Bioaccumulation and fate of pharmaceuticals in a Mediterranean coastal lagoon: Temporal variation and impact of a flash flood event.

Coastal ecosystems are particularly vulnerable to terrestrial inputs from human-impacted areas. The prevalence of wastewater treatment plants, unable to remove contaminants such as pharmaceuticals (PhACs), leads to their continuous input into the marine environment. In this paper, the seasonal occurrence of PhACs in a semi-confined coastal lagoon (the Mar Menor, south-eastern Spain) was studied during 2018 and 2019 by evaluating their presence in seawater and sediments, and their bioaccumulation in aquatic organisms. Temporal variation in the contamination levels was evaluated by comparison to a previous study carried out between 2010 and 2011 before the cessation of permanent discharges of treated wastewater into the lagoon. The impact of a flash flood event (September 2019) on PhACs pollution was also assessed. A total of seven compounds (out of 69 PhACs analysed) were found in seawater during 2018-2019, with a limited detection frequency (<33%) and concentrations (up to 11 ng/L of clarithromycin). Only carbamazepine was found in sediments (ND-1.2 ng/g dw), suggesting an improved environmental quality in comparison to 2010-2011 (when 24 and 13 compounds were detected in seawater and sediments, respectively). However, the biomonitoring of fish and molluscs showed a still remarkable accumulation of analgesic/anti-inflammatory drugs, lipid regulators, psychiatric drugs and ß-blocking agents, albeit not higher than in 2010. The flash flood event from 2019 increased the prevalence of PhACs in the lagoon, compared to the 2018-2019 sampling campaigns, especially in the upper water layer. After the flash flood the antibiotics clarithromycin and sulfapyridine yielded the highest concentrations ever reported in the lagoon (297 and 145 ng/L, respectively), alongside azithromycin in 2011 (155 ng/L). Flash flood events associated with sewer overflows and soil mobilisation, which are expected to increase under climate change scenarios, should be considered when assessing the risks posed by pharmaceuticals to vulnerable aquatic ecosystems in the coastal areas.

Can three-dimensional high-resolution anorectal manometry detect anal sphincter defects in patients with faecal incontinence?

AIM: Endoanal ultrasound (EAUS) is the gold standard for detecting anal sphincter defects in patients with faecal incontinence (FI), while anorectal manometry evaluates sphincter function. Three-dimensional high-resolution anorectal manometry (3D HRAM) is a newer modality with the potential to assess both sphincter function and anatomy. The purpose of the present study was to compare 3D HRAM with 3D EAUS for the detection of anal sphincter defects in patients with FI. METHOD: A linkage analysis was performed between the 3D HRAM and 3D EAUS databases of a tertiary referral centre to identify patients with FI who underwent both 3D EAUS and 3D HRAM. With 3D HRAM, a defect was defined as any pressure measurement below 25 mmHg at rest with at least 18° of continuous expansion. The 3D HRAM findings were compared with those of 3D EAUS. RESULTS: The study cohort included 39 patients with a mean age of 64.7 ± 15.2 years (SD); and 31 (79%) were female. Eight (21%) patients had an anal sphincter defect on EAUS with a median size of 93° (range 40°-136°). Fourteen (36%) had a defect shown by 3D HRAM with a median size of 144° (36°-180°). The sensitivity, specificity and positive and negative predictive values of 3D HRAM in detecting a sphincter defect were 75%, 74%, 43% and 92%, respectively. CONCLUSION: With a negative predictive value of 92%, 3D HRAM may be a useful screening method for ruling out a sphincter defect in patients with FI, thereby avoiding both EAUS and manometry in selected patients.

Repeat Rifaximin for Irritable Bowel Syndrome: No Clinically Significant Changes in Stool Microbial Antibiotic Sensitivity.

