Paper-based microfluidic electro-analytical device (PMED) for magneto-assay automation: Towards generic point-of-care diagnostic devices.

Rapid diagnostic tests (RDTs) for point-of-care (POC) testing of infectious diseases are popular because they are easy to use. However, RDTs have limitations such as low sensitivity and qualitative responses that rely on subjective visual interpretation. Additionally, RDTs are made using paper-bound reagents, which leads to batch-to-batch variability, limited storage stability and detection of only the analytes they were designed for. This work presents the development of a versatile technology, based on short magneto-assays and inexpensive paper-based microfluidic electro-analytical devices (PMEDs). PMEDs were produced locally using low-cost equipment, they were stable at room temperature, easy to use, and provided quantitative and objective results. The devices served to detect alternatively a variety of magneto-assays, granting quantitation of streptavidin-HRP, biotinylated HRP and Pasmodium falciparum lactate dehydrogenase (Pf-LDH) in less than 25 min, using either commercial or customized screen-printed electrodes and measurement equipment. Furthermore, Pf-LDH detection in diluted lysed whole blood displayed a linear response between 3 and 25 ng mL-1, detection and quantification limits ranging between 1 and 3 ng mL-1 and 6-12 ng mL-1, respectively, and provided results that correlated with those of the reference ELISA. In short, this technology is versatile, simple, and highly cost-effective, making it perfect for POC testing.

¿Qué significa una calcificación intratesticular? / What does intratesticular calcification signify in Pediatrics?

El dolor escrotal agudo es una entidad clínica que en un porcentaje elevado de casos va asociado a una necesidad de actuación médica urgente como es la torsión testicular. La ecografía testicular ayuda en el diagnóstico y debido a su mayor uso en las últimas décadas se están realizando hallazgos accidentales como las microlitiasis testiculares. Suele ser un proceso benigno, pero está descrita su asociación con tumores testiculares, por lo que se recomienda un seguimiento clínico y ecográfico periódico (AU)

Acute scrotal pain is a clinical entity that in a high percentage of cases is associated with an urgent need for medical intervention such as testicular torsion. Testicular ultrasound helps diagnosis and incidental findings, as the testicular microlithiasis, are found due to the increased use of ultrasound in recent decades. It is a benign condition, but it has been found associated with testicular tumors, so periodic clinical and ultrasound monitoring is recommended (AU)

Campylobacter epidemiology from breeders to their progeny in Eastern Spain.

While horizontal transmission is a route clearly linked to the spread of Campylobacter at the farm level, few studies support the transmission of Campylobacter spp. from breeder flocks to their offspring. Thus, the present study was carried out to investigate the possibility of vertical transmission. Breeders were monitored from the time of housing day-old chicks, then throughout the laying period (0 to 60 wk) and throughout their progeny (broiler fattening, 1 to 42 d) until slaughter. All samples were analyzed according with official method ISO 10272:2006. Results revealed that on breeder farms, Campylobacter isolation started from wk 16 and reached its peak at wk 26, with 57.0% and 93.2% of positive birds, respectively. After this point, the rate of positive birds decreased slightly to 86.0% at 60 wk. However, in broiler production all day-old chicks were found negative for Campylobacter spp, and the bacteria was first isolated at d 14 of age (5.0%), with a significant increase in detection during the fattening period with 62% of Campylobacter positive animals at the end of the production cycle. Moreover, non-positive sample was determined from environmental sources. These results could be explained because Campylobacter may be in a low concentration or in a non-culturable form, as there were several studies that successfully detected Campylobacter DNA, but failed to culture. This form can survive in the environment and infect successive flocks; consequently, further studies are needed to develop more modern, practical, cost-effective and suitable techniques for routine diagnosis.

Comparison of different sampling types across the rearing period in broiler flocks for isolation of Campylobacter spp.

