Evaluation of routine microbiological techniques for establishing the diagnosis of catheter-related bloodstream infection caused by coagulase-negative staphylococci

Microbiological diagnosis of catheter-related bloodstream infection (CR-BSI) is often based on isolation of indistinguishable micro-organisms from an explanted catheter tip and blood culture, confirmed by antibiograms. Whether phenotypic identification of coagulase-negative staphylococci (CoNS) allows an accurate diagnosis of CR-BSI to be established was evaluated. Eight patients with a diagnosis of CR-BSI had CoNS isolated from pure blood cultures and explanted catheter tips which were considered as indistinguishable strains by routine microbiological methods. For each patient, an additional three colonies of CoNS isolated from the blood and five from the catheter tip were subcultured and further characterized by antibiogram profiles, analytical profile index (API) biotyping and PFGE. PFGE distinguished more strains of CoNS compared to API biotyping or antibiograms (17, 10 and 11, respectively). By PFGE, indistinguishable micro-organisms were only isolated from pure blood and catheter tip cultures in four out of eight (50%) patients thus supporting the diagnosis of CR-BSI. In another patient, indistinguishable micro-organisms were identified in both cultures; however, other strains of CoNS were also present. The remaining three patients had multiple strains of CoNS, none of which were indistinguishable in the tip and blood cultures, thus questioning the diagnosis of CR-BSI. Phenotypic characterization of CoNS lacked discriminatory power. Current routine methods of characterizing a limited number of pooled colonies may generate misleading results as multiple strains may be present in the cultures. Multiple colonies should be studied using a rapid genotypic characterization method to confirm or refute the diagnosis of CR-BSI. © 2007 SGM.

Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naive hosts, P. falciparum parasites of the 3D7 strain produce 1.4 x 10(-)(1)(3) g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/muL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

鄂尔多斯盆地上古生界低渗透砂岩有效储层识别与预测

Sulige Gasfield, with a basically proven reserve as high as one trillion cubic meters, is one giant gas field discovered in China. The major gas -bearing layers are Upper Paleozoic strata with fluvial-lacustrine sedimentary facies. Generally, gas reservoirs in this field are characteristic by "five low" properties, namely low porosity, low permeability, low formation pressure, low productivity and low gas abundance. Reservoirs in this field also feature in a large distribution area, thin single sandbody thickness, poor reservoir physical properties, thin effective reservoir thickness, sharp horizontal and/or vertical changes in reservoir properties as well as poor connectivity between different reservoirs. Although outstanding achievements have been acquired in this field, there are still several problems in the evaluation and development of the reservoirs, such as: the relation between seismic attributes and reservoir property parameters is not exclusive, which yields more than one solution in using seismic attributes to predict reservoir parameters; the wave impedance distribution ranges of sandstone and mudstone are overlapped, means it is impossible to distinguish them through the application of post-stack impedance inversion; studies on seismic petrophysics, reservoir geophysical properties, wave reflection models and AVO features have a poor foundation, makes it difficult to recognize the specific differences between tight sandstone and gas-bearing sandstone and their distribution laws. These are the main reasons causing the low well drilling success rate and poor economic returns, which usually result in ineffective development and utilization of the field. Therefore, it is of great importance to perform studies on identification and prediction of effective reservoirs in low permeable sandstone strata. Taking the 2D and 3D multiwave-multicomponent seismic exploration block in Su6-Su5 area of Sulige field as a study area and He 8 member as target bed, analysis of the target bed sedimentary characteristics and logging data properties are performed, while criteria to identify effective reservoirs are determined. Then, techniques and technologies such as pre-stack seismic information (AVO, elastic impedance, wave-let absorption attenuation) and Gamma inversion, reservoir litological and geophysical properties prediction are used to increase the precision in identifying and predicting effective reservoirs; while P-wave and S-wave impedance, ratio of P/S wave velocities, rock elastic parameters and elastic impedance are used to perform sandstone gas-bearing property identification and gas reservoir thickness prediction. Innovative achievements are summarized as follows: 1. The study of this thesis is the first time that multiwave-multicomponent seismic data are used to identify and predict non-marine classic reservoirs in China. Through the application of multiwave-multicomponents seismic data and integration of both pre-stack and post-stack seismic data, a set of workflows and methods to perform high-precision prediction of effective reservoirs in low permeable sandstone is established systematically. 2. Four key techniques to perform effective reservoir prediction including AVO analysis, pre-stack elastic wave impedance inversion, elastic parameters inversion, and absorption attenuation analysis are developed, utilizing pre-stack seismic data to the utmost and increasing the correct rate for effective reservoir prediction to 83% from the former 67% with routine methods. 3. This thesis summarizes techniques and technologies used in the identification reservoir gas-bearing properties using multiwave-multicomponent seismic data. And for the first time, quantitative analysis on reservoir fluids such as oil, gas, and/or water are carried out, and characteristic lithology prediction techniques through the integration of pre-stack and post-stack seismic prediction techniques, common seismic inversion and rock elastic parameters inversion, as well as P-wave inversion and converted wave inversion is put forward, further increasing the correct rate of effective reservoir prediction in this area to 90%. 4. Ten seismic attribute parameters are selected in the 3D multi-wave area to perform a comprehensive evaluation on effective reservoirs using weighted-factor method. The results show that the first class effective reservoir covers an area of 10.08% of the study area, while the second and the third class reservoirs take 43.8% and 46% respectively, sharply increasing the success rate for appraisal and development wells.

