Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7%), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Molecular analysis of the bacterial diversity in a specialized consortium for diesel oil degradation

Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI.

A contaminação dos oceanos por radionuclídeos antropogênicos

Several hundreds of artificial radionuclides are produced as the result of human activities, such as the applications of nuclear reactors and particle accelerators, testing of nuclear weapons and nuclear accidents. Many of these radionuclides are short-lived and decay quickly after their production, but some of them are longer-lived and are released into the environment. From the radiological point of view the most important radionuclides are cesium-137, strontium-90 and plutonium-239, due to their chemical and nuclear characteristics. The two first radioisotopes present long half life (30 and 28 years), high fission yields and chemical behaviour similar to potassium and calcium, respectively. No stable element exists for plutonium-239, that presents high radiotoxicity, long half-life (24000 years) and some marine organisms accumulate plutonium at high levels. The radionuclides introduced into marine environment undergo various physical, chemical and biological processes taking place in the sea. These processes may be due to physical dispersion or complicated chemical and biological interactions of the radionuclides with inorganic and organic suspend matter, variety of living organisms, bottom sediments, etc. The behaviour of radionuclides in the sea depends primarily on their chemical properties, but it may also be influenced by properties of interacting matrices and other environmental factors. The major route of radiation exposure of man to artificial radionuclides occuring in the marine environment is through ingestion of radiologically contamined marine organisms. This paper summarizes the main sources of contamination in the marine environment and presents an overview covering the oceanic distribution of anthropogenic radionuclides in the FAO regions. A great number of measurements of artificial radionuclides have been carried out on various marine environmental samples in different oceans over the world, being cesium-137 the most widely measured radionuclide. Radionuclide concentrations vary from region to region, according to the specific sources of contamination. In some regions, such as the Irish Sea, the Baltic Sea and the Black Sea, the concentrations depend on the inputs due to discharges from reprocessing facilities and from Chernobyl accident. In Brazil, the artificial radioactivity is low and corresponds to typical deposition values due to fallout for the Southern Hemisphere.

Eletrodos modificados com polímeros perfluorados e sulfonados: aplicações em análises ambientais

Perfluoro and sulfonated ion-exchange polymers are recognized as a very useful material for various mechanistic studies and applications in electrochemistry. These polymers are characterized by high equivalent weights and by a low number of ion-exchange sites interposed between long organic chains. The solubility enables a preparation of stable polyelectrolyte films on the electrode surface. Examples of the determination of trace metals and organic componds in real environmental samples are presented.

Determinação de As em amostras orgânicas de interesse ambiental por espectrometria de absorção atômica com atomização eletrotérmica após combustão em bomba de O2

The toxicity of the major As species present in the environment justifies the effort for quantifying the element in environmental organic samples, which can vary from animal and vegetal tissues to coal and industrial residues. This paper comments about the applicability of the O2 bomb digestion, as a general procedure for all environmental organic materials. A rapid and straightforward method is suggested, which consists in burning the sample in the bomb at high O2 pressure, dissolving the vapours in diluted HNO3 and determining As in the resulting solution by atomic absorption spectrometry with electrothermal atomization. The method was applied to certified materials and plant samples.

Desafios da química analítica frente às necessidades da indústria farmacêutica

The development of liquid chromatography-mass spectrometric (LC-MS) techniques in the last few decades has made possible the analysis of trace amounts of analytes from complex matrices. With LC, the analytes of interest can be separated from each other as well as from the interfering matrix, after which they can be reliably identified thanks to the sensitivity and specificity of MS. LC-MS has become an irreplaceable tool for many applications, ranging from the analysis of proteins or pharmaceuticals in biological fluids to the analysis of toxic substances in environmental samples. In different segments of Brazilian Industry mass spectrometry has an important role, e.g. in the pharmaceutical industry in the development of generic formulations, contributing to the growth of Industry and social inclusion. However, the Brazilian chemists until this moment don't have an effective role in this new segment of the analytical chemistry in Brazil. The present paper shows the actual scenario for mass spectrometry in the pharmaceutical industry, emphasizing the need of a revision of graduation courses to attend the needs of this growing market.

Um novo método para quantificar mercúrio orgânico (Hg orgânico) empregando a espectrometria de fluorescência atômica do vapor frio

In this work a simple and sensitive procedure to extract organic mercury from water and sediment samples, using methylene chloride in acidic media followed by CVAFS quantification has been developed. The method was evaluated for possible interferents, using different inorganic mercury species and humic acid, no effects being observed. The detection limit for organic mercury was 160 pg and 396 pg for water and sediment samples respectively. The accuracy of the method was evaluated using a certified reference material of methylmercury (BCR-580, estuarine sediment). Recovery tests using methylmercury as surrogate spiked with 1.0 up to 30.0 ng L-1 ranged from 90 up to 109% for water samples, whereas for sediments, recoveries ranged from 57 up to 97%.