Campylobacter is the most common bacterial cause of human gastrointestinal disease in most developed countries. It is generally accepted that poultry products are a significant source of foodborne Campylobacter infections in humans. Assessing the effectiveness of any potential intervention at farm level requires monitoring of the Campylobacter status of broiler flocks, using appropriate sampling methods. The aim of this study was to assess the influence of the sample type across the rearing period for the detection of Campylobacter spp. at farm level. During this study, 21 commercial broiler farms were intensively sampled. Each farm was visited and sampled at different times during the rearing period (d 1, 7, 14, 21, 28, 35, and 42). On the first day of rearing, the status of the house and the day-old flock was evaluated, and environmental and cecal samples were collected. During rearing, 4 different sample types were collected: feces with sock swabs (sock swabs), feces directly from the litter (feces), cloacal swabs, and cecal content. All samples were analyzed according to ISO 10272-1:2006 (Annex E) and also by direct culture. The results of this study showed that Campylobacter spp. were detected in all of the sample types on d 14 of rearing. From this point on, the detection increased significantly, with a maximum detection rate by the end of rearing, regardless of the sample type. All samples that were negative upon direct culture were also negative after pre-enrichment. At the end of rearing, the percentage of samples positive for Campylobacter spp. was 71.4% for cecal samples, 61.9% for cloacal swabs, 45.2% for sock swabs, and 69.1% for fecal samples. C. jejuni was detected in all the sample types, with positive rates ranging from 67.1 to 76.0% for cecal samples and cloacal content, respectively. Cecal samples, cloacal swabs, and fecal samples cultured by direct plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA) without pre-enrichment have the same sensitivity for detection of Campylobacter spp. in broiler flocks independent of the day of rearing.

Phylogenetic and structural response of heterotrophic bacteria to dissolved organic matter of different chemical composition in a continuous culture study.

Dissolved organic matter (DOM) and heterotrophic bacteria are highly diverse components of the ocean system, and their interactions are key in regulating the biogeochemical cycles of major elements. How chemical and phylogenetic diversity are linked remains largely unexplored to date. To investigate interactions between bacterial diversity and DOM, we followed the response of natural bacterial communities to two sources of phytoplankton-derived DOM over six bacterial generation times in continuous cultures. Analyses of total hydrolysable neutral sugars and amino acids, and ultrahigh resolution mass spectrometry revealed large differences in the chemical composition of the two DOM sources. According to 454 pyrosequences of 16S ribosomal ribonucleic acid genes, diatom-derived DOM sustained higher levels of bacterial richness, evenness and phylogenetic diversity than cyanobacteria-derived DOM. These distinct community structures were, however, not associated with specific taxa. Grazing pressure affected bacterial community composition without changing the overall pattern of bacterial diversity levels set by DOM. Our results demonstrate that resource composition can shape several facets of bacterial diversity without influencing the phylogenetic composition of bacterial communities, suggesting functional redundancy at different taxonomic levels for the degradation of phytoplankton-derived DOM.

Fuel cell-powered microfluidic platform for lab-on-a-chip applications: Integration into an autonomous amperometric sensing device.

The present paper reports for the first time the integration of a microfluidic system, electronics modules, amperometric sensor and display, all powered by a single micro direct methanol fuel cell. In addition to activating the electronic circuitry, the integrated power source also acts as a tuneable micropump. The electronics fulfil several functions. First, they regulate the micro fuel cell output power, which off-gas controls the flow rate of different solutions toward an electrochemical sensor through microfluidic channels. Secondly, as the fuel cell powers a three-electrode electrochemical cell, the electronics compare the working electrode output signal with a set reference value. Thirdly, if the concentration measured by the sensor exceeds this threshold value, the electronics switch on an integrated organic display. This integrated approach pushes forward the development of truly autonomous point-of-care devices relying on electrochemical detection.

Effect of low-protein diets and single sex on production performance, plasma metabolites, digestibility, and nitrogen excretion in 1- to 48-day-old broilers.

