Standardization of DNA extraction from paraffinized spleen samples: molecular diagnosis of human malaria.

BACKGROUND: Plasmodium vivax is the main species responsible for human malaria in Brazil, and one of its manifestations is splenic malaria, though there are still challenges in its diagnosis. The present study aimed to standardize Plasmodium sp. DNA extraction from histological slices of spleen and diagnosis using real-time qPCR. METHODS: This study performed a microtomy of a paraffin-embedded spleen as a positive control for P. vivax from a patient who had been previously diagnosed with the parasite. The sample was deparaffinized with xylol and ethanol, then DNA extraction was performed with two commercial kits. qPCR was carried out with the Taqman system for detection of Plasmodium sp. and was made species-specific using PvmtCOX1 gene. From 2015 to 2019, 200 spleen samples were obtained from trauma patients subjected to splenectomy in Manaus, Amazonas. All the samples were tested for cell-free human DNA (cfDNA). RESULTS: The deparaffinization and the Plasmodium vivax DNA extraction method was successfully standardized, and the control sample was positive for P. vivax. Of the 200 samples, all qPCRs were negative, but they were positive for human PCR. CONCLUSION: Paraffinization is practical and efficient for the preservation of samples, but the formation of bonds between proteins and DNA makes extraction difficult. Despite this, in this study, it was possible to standardize a method of DNA extraction for detecting P. vivax.

Integrative toolbox to assess the quality of freshwater sediments contaminated with potentially toxic metals.

The Guadiana Basin is a transnational basin, presenting historical contamination with potentially toxic metals (PTM), which origin can be both natural and anthropogenic. This study explores the use of a set of observational, chemical and ecotoxicological assays with Heterocypris incongruens, Vibrio fischeri, Pseudokirchneriella subcapitata, Thamnocephalus platyurus, identifying the most sensitive to be included in a toolbox to analyze the quality of freshwater sediments related to this type of contamination. The study included the analysis of a reservoir and streams sediments of Guadiana basin, in two consecutive years with different climate conditions 2017 (dry year) and 2018 (normal year). The results showed high chemical variability along the basin, with greater contamination with PTM in the reservoir sediments. The calculated Enrichment Factors (EF) indicated high anthropogenic contamination by Cd, followed by Pb (EF > 1.5). The geoaccumulation index (Igeo) revealed that the sediments were severely polluted with Cd, and slightly polluted with Pb and Cu, inducing a higher sublethal toxicity to Heterocypris incongruens. Among the parameters evaluated, and after the use of multivariate statistical techniques, the toolbox for assessing sediments quality, in similar climate and geological conditions, should include the analysis of: meteorology, land use/cover in the area, granulometry, organic matter content, PTM concentrations, contamination indices (e.g., Igeo and EF), and sublethal bioassays with H. incongruens (total sediment analysis) and Vibrio fisheri luminescence inhibition (pore water analysis).

Benthic diatom community dynamics in Mediterranean intermittent streams: Effects of water availability and their potential as indicators of dry-phase ecological status.

The study of intermittent rivers is a critical and timely issue due to their worldwide increase, triggered by several causes including climate change. The need to understand the response of intermittent river biota to water intermittency led us to conduct this study using benthic diatoms collected in southern Portugal. Benthic diatoms were explored in terms of assemblages, diversity indices, the Specific Pollution Sensitivity (SPI) Index, functional metrics (i.e. ecological guilds and life-forms) and conservation status. We verified that changes in water physico-chemical characteristics were highly controlled by flow intermittency, which in turn is directly linked to meteorological variables (air temperature and precipitation). Changes in diatom assemblages reflect the aquatic regime of sites, changes in aquatic states through time and mesohabitats (dry biofilm, samples collected in pools or under flowing conditions). Species richness, on the other hand, did not reflect these differences, whilst Shannon diversity and Pielou's Evenness indices only reflected mesohabitat differences. The SPI distinguished sampling periods, and mesohabitats. The relative abundance of ecological guilds changed with aquatic states, with the low-profile guild dominating in eurheic and arheic conditions (except during Summer), being replaced by motile taxa in summer arheic conditions, reflecting increases in nutrient and siltation. The hypothesis that benthic diatom assemblages in dry biofilm can be used as an indicator of ecological status during the dry-phase was validated, since no differences between the Ecological Quality Ratio determined in dry biofilm collected in Summer 2017 and the previous Spring 2017 in flowing water. A method is proposed for diatom sampling in dry biofilm, contributing to an integrated ecological status evaluation, which considers the dry-phase and enhances the reach of biomonitoring programs.

Vertical distribution of benthic diatoms in a large reservoir (Alqueva, Southern Portugal) during thermal stratification.

Freshwater diatom communities are known to respond to a wide range of environmental factors, however, the depth gradient is usually neglected and few studies are available, especially in large reservoirs. During the ALqueva hydro-meteorological EXperiment (ALEX) field campaign, diatom communities were studied in the margins and in three platforms (from the surface to the bottom of the reservoir) located in the limnetic zone of the Alqueva reservoir, one of the largest artificial lakes in western Europe. A detailed meteorological and physico-chemical characterization of the reservoir was carried out from June to September in Summer 2014, when the reservoir was stratified, to relate these variables with diatom assemblages. Despite the large dimensions of the reservoir, no differences in the water physico-chemical characteristics and diatom descriptors were detected among platforms. Small changes in diatom assemblages, ecological guilds, taxa richness and Shannon diversity index were observed between sampling campaigns. Nevertheless, differences in diatoms were detected along a depth gradient, both in terms of diatom assemblages and ecological guilds. Taxa richness, Shannon diversity index, Pielou's evenness and Specific Pollution sensitivity Index (SPI) also differed with depth, with the lowest values of all indices detected at surface samples, increasing with depth, reaching the highest values at 20 m for taxa richness, Shannon diversity and Pielou's evenness indices.

