Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column , conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM . The present method is therefore suitable for acetylcholine determination in rat brain microdialysates. In the central nervous system, acetylcholine is involved in attention, learning, memory, consciousness, sleep, and control of voluntary movements -5. Dysfuin vivo microdialysis technique is extensively used to monitor the concentration of acetylcholine in the extracellular milieu of designated brain regions in conscious animals [The  animals . This te animals . Typical animals -16. The Liquid chromatography with tandem mass spectrometry was more recently developed as a sensitive but expensive method for acetylcholine determination in microdialysis samples, reaching a detection limit of 0.02-0.03 nM -19. HoweTwo liquid chromatographic methods have been combined with the amperometric determination of acetylcholine: ion-pair chromatography with octadecyl silica columns as the stationary phase  and ion-In the present study an ion-pair liquid chromatography method with amperometric detection was optimized and validated for acetylcholine determination in microdialysis samples. Different mobile phase conditions were compared to improve the sensitivity, long-term enzymatic and chromatographic stability. The polymer-coated octadecyl silica column type used in this study allowed a reliable separation of acetylcholine from choline and other matrix components over 4 months of intensive use.2.2.1.The chromatographic parameters were chosen to obtain an optimal equilibrium between sensitivity and chromatographic stability. A microbore polymer-coated silica column with high endcapping and low octadecyl binding-density was selected to allow chromatographic stability when using a purely aqueous phosphate buffer as the mobile phase. Typically, mobile phases with pH 8.0-8.5 have been used for detection of acetylcholine by liquid chromatography with amperometric detection -15. NeveGiven that the stability of silica-based chromatographic columns is dramatically improved at lower pH , furtherThe efficacy of acetylcholine detection can be improved by reducing the flow rate and increasing the ionic strength of the mobile phase -31, 33. Reversed-phase ion-pair chromatographic separation of quaternary ammonium compounds, such as choline and acetylcholine, is based on the partition of the ion-pairs formed with anionic counter ions, such as 1-hexanesulphonate or 1-octanesulphonate, between the apolar stationary phase and the polar mobile phase.Interactions between unpaired choline and acetylcholine with surface silanol groups of silica-based columns may result in strong adsorption of both analytes to the stationary phase, producing irregular retention times and band-broadening or peak-splitting . This caIn the present study, the aforementioned mobile phase did not yield a statisfactory baseline resolution of acetylcholine from the injection peak on a 1.0×150 mm octadecyl silica column . This il2.2.The chromatographic separation of acetylcholine from choline and other matrix components, the selective conversion of acetylcholine into hydrogen peroxide by the post-column IMER and the low working potential of the enzyme-coated glassy carbon electrode contribute to the high selectivity of the described method. Acetylcholine calibration samples in water yielded a peak with stable peak area, retention time and signal-to-noise (S/N) ratio. The acetylcholine peak could not be detected in the corresponding blanks . CholineHowever, the reliability of acetylcholine determination at low concentration levels has hitherto not been investigated. Therefore, the lower limit of quantification (LLOQ) in water was estimated at 0.3 nM for an S/N ratio of 5 . The LLO2.3.y = ax + b, where y represents peak area and x represents the concentration of acetylcholine. The mean ± SD equations of the calibration curves (n = 5) were y = (56.2 ± 0.5)x + (5.0 ± 2.2) and y = (55.3 ± 0.6)x + (4.9 ± 2.7) for calibration standards prepared in water and quality control samples in microdialysis matrix respectively. The corresponding mean correlation coefficients were 0.9975 and 0.9984. An analysis of variance lack of fit test showed that the calibration curves were linear over the investigated concentration range in water  and in microdialysis matrix . A two-way analysis of variance showed that the slopes were not significantly different . The absence of matrix effects shows that the acetylcholine concentration in microdialysis samples can be effectively determined by interpolation on a linear regression curve prepared in water.Calibration standards and quality control samples spiked with 0.0, 0.3, 1.0, 3.0 and 10.0 nM acetylcholine were prepared in water and microdialysis matrix respectively. The calibration standards were analyzed within one day and peak areas were fitted to a linear regression equation of the format 2.4.The precision and the accuracy of the present method were determined over three consecutive days. Each day five replicate quality control samples, prepared by spiking 0.0, 0.3, 1.0, 3.0 and 10.0 nM acetylcholine in microdialysis matrix, were analyzed for acetylcholine content through interpolation on a calibration curve obtained with calibration standards prepared in water. The relative standard deviation of the calculated concentrations was used as the index of precision. The accuracy of