Development of a DGGE method to explore Legionella communities.

Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires' disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains. The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.

Innovative isotopic method to evaluate bioaccumulation of As and MTEs in Vitis vinifera.

The transfer of metal and metalloid trace elements (MTEs) from contaminated soil to grapevines is a major issue for grape consumption and for the associated health risks. Based on an isotopic approach, we shed light on the concept of MTE bioavailability. The bioavailable fractions are identified by using the Sr-isotope ratio as a proxy for MTEs. This allows us to differentiate three soil reservoirs: the 'current available fraction' in soil water, the 'reserve available fraction' stored in mineral phases of the soil fractions, and the 'non-available fraction'. The reserve available fraction, representing 10 to 60% of bulk soil depending on the MTE, includes the exchangeable, carbonates, humic substance and oxides fractions. The 87Sr/86Sr isotopic signatures of grape berries and vine leaves show an additional source of MTEs, which is imported by foliar uptake and can contribute up to 10% of the MTEs in leaves. In addition, root-uptake and translocation rates show high accumulation rates of Co, Sn and Cu, and low ones for As, Sb, Zn and Cd. A daily intake between 1 and 3 kg of (dry grapes) would reach the benchmark dose level for a 0.5% (BMDL0.5). While such a daily intake of grapes is unreasonable, consumption of other local vegetables and fruit would contribute to the daily intake. Hence, a chronic arsenic exposure is of great concern for human health in mining areas. We outline the importance of geochemical tracers, such as Sr isotopes, when determining the transfer and translocation of MTEs in plants. Our method presents a high-precision evaluation of the bioavailability and bioaccumulation of MTEs, and a better understanding of these processes in plants, thus leading to a better assessment of the environmental risk on human health.

Germline promoter hypermethylation in BRCA1 and BRCA2 genes is not present in hereditary breast cancer patients

Purpose: Germline promoter hypermethylation of BRCA1 and BRCA2 genes is an alternative event of gene silencing that has not been widely investigated in hereditary breast and ovarian cancer (HBOC) syndrome. Methods: We analyzed germline BRCA promoter hypermethylation in HBOC patients with and without BRCA mutations and control subjects, using a recently developed BRCA methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Results: Neither the patients tested nor the control subjects showed germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed. Conclusions: Despite the results achieved at somatic levels by other researchers, these were not confirmed in our study at the germline level. Our results show the need to establish more predictive CpG sites in the BRCA promoter regions to optimize the MS-MLPA assay for the detection of germline hypermethylation as an effective pre-screening tool for whole-BRCA genetic analysis in HBOC, because we can not rule out the existence of germline promoter hypermethylation in BRCA

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Germline promoter hypermethylation in BRCA1 and BRCA2 genes is not present in hereditary breast cancer patients.

PURPOSE: Germline promoter hypermethylation of BRCA1 and BRCA2 genes is an alternative event of gene silencing that has not been widely investigated in hereditary breast and ovarian cancer (HBOC) syndrome. METHODS: We analyzed germline BRCA promoter hypermethylation in HBOC patients with and without BRCA mutations and control subjects, using a recently developed BRCA methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. RESULTS: Neither the patients tested nor the control subjects showed germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed. CONCLUSIONS: Despite the results achieved at somatic levels by other researchers, these were not confirmed in our study at the germline level. Our results show the need to establish more predictive CpG sites in the BRCA promoter regions to optimize the MS-MLPA assay for the detection of germline hypermethylation as an effective pre-screening tool for whole-BRCA genetic analysis in HBOC, because we can not rule out the existence of germline promoter hypermethylation in BRCA.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE): a promising tool to diagnose bacterial infections in diabetic foot ulcers.

AIM: The diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. METHODS: Twenty diabetic patients (types 1 and 2) with foot ulcers (grades 1-4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. RESULTS: Polymerase chain reaction (PCR)-DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR-DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P = 0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. CONCLUSION: DGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.

Monitoring of methotrexate chlorination in water.

