Human brucellosis in Portugal-Retrospective analysis of suspected clinical cases of infection from 2009 to 2016.

Brucellosis is a zoonosis that is emerging in some regions of the world. Although brucellosis is a disease of obligatory declaration and is not eradicated in Portugal, no prevalence data is available in this country. In this study, we retrospectively analyzed the data available at the Reference Laboratory at the Portuguese National Institute of Health during the past 7 years (2009-2016) in order to get insight into the epidemiological scenario of brucellosis in Portugal. A total of 2313 biological samples from patients with clinical suspicion of brucellosis were subjected to immunological techniques for laboratory diagnosis. From 2010 to 2015, a subset of 259 samples was subjected to molecular methods. According to the available data, 167 out of 2313 (7.2%) samples had positive serology for Brucella spp. and 43 out of 259 samples (16.6%) were positive for B. melitensis by real time PCR, being classified as biovar 1 and 3. This study draws attention to the importance of integrating clinical and laboratory data of human cases in order to increase the efficacy of the response measures in case of outbreaks.

An epidemiological approach to characterise the human exposure pathways in a contaminated estuarine environment.

This study's aim was to develop and implement an integrative epidemiologic cross-sectional study that allows identifying and characterising exposure pathways of populations living and working on the shores of a contaminated estuarine environment. Population residing in Carrasqueira, located on the Sado estuary with known contaminated areas was compared to another population on a noncontaminated estuary (Vila Nova de Mil Fontes - VNMF), considered a nonexposed population. Simple random samples of individuals were selected in each study population from the National Health Service Lists: 140 individuals were selected in Carrasqueira and 219 in VNMF. Participation rates were higher in the exposed group (62.5%, n=102 in Carrasqueira and 48.3%, n=100 individuals in VNMF). The same structured questionnaire was used in both populations, including questions on occupational activities, leisure activities, consumption of food (including fish and mollusks from the estuary) and use of water for human intake and agriculture. Results showed that a significantly higher proportion of Carrasqueira participants reported doing tasks in their job that promote direct (48.8% vs 1.2% in VNMF, p-value<0.001) or indirect (30% vs 11.9% in VNMF, p-value=0.004) contact with water from the estuary. Regarding seafood consumption, the exposed population of Carrasqueira had a higher frequency of consumption of cuttlefish (23.5% vs 9% in VNMF, p-value=0.007), sole (22.5% vs 4% in VNMF, p-value<0.001) and clams (18.6% vs 5.0 in VNMF, p-value=0.004). The comparative study design, with exposed and nonexposed populations living on the shores of two different estuaries allowed us to confirm the hypothesis of a higher risk of contamination from the contaminated estuarine environment. The study design and the selection of both populations were adequate for this type of epidemiologic study of potential routes of human contamination in a mixture of contaminated estuarine environment and can be used in other estuarine areas with similar environmental risk.

Crosstalk between inflammation, iron metabolism and endothelial function in Behçet's disease.

Behçet's disease (BD) is a rare chronic vasculitis of unclear etiology. It has been suggested that inflammatory response has an important role in BD pathophysiology. Herein, we aimed to study the interplay between inflammation, iron metabolism and endothelial function in BD and search for its putative association with disease activity. Twenty five patients clinically diagnosed with BD were selected and twenty four healthy age-sex matched individuals participated as controls. Results showed an increase of total number of circulating white blood cells and neutrophils, serum transferrin, total iron binding capacity, mieloperoxidase (MPO), ceruloplasmin (Cp), C reactive protein, ß2 microglobulin and Cp surface expression in peripheral blood monocytes in BD patients comparatively to healthy individuals (p < 0,05). Of notice, the alterations observed were associated to disease activity status. No significant differences between the two groups were found in serum nitric oxide concentration. The results obtained suggest an important contribution from innate immunity in the pathogenesis of this disease. In particular, surface expression of leukocyte-derived Cp may constitute a new and relevant biomarker to understand BD etiology.

Total mercury in infant food, occurrence and exposure assessment in Portugal.