Two experiments were conducted to investigate the effect of low-CP diets supplemented with crystalline amino acids, according to an ideal amino acid ratio, on the performance, plasma metabolites, nutrient digestibility, nitrogen balance, and water intake in male and female chickens from 1 to 48 d of age using a 4-phase feeding program: prestarter (1-7 d), starter (8-21 d), grower (22-35 d), and finisher (36-48 d). Three experimental diets were formulated for each phase: a control diet with a CP level of 24.5, 23.0, 21.5, and 20.5%, respectively, and medium- and low-CP diets containing 1.5 and 3% less than that of the control, respectively, but the same ME and digestible lysine levels. In experiment 1, in males, the reduction in dietary protein content by 3% increased the feed conversion ratio (P < 0.05) in the starter, grower, and finisher phases, and plasma albumin levels were reduced (P < 0.05) during the prestarter, starter, and finisher phases. In females, the same effect on plasma album levels was observed (P < 0.05) only in the starter phase. Sex affected plasma albumin levels during the prestarter, starter, and finisher phases, being higher in females. In experiment 2, the reduction in dietary protein content linearly decreased water consumption in both sexes (P < 0.05). The reduction of dietary protein by 1.5 or 3% reduced nitrogen excretion to the environment by 9.5 and 17% in males and by 11.8 and 14.6% in females, respectively. In summary, the low-CP 4-phase feeding reduced water intake and nitrogen excretion with an adverse effect on the feed conversion ratio in males but not in females. So, single-sex rearing could be used to reduce the environmental impact of chicken farms.

Improved method for bacterial cell capture after flow cytometry cell sorting.

Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

Potential effect of freshwater virus on the structure and activity of bacterial communities in the Marennes-Oléron Bay (France).

Batch culture experiments using viral enrichment were conducted to test the response of a coastal bacterial community to autochthonous (i.e., co-existing) or allochthonous riverine viruses. The effects of viral infections on bacterial dynamics and activity were assessed by epifluorescence microscopy and thymidine incorporation, respectively, whereas the effect of viral infection on bacterial community composition was examined by polymerase chain reaction-single strand conformation polymorphism 16S ribosomal RNA fingerprinting. The percentages of high nucleic acid-containing cells, evaluated by flow cytometry, were significantly correlated (r2=0.91, n=12, p<0.0001) to bacterial production, making this value a good predictor of active cell dynamics along the study. While confinement and temperature were the two principal experimental factors affecting bacterial community composition and dynamics, respectively, additions of freshwater viruses had significant effects on coastal bacterial communities. Thus, foreign viruses significantly reduced net bacterial population increase as compared to the enrichment treated with inactivated virus. Moreover, freshwater viruses recurrently and specifically affected bacterial community composition, as compared to addition of autochthonous viruses. In most cases, the combined treatment viruses and freshwater dissolved organic matter helped to maintain or even enhance species richness in coastal bacterial communities in agreement to the 'killing the winner' hypothesis. Thus, riverine virus input could potentially influence bacterial community composition of the coastal bay albeit with modest modification of bulk bacterial growth.

Polysaccharidase preparations added to a wheat-based diet: effects on performance and digestive parameters of broiler chickens held at three different locations.

1. Three experiments of 39 d were simultaneously conducted to assess the efficacy of three carbohydrase combinations on performance, apparent nutrient digestibility, intestinal morphology and digestive organ relative weights of chickens fed on a wheat-based diet. 2. Experiment 1 was performed under laboratory conditions, with the birds housed in cages. In experiment 2, the birds were placed in floor pens built in a commercial farm, and raised with their contemporaries at the farm. In experiment 3, broilers were reared under industrial management circumstances in commercial farms. A two-phase feeding programme was used: starter from 1 to 21 d of age and finisher from 22 to 39 d. The basal diet without supplementation (BD) and three enzyme preparations (BDF, BDP and BDR) were evaluated. 3. No differences in growth traits were observed among treatments in experiments 1 and 2. 4. When overall growing period was evaluated, all enzyme combinations improved apparent DM ileal digestibility of feed in the three experiments, but significant differences were only found in experiments 2 and 3. For this same period, an increase in apparent CP ileal digestibility of broiler chickens fed supplemented diets against the control group was found in experiment 3. Moreover, a significant greater apparent ileal fat digestibility in the enzyme treatments compared with the control group was shown when broilers were raised under industrial conditions (experiment 3). 5. Enzyme combinations had no effect on villus heights and crypt depths of broilers in experiment 1, whereas, in experiment 2, a greater villus height was found in BDF and BDP treatments than in control birds. 6. There were no differences in digestive organ relative weights among dietary treatments except for pancreas in experiment 1.