Anti-cancer drugs are an important class of pharmaceutical products. Methotrexate (MTX) is a folic acid antagonist used in high doses as antimetabolite in anti-cancer treatment as well as in low doses for the treatment of rheumatoid arthritis and adults' psoriasis. In the past, several anti-cancer drugs, including methotrexate, have been found in the environment. Their presence in water, especially if used for the production of drinking water, is even in low concentrations of particular interest, due to the risk to retrieve them in the consumed water and their high activity and grave effects. But prior to usage as drinking water, raw waters are treated and chlorination is a common practice in several countries. As such a treatment can lead to the formation of organochlorine in water, the study of the fate of MTX during chlorination in a batch trial was carried out. The reaction was monitored by dissolved organic carbon (DOC) and by fluorescence and UV spectroscopy. Investigation of by-products formed was done with liquid chromatography/mass spectrometry (LC/MS). Under the given experimental conditions, Methotrexate was eliminated rapidly (t1/2 around 21 min). However, DOC elimination was incomplete. Monitoring with LC-MS showed the formation of a monochlorinated transformation product of MTX. In silico analysis of the proposed transformation products for different carcinogenic, mutagenic and genotoxic endpoints with different software platforms provided no clear evidence that the possible transformation products after chlorination might be more toxic than the parent compound. However, since a number of alerts is altered after chlorination, it cannot be excluded that the toxicity of these transformation products might be modulated compared with the parent compound.

Assessment of poly-L-lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept.

AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = -0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.

Inter-laboratory exercise on antibiotic drugs analysis in aqueous samples.

An inter-laboratory exercise was organized under the PHARMAS EU project, by the Advanced School of Public Health (EHESP), in order to evaluate the performances of analytical methods for the measurement of antibiotics in waters (surface and tap). This is the first time such an exercise on antibiotics has been organized in Europe, using different kinds of analytical methods and devices. In this exercise thirteen laboratories from five countries (Canada, France, Italy, the Netherlands and Portugal) participated, and a total number of 78 samples were distributed. During the exercise, 2 testing samples (3 bottles of each) prepared from tap water and river water, respectively, spiked with antibiotics, were sent to participants and analyzed over a period of one month. A final number of 77 (98.7%) testing samples were considered. Depending on substances studied by each participant, 305 values in duplicate were collected, with the results for each sample being expressed as the target concentration. A statistical study was initiated using 611 results. The mean value, standard deviation, coefficient of variation, standard uncertainty of the mean, median, the minimum and maximum values of each series as well as the 95% confidence interval were obtained from each participant laboratory. In this exercise, 36 results (6% of accounted values) were outliers according to the distribution over the median (box plot). The outlier results were excluded. In order to establish the stability of testing samples in the course of the exercise, differences between variances obtained for every type of sample at different intervals were evaluated. The results showed no representative variations and it can be considered that all samples were stable during the exercise. The goals of this inter-laboratory study were to assess results variability when analysis is conducted by different laboratories, to evaluate the influence of different matrix samples, and to determine the rate at which participating laboratories successfully completed the tests initiated.

Discharge of pharmaceutical products (PPs) through a conventional biological sewage treatment plant: MECs vs PECs?

Pharmaceuticals for human use are consumed in significant quantities and their occurrence in aquatic systems has been reported by a number of authors. In the context of environmental risk assessment, there is an increasing interest in evaluating the discharge of pharmaceutical products to surface waters through sewage treatment plants (STP). This case study was carried out on a conventional biological treatment plant (Alès, France) and focused on a set of eleven drugs representing the main therapeutic classes. Measured environmental concentrations (MECs) range from the low ng L(-1) to 1.5 microg L(-1) in effluent and up to few hundred ng L(-1) in receiving surface waters. There is a good agreement between MEC and predicted environmental concentration (PEC) values for seven of the eleven investigated drugs in STP effluent. There is not such a good match between PEC and MEC values in surface waters, and this highlights the limits of this approach, at the local scale.

Bocio endémico en la Comunidad Valenciana / Endemic goiter in the autonomous community of Valencia (Spain)

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The discoidin domain receptor 1 as a novel susceptibility gene for schizophrenia.