Commercial infant food labelled as from organic and conventional origin (n = 87) was analysed for total mercury content using a direct mercury analyser (DMA). Median contents of mercury were 0.50, 0.50 and 0.40 µg kg⁻¹ for processed cereal-based food, infant formulae and baby foods, respectively, with a maximum value of 19.56 µg kg⁻¹ in a baby food containing fish. Processed cereal-based food samples showed statistically significant differences for mercury content between organic and conventional analysed products. Consumption of commercial infant food analysed did not pose a risk to infants when the provisionally tolerable weekly intake (PTWI) for food other than fish and shellfish was considered. By the contrary, a risk to some infants could not be excluded when using the PTWI for fish and shellfish. This is the first study reporting contents of total mercury in commercial infant food from both farming systems and the first on exposure assessment of children to mercury in Portugal.

Implication of the RD(Rio) Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal.

Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RD(Rio) long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RD(Rio) deletion (referred to as RD(Rio)) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RD(Rio) sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12loci MIRU-VNTR RD(Rio) signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RD(Rio). This is the first report associating the LAM RD(Rio) sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB.

Primary antibiotic resistance of Helicobacter pylori strains isolated from Portuguese children: a prospective multicentre study over a 10 year period.

OBJECTIVES: The aim of this study was to prospectively assess the pattern of evolution of primary resistance to antibiotics in Helicobacter pylori strains isolated from Portuguese children over a 10 year period (2000-09). METHODS: A total of 1115 H. pylori strains were tested for antibiotic susceptibility to clarithromycin, metronidazole, amoxicillin, ciprofloxacin and tetracycline. RESULTS: H. pylori strains were isolated from children and adolescents [ages 4 months-18 years (mean age 10.17 ±â€Š4.03 years)], comprising 562 (50.4%) boys and 553 (49.6%) girls. Overall, the primary resistance rate was 34.7% to clarithromycin, 13.9% to metronidazole and 4.6% to ciprofloxacin, while 6.9% were resistant to two of these antibiotics simultaneously. Resistance to amoxicillin and to tetracycline was not detected. In general, the resistance rate was not associated with gender or the children's age. European ethnicity, when compared with an African background, was associated with clarithromycin resistance [P = 0.002; odds ratio (OR) = 0.30; 95% confidence interval (CI) 0.14-0.66], while the inverse situation was observed for metronidazole (P < 0.001; OR = 3.50; 95% CI 1.90-6.45). No significant temporal trend was noticed for resistance to clarithromycin and metronidazole, whereas ciprofloxacin and double-resistance rates have significantly increased over time (P = 0.004 and P = 0.05, respectively). CONCLUSIONS: The primary resistance rate of H. pylori strains isolated from Portuguese children to the commonly used anti-H. pylori antibiotics used is high. Additionally, the increasing trend of ciprofloxacin-resistant and double-resistant strains may compromise H. pylori eradication in a high-prevalence population.

Pathogenic fungi: an unacknowledged risk at coastal resorts? New insights on microbiological sand quality in Portugal.

Whilst the potential impact on beach users from microorganisms in water has received considerable attention, there has been relatively little investigation into microbial contaminants in sand. Thirty three beaches across Portugal were analyzed during a five year period (2006-2010) to determine the presence of yeasts, pathogenic fungi, dermatophytes, total coliforms, Escherichia coli and intestinal enterococci in sand. Our results showed that 60.4% of the samples were positive for fungi and that 25.2% were positive for the bacterial parameters. The most frequent fungal species found were Candida sp. and Aspergillus sp., whereas intestinal enterococci were the most frequently isolated bacteria. Positive associations were detected among analyzed parameters and country-regions but none among those parameters and sampling period. Regarding threshold values, we propose 15 cfu/g for yeasts, 17 cfu/g for potential pathogenic fungi, 8 cfu/g for dermatophytes. Twenty-five cfu/g for E. coli, and 10 [corrected] cfu/g for intestinal enterococci.

Air conditioning and intrahospital mortality during the 2003 heatwave in Portugal: evidence of a protective effect.