BACKGROUND: Rifaximin has demonstrated efficacy and safety for diarrhea-predominant irritable bowel syndrome (IBS-D). AIM: To determine the rifaximin repeat treatment effect on fecal bacterial antibiotic susceptibility. METHODS: Patients with IBS in Trial 3 (TARGET 3) study who responded to open-label rifaximin 550 mg three times daily for 2 weeks, with symptom recurrence within 18 weeks, were randomized to double-blind treatment: two 2-week repeat courses of rifaximin or placebo, separated by 10 weeks. Prospective stool sample collection occurred before and after open-label rifaximin, before and after the first repeat course, and at the end of the study. Susceptibility testing was performed with 11 antibiotics, including rifaximin and rifampin, using broth microdilution or agar dilution methods. RESULTS: Of 103 patients receiving open-label rifaximin, 73 received double-blind rifaximin (n = 37) or placebo (n = 36). A total of 1429 bacterial and yeast isolates were identified, of which Bacteroidaceae (36.7%) and Enterobacteriaceae (33.9%) were the most common. In the double-blind phase, Clostridium difficile was highly susceptible to rifaximin [minimum inhibitory concentration (MIC) range 0.008-1 µg/mL] and rifampin (MIC range 0.004-0.25 µg/mL). Following double-blind rifaximin treatment, Staphylococcus isolates remained susceptible to rifaximin at all visits (MIC50 range ≤0.06-32 µg/mL). Rifaximin exposure was not associated with long-term cross-resistance of Bacteroidaceae, Enterobacteriaceae, and Enterococcaceae to rifampin or nonrifamycin antibiotics tested. CONCLUSIONS: In this study, short-term repeat treatment with rifaximin has no apparent long-term effect on stool microbial susceptibility to rifaximin, rifampin, and nonrifamycin antibiotics. CLINICALTRIALS. GOV IDENTIFIER: NCT01543178.

Cytotoxic cell involvement in human cutaneous leishmaniasis: assessments in active disease, under therapy and after clinical cure.

Cutaneous leishmaniasis (CL) is an important public health issue worldwide. The control of Leishmania infection depends on cellular immune mechanisms, and the inflammatory response may contribute to pathogenesis. A beneficial role of CD8(+) T lymphocytes has been proposed; nevertheless, other studies suggest a cytotoxic role of CD8(+) T lymphocytes involved in tissue damage, showing controversial role of these cells. The goal of the current study was to understand the immunopathology of CL and determine the profile of cytotoxic cells--such as CD4(+) T, natural killer and natural killer T cells--that might be involved in triggering immunological mechanisms, and may lead to cure or disease progression. The frequencies of cytotoxic cell populations in peripheral blood, obtained from patients with active disease, during treatment and after clinical healing, were assessed by flow cytometry. Cytotoxicity could not be related to a deleterious role in Leishmania braziliensis infection, as patients with active CL showed similar percentages of degranulation to healthy individuals (HI). Cured patients exhibited a lower percentage of degranulating cells, which may be due to a downregulation of the immune response. The understanding of the immunopathological mechanisms involved in CL and the commitment of cytotoxic cells enables improvements in therapeutic strategies.

Screening for fragile X syndrome in males from specialized institutions in the northeast region of Brazil.

The objective of this study was to perform a study of fragile X syndrome (FXS) in São Luís, Maranhão, in males residing in five specialized institutions. Two hundred thirty-eight males with intel-lectual disability of unknown etiology participated in this study. Blood samples were processed and stored until DNA extraction. Screening for FMR1 gene mutations was performed using non-isotopic polymerase chain reaction amplification and DNA sequencing using an ABI Prism 3130 automated sequencer. Two individuals (0.84%) were positive for FMR1 mutations. One had a mutation due to expansion of the CGG repeat beyond normal levels and the other had a deletion in exon 1 of the FMR1 gene, which was confirmed by sequencing. Both probands were over 18 years old, which demonstrates the late diagnosis of the condition in these individuals and reinforces the need to implement ef-fective programs for early diagnosis of FXS in the state of Maranhão. We found that FXS might be transmitted in the families of the two indi-viduals bearing the mutation, and that it is important to understand the mutation dynamics to provide better counseling to the family members of these two individuals.