Evidence suggests that myelin alterations could predispose to schizophrenia. Reduced expression of several myelin genes has been observed in schizophrenia patients. Recently, we identified the discoidin domain receptor 1 (DDR1; located at human chromosome 6p21.3) as a myelin gene in the mouse model and in a human oligodendroglial cell line. In the present study we screened for single nucleotide polymorphisms (SNPs) in the DNA from 100 schizophrenia patients. We identified a novel mutation within exon 10 that produces the amino-acid substitution N502S in the a-d isoforms, and M475V in the e isoform. However the frequency of the mutation (2%) was similar in schizophrenia patients and in control subjects. In a case-control assessment with 389 schizophrenic patients and 615 controls, we identified one SNP (SNP9, rs1049623) associated with schizophrenia (odds ratio=1.44, 95% confidence interval: 1.15-1.79, adjusted P=0.0016). This association was confirmed in haplotype analysis; the SNPs 9-10-11 (rs1049623, rs2267641 and rs2239518) haplotype remaining significant even after adjustment for multiple testing (adjusted P=0.0136). Of note was a strong gender dependence in the association, that is, statistical significance restricted to men (adjusted P-value=0.0002). Regression analysis of DDR1 mRNA expression in peripheral blood lymphocytes from schizophrenia patients showed that the presence of the G allele significantly decreased the relative number of mRNA copies in a dose-dependent manner (P=0.003). These data suggest that the risk haplotype tags a cis-acting variant involved in the transcription regulation system of the gene. In conclusion, we propose the DDR1 as a new susceptibility gene for schizophrenia.

[Endemic goiter and iodine deficiency: are they still a reality in Spain]. / Bocio endémico y déficit de yodo: 'sigue siendo una realidad en España?

OBJECTIVE: To estimate the prevalence of goiter and iodine deficiency in a health district in the Autonomous Community of Valencia, given the absence of data in this region of Spain. MATERIAL AND METHODS: We conducted a descriptive, epidemiologic, cross-sectional study, stratified by age and sex, in four interior regions of the province of Valencia. We selected students aged from 6 to 14 years old in 20 centers. Thyroid examination was performed by means of palpation and inspection (goiter > or = 0B). Urinary iodine excretion was analyzed in a routine urine sample. Sociodemographic and anthropometric data, as well as nutritional iodine status, were recorded in a standardized survey. In children with goiter, thyroid-stimulating hormone (TSH), free T4, and antithyroid antibodies were determined. RESULTS: We studied 928 children (478 boys and 450 girls). The prevalence of goiter was 33.7 % (95 % CI: 30.7-36.9 %). There were no significant differences in the prevalence of goiter by age or sex, but an inverse correlation was detected between the prevalence of goiter and parental socioeconomic position. Mean urinary iodine excretion was 155 .g/l, with no significant correlation with the prevalence of goiter. In children with goiter, 13 had positive antithyroid antibodies, 18 had high TSH (subclinical hypothyroidism), and one had suppressed TSH (subclinical hyperthyroidism). CONCLUSIONS: There is endemic goiter in the region studied. Urinary iodine levels were in the normal range and could be interpreted as indicating a transition phase to an improvement in iodine deficiency. Autoimmune diseases would only explain 4 % of cases of goiter.

Bocio endémico y déficit de yodo: ¿sigue siendo una realidad en España? / Endemic goiter and iodine deficiency: are they still a reality in Spain?

Objetivo Determinar la prevalencia de bocio y déficit de yodo en comarcas del interior de la Comunidad Valenciana, dada la ausencia de datos en esta comunidad autónoma. Material y métodos Se trata de un estudio epidemiológico, descriptivo, transversal, estratificado por sexo y edad en cuatro comarcas del interior de la provincia de Valencia. Se estudiaron escolares de 6 a 14 años en 20 centros. La exploración tiroidea se realizó mediante palpación e inspección (bocio ≥ 0B). Se determinó la yoduria en una muestra de orina casual. Se recogieron datos de filiación, antropométricos, y una encuesta sobre el estado nutricional de yodo. En niños con bocio se determinó la hormona tiroestimulante (TSH), la tiroxina libre (T4L) y los anticuerpos antitiroideos. Resultados Se estudiaron 928 niños (478 varones y 450 mujeres). La prevalencia de bocio fue de 33,7 % (IC 95 %: 30,7-36,9). No se hallaron diferencias significativas por edad ni por sexo, aunque sí una correlación inversamente proporcional entre prevalencia de bocio y nivel instrucción paternos. La mediana de yoduria fue 155 μg/l, sin observar una correlación significativa con la prevalencia de bocio. Entre los niños con bocio 13 presentaron los anticuerpos antitiroideos positivos, 18 la TSH elevada (hipotiroidismo subclínico) y uno la TSH suprimida (hipertiroidismo subclínico). Conclusiones Existe una endemia bociosa en las comarcas estudiadas de la Comunidad Valenciana. Los valores de yodurias normales podrían interpretarse como una fase de transición hacia una mejoría del déficit de yodo en esta zona. La patología autoinmune explicaría sólo un 4 % de bocios