Biomonitoring of intermittent rivers and ephemeral streams in Europe: Current practice and priorities to enhance ecological status assessments.

Intermittent rivers and ephemeral streams (IRES) are common across Europe and dominate some Mediterranean river networks. In all climate zones, IRES support high biodiversity and provide ecosystem services. As dynamic ecosystems that transition between flowing, pool, and dry states, IRES are typically poorly represented in biomonitoring programmes implemented to characterize EU Water Framework Directive ecological status. We report the results of a survey completed by representatives from 20 European countries to identify current challenges to IRES status assessment, examples of best practice, and priorities for future research. We identify five major barriers to effective ecological status classification in IRES: 1. the exclusion of IRES from Water Framework Directive biomonitoring based on their small catchment size; 2. the lack of river typologies that distinguish between contrasting IRES; 3. difficulties in defining the 'reference conditions' that represent unimpacted dynamic ecosystems; 4. classification of IRES ecological status based on lotic communities sampled using methods developed for perennial rivers; and 5. a reliance on taxonomic characterization of local communities. Despite these challenges, we recognize examples of innovative practice that can inform modification of current biomonitoring activity to promote effective IRES status classification. Priorities for future research include reconceptualization of the reference condition approach to accommodate spatiotemporal fluctuations in community composition, and modification of indices of ecosystem health to recognize both taxon-specific sensitivities to intermittence and dispersal abilities, within a landscape context.

Non-perennial Mediterranean rivers in Europe: Status, pressures, and challenges for research and management.

Non-perennial rivers and streams (NPRS) cover >50% of the global river network. They are particularly predominant in Mediterranean Europe as a result of dry climate conditions, climate change and land use development. Historically, both scientists and policy makers underestimated the importance of NRPS for nature and humans alike, mainly because they have been considered as systems of low ecological and economic value. During the past decades, diminishing water resources have increased the spatial and temporal extent of artificial NPRS as well as their exposure to multiple stressors, which threatening their ecological integrity, biodiversity and ecosystem services. In this paper, we provide a comprehensive overview of the structural and functional characteristics of NPRS in the European Mediterranean, and discuss gaps and problems in their management, concerning their typology, ecological assessment, legislative and policy protection, and incorporation in River Basin Management Plans. Because NPRS comprise highly unstable ecosystems, with strong and often unpredictable temporal and spatial variability - at least as far as it is possible to assess - we outline the future research needs required to better understand, manage and conserve them as highly valuable and sensitive ecosystems. Efficient collaborative activities among multidisciplinary research groups aiming to create innovative knowledge, water managers and policy makers are urgently needed in order to establish an appropriate methodological and legislative background. The incorporation of NPRS in EU-Med River Basin Management Plans in combination with the application of ecological flows is a first step towards enhancing NPRS management and conservation in order to effectively safeguard these highly valuable albeit threatened ecosystems.

Cyanobacteria toxicity: potential public health impact in South Portugal populations.

Cyanobacteria are prokaryotic, plantlike organisms present in lakes, recreational waters, and reservoirs, and often dominate phytoplankton communities in warm, nutrient-enriched hard waters. A stable water column rich in certain nutrients, especially nitrogen and phosphorus, is associated with favorable environmental conditions that support development of cyanobacterial population maxima or "blooms." Under specific conditions, cyanobacteria produce toxins that are responsible for acute poisoning and death of animals and humans. The main aim of this study was to correlate the presence of cyanobacteria blooms with potential toxicity to humans as a public health issue. In Portugal, seven reservoirs located in the southern region were selected and studied between 2000 and 2008. Reservoirs were characterized by physical and chemical aspects, and identification of phytoplankton communities. In the case of cyanobacterial blooms, toxins that affected the liver, nervous system, and skin were detected, namely, Microcystis aeruginosa, Aphanizomenon spp., and Oscillatoria. These findings suggest the presence of a potential risk for public health, and indicate the need to implement mitigation measures in all studied reservoirs. These measures may involve (1) water eutrophication control to avoid blooms, (2) appropriate treatment of water for human consumption, and (3) public warnings or information to those individuals that use these reservoirs for several recreational activities.

Role of the sediments of two tropical dam reservoirs in the flux of metallic elements to the water column.

In tropical climates, the high rainfall and temperature, throughout the annual cycle, allow high leaching rates of metallic elements from the basin upstream, which accumulate in the reservoirs. However, the concentration of these elements in natural waters is usually lower than expected, due to the ease of adsorption and co-precipitation in solid phases. We have studied two tropical dam reservoirs in Brazil, Três Marias (Minas Gerais) and Tucuruí (Pará), with the aim of understanding the correlation between physical-chemical parameters of the water column, chemical and mineralogical characteristics of the accumulated material and the solubility, mobilization and precipitation of metals in reservoirs. Metals speciation performed in selected samples determined that metallic micronutrients are preferentially adsorbed or retained through precipitation/co-precipitation onto fine-size charged crystalline/amorphous Fe-oxides. Under the prevailing reducing and low pH conditions of the bottom reservoirs, some adsorbed metals (particularly Fe and Mn) are easily released from their metal bearing-phases and mobilized to the aqueous phase of sediments, which show high levels of soluble forms of these elements. However, the solubilization process and the release to the water column are not very extensive, as abundances of metals such as Fe, Mn, Zn and Cu in water are low, although increasing with depth.