Levels of mannose-binding lectin in individuals with visceral leishmaniasis in the northeast region of Brazil.

Visceral leishmaniasis (VL) is one of the seven priority endemic diseases in the world. The clinical outcome of many infections is not only dependent on the pathogenic organism, but also on the genetic variability of the host susceptibility to infection. Mannose-binding lectin (MBL) is a protein that plays an important role in the innate immune system. The aim of this study was to compare the serum levels of MBL between healthy controls and carriers of VL. The VL cases were recruited randomly from the main hospitals and referral outpatient clinics for VL in São Luís, and from home visits. Determination of MBL protein levels was performed by enzyme-linked immunosorbent assay. Of the 161 patients with VL and the 161 healthy controls, 60.9 and 67.1% had high levels of MBL, respectively. There was no significant difference in MBL levels between cases and controls. Low socioeconomic status and living conditions are conducive to the occurrence of VL. Owing to the small number of existing studies, it is extremely important to conduct further studies on MBL levels and susceptibility to VL, especially in regions where the disease is endemic, such as Maranhão, Brazil.

Occurrence and distribution of contaminants of legacy and emerging concern in surface waters of two Western Mediterranean coastal areas: Mar Menor Lagoon and Ebro Delta.

The occurrence and distribution of UV filters, plastic additives, synthetic musks, other personal care products (Other PCPs), triazines, polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs), organophosphorus pesticides (OPPs), polychlorinated biphenyls (PCBs) and other current-use pesticides (Other CUPs) were characterised during summer 2018 and winter 2019 in surface waters of two sensitive areas of the Spanish coast located on the Mediterranean Sea (Mar Menor lagoon and Ebro Delta). Sixty-three organic contaminants out of a total of 100 compounds were detected, thus confirming the presence of all groups of pollutants studied in surface water at concentrations of ng/L. Both areas are affected by agricultural, urban and recreational activities, PCPs (mainly UV filters) being the predominant compounds found in both seasons which showed significant increases in concentrations in summer. The contaminants found at the highest concentrations were octocrylene, homosalate and ethylhexyl salicylate, which showed risk quotients higher than 1, indicating a potential risk to aquatic organisms, particularly in summer.

Floating plastics as integrative samplers of organic contaminants of legacy and emerging concern from Western Mediterranean coastal areas.

This study investigates the role of floating plastics as integrative samplers of organic contaminants. To this end, plastics items were collected in two Western Mediterranean coastal areas: the Mar Menor lagoon, and the last transect of Ebro river. Floating plastics were identified and characterized by attenuated total reflection Fourier-transform infrared spectrometry. Then, organic contaminants were extracted from plastic items by ultrasonic extraction with methanol, and the concentrations of 168 regulated and emerging contaminants were analysed. These compounds were analysed by stir bar sorptive extraction coupled to gas chromatography-mass spectrometry (GC-MS), except for bisphenol analogues, which were analysed with a ultraperformance liquid chromatography pump coupled to a triple quadrupole mass spectrometer (UHPLC-MS/MS), and pharmaceutical compounds, determined by UPLC coupled to hybrid triple quadrupole-linear ion trap mass spectrometer (UPLC-MS/MS). All the contaminants groups considered were detected in the samples, being particularly relevant the contribution of plastic additives. The most frequently detected contaminants were UV-filters, PAHs, pharmaceuticals and synthetic musks. Apart from plasticizers, the individual contaminants octocrylene, homosalate, galaxolide, salycilic acid and ketoprofen were frequently detected in plastics items. The results pointed out to urban and touristic activities as the main sources of pollution in the coastal areas investigated. The utility of floating plastics as integrative samplers for the detection of organic contaminants in aquatic ecosystems has been demonstrated.