Objective To estimate the prevalence of goiter and iodine deficiency in a health district in the Autonomous Community of Valencia, given the absence of data in this region of Spain. Material and methods We conducted a descriptive, epidemiologic, cross-sectional study, stratified by age and sex, in four interior regions of the province of Valencia. We selected students aged from 6 to 14 years old in 20 centers. Thyroid examination was performed by means of palpation and inspection (goiter ≥ 0B). Urinary iodine excretion was analyzed in a routine urine sample. Sociodemographic and anthropometric data, as well as nutritional iodine status, were recorded in a standardized survey. In children with goiter, thyroid-stimulating hormone (TSH), free T4, and antithyroid antibodies were determined. Results We studied 928 children (478 boys and 450 girls). The prevalence of goiter was 33.7 % (95 % CI: 30.7-36.9 %). There were no significant differences in the prevalence of goiter by age or sex, but an inverse correlation was detected between the prevalence of goiter and parental socioeconomic position. Mean urinary iodine excretion was 155 μg/l, with no significant correlation with the prevalence of goiter. In children with goiter, 13 had positive antithyroid antibodies, 18 had high TSH (subclinical hypothyroidism), and one had suppressed TSH (subclinical hyperthyroidism). Conclusions There is endemic goiter in the region studied. Urinary iodine levels were in the normal range and could be interpreted as indicating a transition phase to an improvement in iodine deficiency. Autoimmune diseases would only explain 4 % of cases of goiter

[Routine preoperative screening of asymptomatic patients: beliefs and attitudes of anesthesiologists in the Canary Islands, Spain]. / Pruebas preoperatorias rutinarias en población asintomática: opiniones y actitudes de los profesionales en Canarias.

OBJECTIVES: To identify factors that explain anesthesiologists' inappropriate use of preoperative tests in asymptomatic patients scheduled for elective surgery. MATERIAL AND METHODS: A validated questionnaire was sent to anesthesiologists at university hospitals in the Canary Islands. Information about preoperative testing patterns and reasons for selecting tests was gathered. RESULTS: The questionnaire was self-administered by 66 anesthesiologists (68% of the total). Scientific knowledge was not the reason why most respondents ordered preoperative tests in asymptomatic individuals. That was the opinion of 95% of anesthesiologists for chest x-rays, 82% for electrocardiograms, and 68% for laboratory tests. Clinical history and a medical examination gave sufficient information for selecting patients in need of specific tests in the opinion of 77.19% of the respondents. Half did not agree that routine electrocardiograms and laboratory tests should be abandoned. The justification for these tests was coverage of medical malpractice liability for 68.42%. Most considered that the need to order preoperative tests in asymptomatic patients increased after age 40. CONCLUSIONS: Although anesthesiologists admit that their request for preoperative tests in asymptomatic individuals is not supported by scientific evidence, the quest for safety and legal protection from the consequences of potential adverse consequences of providing anesthesia affects the selection of preoperative tests for asymptomatic patients.

Pruebas preoperatorias rutinarias en población asintomática: opiniones y actitudes de los profesionales en Canarias / Routine preoperative screening of asymptomatic patients: beliefs and attitudes of anesthesiologists in the Canary Islands, Spain

OBJETIVO: Identificar algunas razones que contribuyen a explicar el uso inapropiado, por parte de los anestesiólogos, de las pruebas preoperatorias en población asintomática que va a ser sometida a cirugía electiva. MATERIAL Y MÉTODO: Un cuestionario validado fue enviado a los anestesiólogos de los hospitales universitarios de Canarias. Se recogió información sobre el patrón de uso y sobre las razones que justifican la selección de pruebas preoperatorias. RESULTADOS: El cuestionario fue autocumplimentado por 66 anestesiólogos (68% del total). Para la mayoría, el conocimiento científico disponible no avala la realización de pruebas preoperatorias en personas asintomáticas. Ésta era la opinión del 95% de los anestesiólogos para la radiografía de tórax; del 82% para el ECG y del 68% para las pruebas de laboratorio. Para el 77,19% de los participantes, la historia clínica y el examen médico suministran suficiente información para seleccionar los pacientes que precisen pruebas específicas. El 50% no está de acuerdo en abandonar la solicitud rutinaria del ECG y de las pruebas de laboratorio. Para el 68,42%, la solicitud de estas pruebas se justifica para cubrir la responsabilidad médico/legal. La mayoría consideran que a partir de los 40 años de edad aumenta la necesidad de realizar pruebas preoperatorias en población asintomática. CONCLUSIONES: Si bien la mayoría de anestesiólogos admiten que la solicitud de pruebas preoperatorias en población asintomática no está respaldada por el conocimiento científico, la búsqueda de seguridad y protección legal frente a las consecuencias adversas potenciales del acto anestésico condiciona la toma de decisiones en la selección de pruebas preoperatorias en población asintomática