Planilha de validação: uma nova ferramenta para estimar figuras de mérito na validação de métodos analíticos univariados

This work presents the VALIDATION SPREADSHEET, a public domain tool that can be used to evaluate the figures of merit for univariate analytical methods. A real example of BTEX determination in environmental samples is included to illustrate its use. The spreadsheet has been developed for Excel® and Open Office®, and is available on the internet at http//lqta.iqm.unicamp.br.

Controle da qualidade na determinação potenciométrica de sulfetos volatilizáveis por acidificação (SVA) utilizando adições de padrão

This paper presents a quality assurance and quality control protocol in order to ensure the reliability of analytical results for the determination of acid volatile sulfides in environmental samples by potentiometry. The monitoring of the ion selective sensor readings must be realized continually and particularly in this situation where the analyte is susceptible to transformation and volatilization. Potentiometry with the standard addition was presented as a rapid, accurate, precise, feasible as well as a low cost technique. Concentrations of the analyte in the range of 0.49 to 1.02 mg L-1 were tested with percentage recoveries varying from 89 to 113%.

Construção e aplicação de um minissensor de filme de bismuto utilizando materiais de baixo custo para determinações voltamétricas in loco

The construction of a low cost mini sensor containing a bismuth-film electrode (BiFE), as work electrode, a silver electrode as pseudo reference electrode, and copper as counter electrode is proposed. The application of this mini sensor using a low cost electrochemical cell for in loco voltammetric determinations of inorganic and organic analytes is also described.

LOW COST ANALYZER FOR THE DETERMINATION OF PHOSPHORUS BASED ON OPEN-SOURCE HARDWARE AND PULSED FLOWS

The need for automated analyzers for industrial and environmental samples has triggered the research for new and cost-effective strategies of automation and control of analytical systems. The widespread availability of open-source hardware together with novel analytical methods based on pulsed flows have opened the possibility of implementing standalone automated analytical systems at low cost. Among the areas that can benefit from this approach are the analysis of industrial products and effluents and environmental analysis. In this work, a multi-pumping flow system is proposed for the determination of phosphorus in effluents and polluted water samples. The system employs photometric detection based on the formation of molybdovanadophosphoric acid, and the fluidic circuit is built using three solenoid micropumps. The detection is implemented with a low cost LED-photodiode photometric detection system and the whole system is controlled by an open-source Arduino Uno microcontroller board. The optimization of the timing to ensure the color development and the pumping cycle is discussed for the proposed implementation. Experimental results to evaluate the system behavior are presented verifying a linear relationship between the relative absorbance and the phosphorus concentrations for levels as high as 50 mg L-1.

Uses of nitrate ion sensitive electrodes

Some models of ion-selective electrodes (ISE) and other methods have been elaborated, to quantify nitrate levels in environmental samples (water, fruits, vegetables and others), using direct potentiometry

Amphoteric behavior of di-2-pyridyl ketone benzoylhydrazone in ethanol-water mixtures

The ligand di-2-pyridyl ketone benzoylhydrazone (DPKBH) is widely used for the determination of transition metal ions in environmental samples. Due to its low solubility in water it is used in aqueous-ethanol (1:1) solvent and for higher sensitivity the pH must be properly adjusted. The properties of DPKBH solutions must be known at different ethanol-water percentages in order to achieve higher sensitivity and/or selectivity for metal analysis. The acid-base behavior of this reagent in aqueous-ethanol solvent and the dissociation/ionization constants (pK1 and pK2) of DPKBH have been determined in different aqueous-ethanol solvent mixtures (10, 20, 30 and 50 % V/V of ethanol) from potentiometric titrations at 25.0 ± 0.1° C. As the amount of ethanol increases from 10 to 30% the pK1 and pK2 values increased, but they decreased in 50% of the organic solvent. The results are correlated with the medium composition and its effects.

Molecular epidemiology and extended-spectrum β-lactamases production of Klebsiella pneumoniae isolated from three dairy herds

The objectives of this study were to isolate Klebsiella pneumoniae from different sources in three dairy cattle herds, to use the pulsed-field gel electrophoresis (PFGE) to measure genotypic similarities between isolates within a dairy herd, to verify the production of extended-spectrum β-lactamases (ESBLs) by the double-disk synergy test (DDST), and to use the PCR to detect the main ESBLs subgroups genes. Three dairy farms were selected based on previous mastitis outbreaks caused by K. pneumoniae. Milk samples were collected from lactating cows and from the bulk tank. Swabs were performed in different locations, including milking parlors, waiting room, soil, animal's hind limbs and rectum. K. pneumoniae was isolated from 27 cases of intramammary infections (IMI) and from 41 swabs. For farm A isolates from IMI and bulk tank were considered of the same PGFE subtype. One isolate from a bulk tank, three from IMI cases and four from environmental samples were positive in the DDST test. All eight DDST positive isolates harbored the bla shv gene, one harbored the bla tem gene, and three harbored the bla ctx-m gene, including the bulk tank isolate. Our study confirms that ESBL producing bacteria is present in different locations in dairy farms, and may be responsible for IMI. The detection of ESBLs on dairy herds could be a major concern for both public and animal health.

Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.