentrations of posaconazole in different compartments of peripheral blood.

Therapeutic drug monitoring (TDM) of antifungal plasma concentrations is increasingly recommended. However, data on antifungal concentrations in the other compartments of the peripheral blood are limited. Hence, we collected 23 blood samples from 14 patients receiving posaconazole for prophylaxis of fungal infections. These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. The intracellular posaconazole concentrations of the obtained cells, i.e., the peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs), and red blood cells (RBCs), were determined by liquid chromatography-tandem mass spectrometry. The intracellular concentrations of the PBMCs and PMNs were significantly higher than those of surrounding media (P < 0.001). The ratios between the intracellular and extracellular concentrations (C/E) were 22.5 +/- 21.2, 7.66 +/- 6.50, and 0.09 +/- 0.05 for the PBMCs, PMNs, and RBCs, respectively. Posaconazole reaches high concentrations within human PBMCs and PMNs and is, to a lesser extent, present in RBCs. The high intracellular concentrations might contribute to posaconazole efficacy and distribution.

Quantitation of azoles and echinocandins in compartments of peripheral blood by liquid chromatography-tandem mass spectrometry.

A rapid turnaround is a prerequisite of therapeutic drug monitoring (TDM). For antifungals, this need is still unmet, since hardly any method has been established to simultaneously quantitate concentrations of different antifungal classes. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed allowing quantitation of anidulafungin (ANF), caspofungin (CSF), isavuconazole (ISC), micafungin (MCF), posaconazole (PSC), and voriconazole (VRC). Quantitation was successful with diluted plasma samples, peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMN), and erythrocytes (RBC). A triple quadrupole mass spectrometer in selected reaction monitoring mode was used with positive electrospray ionization. Cells and calibration standards were extracted with acetonitrile containing internal standard. Internal standards were a CSF derivate for echinocandins and itraconazole for triazoles. Chromatographic separation of the supernatant was achieved by a gradient method facilitating a BetaBasic C4 column. Analytes were quantified in a single 8-min run. Calibration curves were linear and fitted using least squares with a weighting factor of the reciprocal concentration. Limits of detection (ng/ml) were ANF, 8.3; CSF, 31.5; ISC, 1.5; MCF, 97.7; PSC, 3.3; and VRC, 1.4. The lower limits of quantitation (ng/ml) were ANF, 64; CSF, 108; ISC, 4.5; MCF, 160; PSC, 10; and VRC, 4.2. Intraday precisions ranged from 6.3% to 8.8% for azoles and 8.8% to 15.4% for echinocandins. Intraday and interday accuracies (percent bias) of all analytes were within 13.8%. The method was established as standard practice for the quantitation of intracellular antifungal concentrations and optimizes TDM by applying a rapid single method for 6 antifungals.