OBJECTIVES: To identify factors that explain anesthesiologists' inappropriate use of preoperative tests in asymptomatic patients scheduled for elective surgery. MATERIAL AND METHODS: A validated questionnaire was sent to anesthesiologists at university hospitals in the Canary Islands. Information about preoperative testing patterns and reasons for selecting tests was gathered. RESULTS: The questionnaire was self-administered by 66 anesthesiologists (68% of the total). Scientific know-ledge was not the reason why most respondents ordered preoperative tests in asymptomatic individuals. That was the opinion of 95% of anesthesiologists for chest x-rays, 82% for electrocardiograms, and 68% for laboratory tests. Clinical history and a medical examination gave sufficient information for selecting patients in need of specific tests in the opinion of 77.19% of the respondents. Half did not agree that routine electrocardiograms and laboratory tests should be abandoned. The justification for these tests was coverage of medical mal-practice liability for 68.42%. Most considered that the need to order preoperative tests in asymptomatic patients increased after age 40. CONCLUSIONS: Although anesthesiologists admit that their request for preoperative tests in asymptomatic individuals is not supported by scientific evidence, the quest for safety and legal protection from the consequences of potential adverse consequences of providing anesthesia affects the selection of preoperative tests for asymptomatic patients

Monitoring of phenol photodegradation by ultraviolet spectroscopy.

Advanced oxidation processes (AOPs) have been developed as an emerging technology for hazardous organic treatment in industrial wastewater. In this paper, the contribution of ultraviolet (UV) spectroscopy to follow phenol photodegradation was studied in a laboratory photochemical reactor equipped with a low pressure mercury lamp. It has been observed that a multicomponent approach is efficient for the evolution estimation of the initial product or intermediate compounds formed during the photodegradation.

UV monitoring of sugars during wine making.

A UV/UV system for the spectrophotometric determination of sugars during wine production is described. The methodology is based on the formation of a UV-absorbing byproduct produced by the photodegradation of sugars. Under the given experimental conditions, the influence of increasing the amount of acids and alcohol is negligible. The measurement by UV/UV is compared with FTIR as reference method and validated according to the French norm AFNOR XPT 90 210.

Serum DNA as a tool for cancer patient management.

PURPOSE: Genetic analysis has shown that cell-free circulating DNA in plasma or serum of cancer patients shares similar genetic alterations to those described in the corresponding tumor. One of the most important alterations involved in carcinogenesis is aberrant promoter methylation. The interest in this field has grown due to the implementation of the methylation-specific PCR (MSP) assay. The main objective of this study is to analyze the methylation status of different genes in tumor and serum DNA obtained at the time of surgery in two different tumor models (glioblastoma [GBM] and non-small-cell lung cancer [NSCLC]) and their relationship to clinico-pathological characteristics and response to chemotherapy. MATERIAL AND METHODS: Using MSP assay, we assessed the methylation status of MGMT, RASSF1A, p16, DAPK, TMS-1 in tumor and serum DNA obtained at time of surgery or stereotactic biopsy from 28 GBM patients and from 51 NSCLC patients. RESULTS: In GBM patients, the prevalence of MGMT, p16, DAPK, and RASSF1A promoter methylation was 38.1%, 66.7%, 52.4%, 57.1%, respectively, in glioma tissue, and 39.3%, 53.6%, 34.3%, 50%, respectively, in serum. A high correlation between methylation in tumor and serum (Spearman test p = 0.0001) was observed. In NSCLC patients, RASSF1A, DAPK and TMS-1 were methylated in 34%, 45% and 35% tumors, respectively, and in 34%, 40% and 34% serum, respectively. A good correlation was found between alterations found in tumor and serum (Spearman test p = 0.0001). CONCLUSIONS: The study of serum or plasma DNA has opened new roads for translational research and new strategies for molecular diagnosis. Due to the similarities of alterations found in serum DNA and primary tumor, we can use this tool to calculate the risk of local or distant recurrence and its relationship with survival and its value in patient follow-up to evaluate response to therapy.