Challenges in mercury speciation and fractionation in soil and sediment

This investigation focused on the development, test and validation of methodologies for mercury fractionation and speciation in soil and sediment. After an exhaustive review of the literature, several methods were chosen and tested in well characterised soil and sediment samples. Sequential extraction procedures that divide mercury fractions according to their mobility and potential availability in the environment were investigated. The efficiency of different solvents for fractionation of mercury was evaluated, as well as the adequacy of different analytical instruments for quantification of mercury in the extracts. Kinetic experiments to establish the equilibrium time for mercury release from soil or sediment were also performed. It was found that in the studied areas, only a very small percentage of mercury is present as mobile species and that mobility is associated to higher aluminium and manganese contents, and that high contents of organic matter and sulfur result in mercury tightly bound to the matrix. Sandy soils tend to release mercury faster that clayey soils, and therefore, texture of soil or sediment has a strong influence on the mobility of mercury. It was also understood that analytical techniques for quantification of mercury need to be further developed, with lower quantification limits, particularly for mercury quantification of less concentrated fractions: water-soluble e exchangeable. Although the results provided a better understanding of the distribution of mercury in the sample, the complexity of the procedure limits its applicability and robustness. A proficiency-testing scheme targeting total mercury determination in soil, sediment, fish and human hair was organised in order to evaluate the consistency of results obtained by different laboratories, applying their routine methods to the same test samples. Additionally, single extractions by 1 mol L-1 ammonium acetate solution, 0.1 mol L-1 HCl and 0.1 mol L-1 CaCl2, as well as extraction of the organometallic fraction were proposed for soil; the last was also suggested for sediment and fish. This study was important to update the knowledge on analytical techniques that are being used for mercury quantification, the associated problems and sources of error, and to improve and standardize mercury extraction techniques, as well as to implement effective strategies for quality control in mercury determination. A different, “non chemical-like” method for mercury species identification was developed, optimised and validated, based on the thermo-desorption of the different mercury species. Compared to conventional extraction procedures, this method has advantages: it requires little to no sample treatment; a complete identification of species present is obtained in less than two hours; mercury losses are almost neglectable; can be considered “clean”, as no residues are produced; the worldwide comparison of results obtained is easier and reliable, an important step towards the validation of the method. Therefore, the main deliverables of this PhD thesis are an improved knowledge on analytical procedures for identification and quantification of mercury species in soils and sediments, as well as a better understanding of the factors controlling the behaviour of mercury in these matrices.

Histopatologia de fígado, rim e baço de piaractus mesopotamicus, prochilodus lineatus e pseudoplatystoma fasciatum parasitados por myxosporídios, capturados no Rio Aquidauana, Mato Grosso do Sul, Brasil

Este estudo descreveu a histopatologia de rim, baço e fígado de Piaractus mesopotamicus, Prochilodus lineatus e Pseudoplatystoma fasciatum, parasitados por mixosporídios, capturados no Rio Aquidauana, MS. Após necropsia, amostras do fígado, rim cefálico e baço foram colhidas, fixadas em formalina a 10 % tamponada e processadas de acordo com a rotina histológica. Os cortes foram feitos à espessura de 5 μm e corados com hematoxilina-eosina. Foram encontrados Myxobolus porofilus em P. lineatus, M. colossomatis em P. mesopotamicus e Myxobolus spp. nas três espécies de hospedeiros. Cistos de mixosporídios no exame histopatológico foram vistos no fígado e baço de P. mesopotamicus. Mais de 50% das amostras de fígado de P mesopotamicus e P lineatus apresentou hepatodistrofia difusa. Mais de 80 % das amostras de fígado de P. fasciatum apresentou formações hialinas concêntricas e esteatose em 50% das amostras. em 95,23 % das amostras de rins de P. mesopotamicus, foram observadas alterações teciduais, e em mais de 60 % dos casos nefrodistrofia difusa moderada e congestão de sinusóides glomerulares. Alterações teciduais nas amostras de rins de P. lineatus foram observadas em menos de 20 % da amostra. No baço dos peixes ora examinados não foram encontradas lesões dignas de relato.