Combined exposure of the bivalve Mytilus galloprovincialis to polyethylene microplastics and two pharmaceuticals (citalopram and bezafibrate): Bioaccumulation and metabolomic studies.

Pharmaceuticals and microplastics constitute potential hazards in aquatic systems, but their combined effects and underlying toxicity mechanisms remain largely unknown. In this study, a simultaneous characterization of bioaccumulation, associated metabolomic alterations and potential recovery mechanisms was performed. Specifically, a bioassay on Mediterranean mussels (Mytilus galloprovincialis) was carried out with polyethylene microplastics (PE-MPLs, 1 mg/L) and citalopram or bezafibrate (500 ng/L). Single and co-exposure scenarios lasted 21 days, followed by a 7-day depuration period to assess their potential recovery. PE-MPLs delayed the bioaccumulation of citalopram (lower mean at 10 d: 447 compared to 770 ng/g dw under single exposure), although reaching similar tissue concentrations after 21 d. A more limited accumulation of bezafibrate was observed overall, regardless of PE-MPLs co-exposure (

Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis.

Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4(+) T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4(+) T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Multifunctional CD4(+) T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4(+) T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis.

A holistic assessment of the sources, prevalence, and distribution of bisphenol A and analogues in water, sediments, biota and plastic litter of the Ebro Delta (Spain).

Bisphenol A (BPA) is one of the main ubiquitous compounds released from plastics in the environment. This compound, considered an endocrine disruptor, poses a risk to aquatic wildlife and human population, being included in multiple environmental monitoring programmes. Following the regulations restricting BPA use in the last years, BPA-like chemicals have been produced and used as BPA substitutes. However, they are not commonly included in monitoring programs yet and their presence is thus misrepresented, despite showing similar endocrine disrupting potential. In this work, an analytical method for analysing bisphenol A and five of its analogues (Bisphenol S, B, F, AF and Tetrabromobisphenol A) is described, validated for water (riverine, sea and wastewater), sediment, and biota (fish and biofilm) and applied to monitor their presence in the Ebro River Delta (NE Spain). In addition, plastic litter was also collected to evaluate their role as potential source of bisphenols. All compounds except BPF were detected in the analysed samples. Wastewater treatment plants (WWTPs) were discarded as major sources of BPs into the natural aquatic environment, as no BPs were detected in treated effluents. Indeed, the high levels of BPs in the natural environment could be related with direct discharge of raw wastewater from small rural population nucleus. The analysis of riverine plastic leachates yielded 4 out of the 6 BPs analysed, strengthening the hypothesis that plastic debris are also a source of BPs in the natural environment. Whereas Bisphenol S and BPA were detected in water and, to a limited extent, in biota, less polar analogues (mainly BPAF and TBBPA) were not found in any of the water samples. Instead, these hydrophobic BPs were found in fish tissues and biofilm, pointing out plastics and microplastics as their possible vectors. Finally, biofilm demonstrated its potential as sentinel of chemical contamination in freshwater environment.

Genetic analysis of LRRK2 functional domains in Brazilian patients with Parkinson's disease.

BACKGROUND AND PURPOSE: Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been associated with Parkinson's disease (PD), and the majority of the pathogenic variants are located in the ROC and MAPKKK domains. METHODS: Exons 29-31 and 38-44 (ROC and MAPKKK domains) were sequenced in 204 patients with PD, mostly Brazilian. RESULTS: We identified four polymorphisms, a novel silent variant p.R1398R and four substitutions: p.T1410M, p.G2019S, p.Y2189C and the novel variant p.C2139S. CONCLUSIONS: The most prevalent mutation was the p.G2019S (2.4%). We consider that the p.T1410M and the p.Y2189C variants are probably polymorphisms and that the p.C2139S mutation is potentially pathogenic.