OBJECTIVES: The objective of the study was to analyse the association between the presence of air conditioning in hospital wards and the intrahospital mortality during the 2003 heatwave, in mainland Portugal. METHODS: Historical cohort study design including all patients aged 45 or more who were hospitalised in the 7 days before the heatwave. The outcome was survival during the 18 days the heatwave lasted and during the 2 days after the end of the heatwave. A comparison group was also selected in four analogous periods without any heatwave event during January to May 2003. Data were obtained from the 2003 hospital discharges database. Air conditioning presence in hospital wards was determined using a survey sent to hospital administrations. A Cox-regression model was used to estimate the confounder-adjusted HR of death, during the heatwave and the comparison period, in patients in wards with air conditioning (AC+) versus patients in wards without air conditioning (AC-). RESULTS: 41 hospitals of mainland Portugal (49% of all hospitals in mainland Portugal) participated, and 2093 patients were enrolled. The overall confounder-adjusted HR of death in AC+ patients versus AC- patients was 0.60 (95% CI 0.37 to 0.97) for the heatwave period and 1.05 (95% CI 0.84 to 1.32) for the comparison group. CONCLUSIONS: The study found strong evidence that, during the August 2003 heatwave, the presence of air conditioning in hospital wards was associated with an increased survival of patients admitted before the beginning of the climate event. The reduction of the risk of dying is estimated to be 40% (95% CI 3% to 63%).

Ceruloplasmin expression by human peripheral blood lymphocytes: a new link between immunity and iron metabolism.

Ceruloplasmin (CP) is a multicopper oxidase involved in the acute phase reaction to stress. Although the physiological role of CP is uncertain, its role in iron (Fe) homeostasis and protection against free radical-initiated cell injury has been widely documented. Previous studies showed the existence of two molecular isoforms of CP: secreted CP (sCP) and a membrane glycosylphosphatidylinositol (GPI)-anchored form of CP (GPI-CP). sCP is produced mainly by the liver and is abundant in human serum whereas GPI-CP is expressed in mammalian astrocytes, rat leptomeningeal cells, and Sertolli cells. Herein, we show using RT-PCR that human peripheral blood lymphocytes (huPBL) constitutively express the transcripts for both CP molecular isoforms previously reported. Also, expression of CP in huPBL is demonstrated by immunofluorescence with confocal microscopy and flow cytometry analysis using cells isolated from healthy blood donors with normal Fe status. Importantly, the results obtained show that natural killer cells have a significantly higher CP expression compared to all other major lymphocyte subsets. In this context, the involvement of lymphocyte-derived CP on host defense processes via its anti/prooxidant properties is proposed, giving further support for a close functional interaction between the immune system and the Fe metabolism.

Lymphocyte ceruloplasmin and Behçet's disease.

INTRODUCTION: Behçet's disease (BD) is a rare chronic inflammatory disorder of unknown aetiology. However, it has been postulated that a dysregulation of the prooxidant/antioxidant balance may be important to its pathogenesis. Ceruloplasmin (CP) is an acute phase protein expressed at the surface of peripheral blood lymphocytes (PBL) with antioxidant properties and with a relevant role in iron (Fe) metabolism. OBJECTIVES: To study CP expression at the surface of PBL (PBLCP) in patients with BD. MATERIAL AND METHODS: We measured serum CP and PBLCP obtained from BD patients (n=10) and respective controls (n=10) using nephelometry and flow cytometry techniques, respectively. Additionally, haematological parameters, biochemical Fe metabolism markers [serum Fe, serum ferritin, serum transferrin, total Fe binding capacity (TIBC), transferrin saturation] and non-specific markers of inflammation [serum C reactive protein (CRP), beta2-microglobulin] were measured in all individuals. RESULTS: Despite the absence of significant differences between the two study groups when comparing serum CP, a significant difference in PBLCP was found in BD patients mainly due to a significant decrease of CP expression at the surface of CD3-CD56+ lymphocytes. Also, a significant decrease of PBLCP was observed in patients treated with azathioprine compared to patients that were not being treated with this drug. CONCLUSIONS: According to this study, we suggest that the significant decrease of PBLCP observed in BD patients might be due to azathioprine treatment and not directly related to the pathophysiology of BD.