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of São Paulo, Brazil

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (So Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

Hematological and cerebrospinal fluid changes in cattle naturally and experimentally infected with the bovine herpesvirus 5

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of Citrus aurantium l. essential oil on muscle regeneration in mdx mice

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder characterized by the progressive loss of muscular strength. Mdx mutant mice show a marked deficiency in dystrophin, which was related to muscle membrane stability. The aim of this study was to verify the possible protective anti-inflammatory effect of citrus oil on mdx muscle fibers. Thus, adult male and female mdx mice (014/06-CEEA) were divided into control and citrus-treated. After 60 days of treatment, one ml of blood was collected for creatine kinase (CK) test. Diaphragm, sternomastoideus, anterior tibial and gastrocnemius muscles were removed and processed according to histological routine methods. The observed alterations indicate a direct effect of citrus. Recent studies have improved the diagnosis of muscular diseases but with no definitions of efficient treatments. Intervention with several therapies is important to many patients presenting muscular dystrophy, which enables them to live longer and be more active, while there is no development of gene therapies.

Detection of Listeria sp in meat and meat products using TECRA Listeria visual immunoassay and biocontrol visual immunoprecipitate assay for Listeria immunoassays and a cultural procedure

The routine methods for detecting Listeria sp. in foods are time consuming and involve using selective enrichments and plating on agars. In this study, the presence of Listeria sp. in 120 meat and meat product samples was investigated by two rapid immunoassays (TECRA Listeria Visual Immunoassay [VIA] and BioControl Visual Immunoprecipitate Assay [VIP] for Listeria) and a cultural procedure. The cultural method of detecting Listeria sp. followed Canada's Health Protection Branch Method, and the rapid tests followed the manufacturers' instructions. The agreement between the cultural and the rapid tests was established at a confidence limit of 95%. Seventy-nine samples (65.8%) were Listeria sp. positive in at least one of the three tests. There was no statistically significant difference between the cultural procedure and any of the rapid immunoassays. The agreement rates between the VIA and the cultural method and between the VIP and the cultural method were 87 and 84%, respectively. Both tests - the VIA and VIP - proved to be rapid, efficient and easy to perform.

Thermoanalytical study of praziquantel-loaded PLGA nanoparticles

Polymeric nanoparticles have received great attention as potential controlled drug delivery systems. Biodegradable polymers has been extensively used in the development of these drug carriers, and the polyesters such as polylactic acid, polyglycolic acid and their copolymers as poly-lactide-co- glycolide are the most used, considering its biocompatibility and biodegradability. Thermal analysis techniques have been used for pharmaceutical substances for more than 30 years and are routine methods for screening drug-excipient interactions. The aim of this work is to use thermal analysis to characterize PLGA nanoparticles containing a hydrophobic drug, praziquantel. The results show that the drug is in an amorphous state or in disordered crystalline phase of molecular dispersion in the PLGA polymeric matrix and that the microencapsulation process did not interfere with the chemical structure of the polymer, mantaining the structural drug integrity.

Padronização da técnica de multiplex PCR para a detecção de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em amostras de leite bovino, obtidas de tanques de expansão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação de testes sorológicos no diagnóstico da brucelose suína em amostras provenientes de um frigorífico e de um rebanho naturalmente infectado do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports

Phosphatidylethanol (PEth) is a direct ethanol metabolite, and has recently attracted attention as biomarker of ethanol intake. The aims of the current study are: (1) to characterize the normalization time of PEth in larger samples than previously conducted; (2) to elucidate potential gender differences; and (3) to report the correlation of PEth with other biomarkers and self-reported alcohol consumption. Fifty-seven alcohol-dependent patients (ICD 10 F 10.25; 9 females, 48 males) entering medical detoxification at three study sites were enrolled. The study sample was comprised of 48 males and 9 females, with mean age 43.5. Mean gamma glutamyl transpeptidase (GGT) was 209.61 U/l, average mean corpuscular volume (MCV) was 97.35 fl, mean carbohydrate deficient transferrin (%CDT) was 8.68, and mean total ethanol intake in the last 7 days was 1653 g. PEth was measured in heparinized whole blood with a high-pressure liquid chromatography method, while GGT, MCV and %CDT were measured using routine methods. PEth levels at day 1 of detoxification ranged between 0.63 and 26.95 micromol/l (6.22 mean, 4.70 median, SD 4.97). There were no false negatives at day 1. Sensitivities for the other biomarkers were 40.4% for MCV, 73.1% for GGT and 69.2% for %CDT, respectively. No gender differences were found for PEth levels at any time point. Our data suggest that PEth is (1) a suitable intermediate term marker of ethanol intake in both sexes; and (2) sensitivity is extraordinary high in alcohol dependent patients. The results add further evidence to the data that suggest that PEth has potential as a candidate for a sensitive and specific biomarker, which reflects longer-lasting intake of higher amounts of alcohol and seemingly has the above mentioned certain advantages over traditional biomarkers.