High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry.

AIMS: To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. METHODS AND RESULTS: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1). CONCLUSIONS: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.

Effectiveness of virosomal subunit influenza vaccine in preventing influenza-related illnesses and its social and economic consequences in children aged 3-14 years: a prospective cohort study.

To evaluate the effectiveness of a virosomal subunit influenza vaccine in preventing influenza-related illnesses and its social and economic consequences in children aged 3-14 years, a prospective cohort study was carried out during the 2004-2005 influenza season in 11 private pediatric clinics in the Barcelona metropolitan area. One dose of a virosomal subunit inactivated influenza vaccine (Inflexal V Berna) was given during September and October 2004 to healthy children aged 3-14 years attended in 5 of the 11 clinics. Who comprised the vaccinated cohort (n=966). The non-vaccinated cohort (n=985) was comprised of children attended in the other six clinics. Informed consent was obtained from all parents. The follow up was performed between 1 November 2004 and 31 March 2005. Using a self-administered questionnaire, information was collected from parents or guardians on any type of acute, febrile respiratory illness suffered by their children during the study period, including antibiotic use, and absence from school or work-loss of parents as a result of the illness. RT-PCR (influenza A+B+C) was carried out on pharyngeal and nasal samples obtained from children attended by pediatricians during this period in these clinics with the following symptoms: fever> or =38.5 degrees lasting at least 72h, cough or sore throat (influenza-like illness). Adjusted vaccination effectiveness was 58.6% (95% CI 49.2-66.3) in preventing acute febrile respiratory illnesses, 75.1% (95% CI 61.0-84.1) in preventing cases of influenza-like illnesses and 88.4% (95% CI 49.2-97.3) in preventing laboratory-confirmed cases of influenza A. The adjusted vaccination effectiveness in reducing antibiotic use (18.6%, 95% CI -4.2 to 3.64), absence from school (57.8%, 95% CI 47.9-65.9) and work-loss of parents (33.3%, 95% CI 8.9-51.2) in children affected by an acute febrile respiratory illness was somewhat lower. Vaccination of children aged 3-14 years in pediatric practices with one dose of virosomal subunit inactivated influenza vaccine has the potential to considerably reduce the health and social burdens caused by influenza-related illnesses.

Effect of formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chickens.

1. The effect of dietary formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chickens was studied. 2. An experiment with 120 Ross male broiler chickens was conducted from 1 to 42 d of age at the laboratory. There were 4 treatment groups: control (C), 10 mg/kg feed avilamycin (AV) and formic acid at two concentrations, 5 and 10 g/kg feed (FA5 and FA10, respectively). 3. No differences in weight gain, feed intake or feed conversion ratio were observed in male broiler chickens fed on the different diets. 4. An effect of the additives on ileal dry matter (DM) digestibility at 42 d of age was detected with the finisher diets; AV and 10 g/kg of feed formic acid slightly improved ileal DM digestibility compared to the other treatment groups. 5. Jejunum pH was not affected when 5 or 10 g/kg formic acid was added, and the results do not clearly show a positive effect of formic acid on the intestinal histomorphology. 6. No differences were noticed for blood metabolites in the different diets, and the levels of calcium and phosphorus in the plasma were not altered by formic acid addition. 7. In conclusion, when using broiler chickens under conditions of good hygiene, dietary formic acid did not have a clear positive effect on performance, intestinal histomorphology or plasma metabolite levels; however, there was a slight positive effect on the ileal digestibility of nutrients.

Different marine heterotrophic nanoflagellates affect differentially the composition of enriched bacterial communities.