the method was determined by comparing the calculated concentrations with their nominal values. At the LLOQ level the repeatability and inter-day precision were less than 20% and the accuracy within 80-120%. At all other levels the repeatability and inter-day precision were less than 15% and the accuracy within 85-115%. This demonstrates that the described method is precise and accurate for determination of acetylcholine in microdialysis samples within the range 0.3 – 10.0 nM with 0.3 nM the LLOQ, as defined by the FDA guideline for bioanalytical validation . The res2.5.Calibration samples containing acetylcholine in water or quality control samples in microdialysis matrix were stable for at least 48 h when stored at 4°C in the autosampler . After 32.6.2=0.17) with an average drop in sensitivity of 0.20 ± 0.06 % per day (2=0.01) with an average drop in sensitivity of 0.12 ± 0.18 % per day  and CaCl2·6H2O from Fluka . All the chemicals used were of analytical grade. Water was purified using a Seralpur Pro 90 CN purification system .Acetylcholine chloride, choline chloride, 1-hexanesulphonic acid sodium salt and 1-octanesulphonic acid sodium salt were purchased from Sigma-Aldrich Chemie . Tetramethylammonium bromide, Na3.2.2EDTA·2H2O and 0.5% v/v ProClin 150 in water. The mobile phase was filtered before use over a Porafil 0.2 μm membrane filter . The chromatographic setup was maintained at an ambient temperature of 20°C. When the system was not in use the mobile phase was recycled at a flow rate of 0.02 mL/min.The liquid chromatography system consisted of a Gilson 307 pump  with a Gilson 805 manometric module and a Gilson 231 XL injector with a Gilson 832 temperature regulator maintaining sample temperature at 4°C. Injections of 20 μL were made in an internal loop mode on a 30 μL injection loop. The needle and loop wash solvent was water with 0.5% v/v of the microbiocide ProClin 150 . The mobile phase was degassed with an external Degasys DG-1210 vacuum degasser  and pumped at a flow rate of 0.075 mL/min. A 0.5 μm inline filter  was placed after the injector. The separation of cholines was obtained by isocratic reversed phase ion-pair chromatography on a Shiseido Capcell Pak C18 AQ with 150 mm column length, 1 mm internal diameter and 5 μm particle size . The mobile phase was a 50 mM phosphate buffer at pH 6.5, containing 1 mM sodium 1-octanesulphonate, 2 mM tetramethylammonium bromide, 0.5 mM Na3.3.A postcolumn 50×1 mm acetylcholine oxidase and choline esterase IMER  was used to convert acetylcholine into hydrogen peroxide. A horseradish peroxidase osmium-redoxpolymer  coated cross-flow glassy carbon electrode with 2 mm surface diameter  was used to detect hydrogen peroxide. The electrode was attached to an LC-4C amperometric detector  operated in reductive mode at +100 mV, 0.2 nA/V sensitivity and 0.1 Hz signal filtration. The enzyme reactors and the amperometric detector were maintained at an ambient temperature of 20°C. Before each new coating, the electrode surface was cleaned with water, rinsed with acetone and dried in a dust free box. The electrode surface was coated with 0.5 μl of a coating surfactant  to facilitate spreading and adhesion of the osmium-peroxidase redoxpolymer to the electrode surface. During method development a single and a double enzyme coating protocol were compared. After letting the surfactant dry for 15 min, a layer of 0.5 μL horseradish peroxidase osmium-redoxpolymer was applied onto the electrode surface. After 15 minutes drying, a second layer of 0.5 μL horseradish peroxidase osmium-redoxpolymer was applied onto the electrode surface. The redoxpolymer was left to harden for 4 h before use.3.4.2 in water. Microdialysis matrix for the preparation of quality control samples were continuously collected from 6 different rats into 2 mL polypropylene tubes  and stored at -20°C until use. Microdialysis samples for routine analysis were collected every 20 min into 0.7 mL polypropylene tubes  and stored at 4°C until analyzed within 48 h.Protocols for the experiments on freely moving rats were carried out according to the European directive 86/609/EEC with the corresponding national guidelines and approved by the Ethical Committee for Animal Experimentation of the Faculty of Medicine and Pharmacy of the Vrije Universiteit Brussel. Male Wistar rats were obtained from Charles-River Laboratories  and weighed 275 g to 320 g at the time of surgery. General anaesthesia was induced by an intraperitoneal injection of 60 mg/kg ketamine  and 5 mg/kg diazepam . Body temperature was maintained at 37°C during surgery using a heating pad. Intracerebral guide cannulae  with replaceable dummy cannulae were stereotaxically implanted above the hippocampus 4.6 mm lateral from midline, 5.6 mm caudal from bregma and 3.6 mm ventral from dura using flat skull coordinates . The guide cannulae were fixed to the skull with two achor screws  and carboxylate dental cement . Post-operative analgesia was provided with an intraperitoneal injection of 4 mg/kg ketoprofen . The dummy cannulae were replaced by concentric microdialysis probes with 4 mm membrane length  immediately after surgery and the animals were allowed to recover overnight in a microdialysis bowl with free access to food and water. One hour before the start of the experiment the microdialysis probe was perfused at a flow rate of 1.5 μL/min with a modified Ringer's solution containing 147 mM NaCl, 4 mM