Redefining the role of lymphocytes in gastroesophageal reflux disease and eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) and reflux esophagitis (RE) overlap clinically and histologically. RE is characterized by epithelial infiltration with small numbers of neutrophils and eosinophils, EoE by a prominent eosinophilic infiltrate. Lymphocytic esophagitis (LE), a new entity characterized by peripapillary lymphocytosis, questions the role lymphocytes play in esophageal inflammation. We test the hypothesis that lymphocyte infiltration in RE differs from EoE. One blinded pathologist read esophageal biopsies from 39 RE and 39 EoE patients. Both groups demonstrated significant numbers of lymphocytes (RE 22.7 +/- 2.2/HPF, EoE 19.8 +/- 1.8/HPF). Eosinophils/HPF in RE and EoE were 2.8 +/- 0.7 and 74.9 +/- 8.2, respectively (P < 0.001). Neutrophils were uncommon in RE (0.26 +/- 0.16/HPF) and EoE (0.09 +/- 0.04; P = 0.07). Eight of the 39 RE specimens had >or=50 lymphocytes in >or=1 HPF. Two were consistent with LE. There was an inverse correlation between numbers of eosinophils and lymphocytes in EoE (R = -0.47; P = 0.002), and no correlation between them in RE (R = 0.18; P = 0.36). The patients with EoE who used antireflux medications had fewer lymphocytes (16.3 +/- 1.3 vs 22.2 +/- 2.3/HPF; P = 0.030) and eosinophils (55.6 +/- 5.2 vs 76.0 +/- 8.7/HPF; P = 0.042) than those who did not. The pathological role of lymphocytes in RE and EoE may be underestimated. Our observation that 5% of the RE specimens meet histopathological criteria for LE potentially blurs the line between these entities. The observation that eosinophil counts are lower in EoE when antireflux meds are used supports the notion that reflux plays a role in the clinical expression of EoE.

Beta3-adrenergic receptor polymorphism is related to cardiometabolic risk factors in obese Brazilian subjects.

We determined whether ADRbeta3 polymorphism is associated with obesity-related traits in 140 obese patients. Molecular analysis was performed by PCR and RFLP. Individuals carrying the Arg64 allele had a lower waist-to-hip ratio, higher adiponectin and high-density lipoprotein cholesterol levels, and a tendency towards lower blood pressure. In contrast, Trp64/Trp64 carriers were at greater risk for dysmetabolic phenotypes (odds ratio = 2.88, P = 0.03).

The impact of folate pathway polymorphisms combined to nutritional deficiency as a maternal predisposition factor for Down syndrome.

Polymorphisms in genes encoding folate metabolizing enzymes have been linked to an increased risk of maternal chromosomal nondisjunction in several populations. With the purpose of evaluating this relationship, we compared the frequencies of 677C>T and 1298A>C polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR) and 66A>G in the methionine synthase reductase gene (MTRR) between 103 young mothers of Down syndrome (DS) individuals and 108 control mothers, whose offspring was karyotypically normal, correlating it with an estimative of folate and - related micronutrients levels intake. Maternal and paternal transmission frequencies of MTHFR 677T allele were also examined to access potential parent-of-origin effects. PCR-RFLP for genomic DNA was accomplished and allele/genotype frequencies differences were determined using the x(2) test, whereas pattern of transmission of the MTHFR 677 allele was analyzed by transmission disequilibrium test. None of the polymorphisms seemed to be more frequent in case mothers than in controls, either individually or combined. The estimative of nutritional intake revealed that folate consumption median was inadequate in both groups, whereas methionine and zinc consumption medians were significantly greater in control mothers. It suggests that such interaction between genetic profile and environment could predispose this sub group of women to have a DS child. Additional studies focusing the interaction between nutritional intakes, biochemical data and folate pathway polymorphisms are needed to confirm the present results. The possibility of neutralize the biochemical negative effects of folate-related polymorphisms through oral supplementation could provide new targets for DS prevention.