We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates: Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1); after that the FL1 decreased when bacteria attained maximal concentrations; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.

A sensitive and rapid method to determine the viability of freeze-dried bacterial cells.

AIMS: To determine the potential use of flow cytometry for viability asssessment of freeze-dried bacterial cells. METHODS AND RESULTS: Escherichia coli CIP 54.8T and Vibrio metschnikovii CIP 104262 were analysed. The viability of freeze-dried cells resuspended in a nutrient broth was evaluated by culture whereas activity was determined by flow cytometry analysis of both esterase activity and cell death. Activity assessment by flow cytometry was found to be a rapid and good indicator of cell viability and was very efficient for quality control. For V. metschnikovii the fraction of active cells varies greatly depending on the freeze-drying procedure and within a given procedure. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial activity assessment by flow cytometry is very efficient for the control of freeze-dried cells.

[Hemorrhagic digestive metastases from testicular choriocarcinoma]. / Métastases digestives hémorragiques du choriocarcinome testiculaire.

The metastasis of testicular choriocarcinoma are often hemorrhagic, primarily of cerebral or pulmonary seat. The secondary digestive localizations are rare and of bad forecast when they bleed. The surgical operation by laparotomy allows the topographic diagnosis and the treatment, but was made responsible for hemorrhagic decompensation of other metastatic localizations engaging the vital forecast.

[Esophageal cancers]. / Les cancers de l'oesophage.

Although slightly declining in France, the incidence rate of esophageal cancer remains amongst the highest seen in Europe, especially in Brittany, in the North as well as in Normandy. Alcohol and tobacco consumption remains the main risk factor for esophageal cancer in Western countries. Positive diagnosis of esophageal cancer is made by upper gastrointestinal endoscopy combined with biopsies. At present, surgery and definitive radiochemotherapy are two therapeutic options offering a chance to cure even though surgery remains the more frequently used treatment. Five-year-survival rate after apparently curative surgical resection or definitive radiochemotherapy remains only 20% in most population-based series. The studies that have examined the role of adjuvant treatments after surgical resection, have failed to demonstrate any improvement in overall or relapse-free survival. The pre-operative cytotoxic combined modality approaches with radiochemotherapy have shown improved relapse-free survival but still remains experimental. Finally, the symptomatic treatment of dysphagia might not be ignored either in locally, locally advanced, or in metastatic disease.

Evaluation of ChemChrome V6 for bacterial viability assessment in waters.

The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In contrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.

Genotypic analysis of thiopurine S-methyltransferase in patients with Crohn's disease and severe myelosuppression during azathioprine therapy.

BACKGROUND & AIMS: Myelosuppression in patients with Crohn's disease (CD) treated with azathioprine has been attributed to low activity of thiopurine S-methyltransferase (TPMT). Allelic variants of the TPMT gene responsible for changes in the enzyme activity have been characterized. We investigated the distribution of mutant alleles associated with TPMT deficiency in patients with CD and myelosuppression during azathioprine/6-mercaptopurine therapy. METHODS: Forty-one patients with CD were included. They developed leukopenia or thrombocytopenia during azathioprine or 6-mercaptopurine treatment. Polymerase chain reaction-based methods were used to search for mutations associated with TPMT deficiency. RESULTS: Four patients (10%) had 2 mutant alleles associated with TPMT deficiency, 7 (17%) had 1 mutant allele, and 30 (73%) had no known TPMT mutation. The delay between administration of the drug and occurrence of bone marrow toxicity was less than 1.5 months in the 4 patients with 2 mutant alleles, and ranged from 1 to 18 months in patients with 1 mutant allele and from 0.5 to 87 months in patients with normal genotype. CONCLUSIONS: Twenty-seven percent of patients with CD and myelosuppression during azathioprine therapy had mutant alleles of the TPMT gene associated with enzyme deficiency. Myelosuppression is more often caused by other factors. Continued monitoring of blood cell counts remains mandatory in patients treated with azathioprine.

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters.

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.