KCl and 2.3 mM CaCl3.5.Stock solutions of acetylcholine chloride (1 M) and choline chloride (1 M) were prepared daily and refrigerated at 4°C. The analytes were weighed using a calibrated balance . Acetylcholine calibration standards and quality control samples were prepared by diluting the stock solution in water or microdialysis matrix respectively. Microdialysis matrices from two rats were thawed and pooled on each day of validation. All dilutions were made using calibrated micropipettes  in 0.7 mL polypropylene tubes . Samples for routine analysis were directly collected into 0.7 mL polypropylene tubes  and analyzed without further manipulation.3.6.Statistics were performed with the GraphPad 4 software. The linear regression method was used to calculate the calibration curves. Linearity of the calibration curves was evaluated using an analysis of variance lack of fit test. Differences in the curves obtained with calibration standards prepared in water and quality control samples prepared in microdialysis matrix were analyzed using a two-way analysis of variance. The repeatability and inter-day precision of the method were calculated over three days of validation using one-way analysis of variance. The accuracy was calculated as the average accuracy obtained over three consecutive days of validation. Values for p < 0.05 were considered significant.4.To our knowledge this is the first report in which a liquid chromatography system with amperometric detection is validated in the concentration range required for acetylcholine determination in rat brain microdialysis samples. The present assay balanced sensitivity to enzymatic and chromatographic stability and was precise and accurate for the quantification of acetylcholine in microdialysis samples without the use of cholinesterase inhibitors. The mobile phase with pH to 6.5 allowed for adequate resolution of acetylcholine from choline and other matrix components for up to 4 months on a microbore polymer-coated silica column with high endcapping and low octadecyl binding-density. However, the present method was unable to resolve choline from the injection peak. The acetylcholine esterase and choline oxidase IMER was stable up to 2-3 months and the enzyme coating was stable up to 1-2 months. With adequate maintenance this system allows the routine analysis of acetylcholine in microdialysis samples.


Cutaneous disorders can precede or follow the initiation of hemodialysis treatment. We evaluated the prevalence of various dermatological manifestations in patients undergoing hemodialysis at least twice a week for minimum of three months at our center. Patients were excluded if they were undergoing hemodialysis less than twice a week or on hemodialysis secondary to ESRD following graft dysfunction. One hundred and forty-three patients were evaluated. Among them, there were 113 male and 30 females. Among the skin changes, pruritus accounted for 56%, Xerosis was observed in 52%, Diffuse blackish hyper pigmentation was seen in 40%. Skin infections was seen in 53% of patients, of these fungal, bacterial and viral infections were 27.2%, 14.6%, and 11.2%, respectively. Kyrle's disease was observed only in 6.9%. Other skin manifestations include eczema 4.8%, psoriasis 2.7%, and drug rash 2.1%. Nail changes were observed in 46 patients of whom 27 patients had onychomycosis. Other changes include discoloration, onycholysis, and splinter hemorrhages. Hair changes were observed in 21.7%. Mucosal changes were seen in 27.3%. In our study, pruritus, xerosis, and pigmentation were higher among skin changes. Recognition and management of some of these dermatological manifestations vastly reduce the morbidity and improve the quality of life. Chronic kidney disease (CKD) is a progressive loss of kidney function over a period of months or years through five stages. The number of patients with end-stage renal disease (ESRD) in India is increasing with an estimated annual incidence of about 100 per million populations . About 5Patients undergoing regular hemodialysis at least twice a week for a minimum of three months were evaluated. Patients were excluded if they were undergoing hemodialysis less than twice a week or on hemodialysis secondary to ESRD following graft dysfunction. All patients underwent clinical examination, and relevant investigations were recorded. Dermatological evaluation and confirmation of the presenting lesions were done by qualified dermatologist. Microscopic evaluation for fungal infections and histopathologic evaluation for Kyrle disease was performed.All the data was entered in Microsoft Excel spread sheet. Statistical analysis was done by using Graphpad Prism, version 4, USA. Continuous data was described as arithmetic mean and standard deviation and categorical data as actual numbers and percentages. One hundred and forty-three patients undergoing regular hemodialysis twice a week were evaluated. Among them, there were 113 male and 30 females (3.67 : 1). The mean age of these patients was 43.8 ± 13.0 yrs (14–70). The mean values of serum creatinine, blood urea, hemoglobin, uric acid, and kt/v of these patients were 9.4 ± 6.3 mg/dL, 133.5 ± 46.9 mg/dL, 7.17 ± 1.7 gm, 7.0 ± 1.8 mg/dL, and 1.2 ± 0.1, respectively. The mean duration of dialysis was 23.0 ± 14.1 months. The causes of ESRD and cutaneous manifestations were described in Among the skin changes, pruritus accounted for 56% of the total study population. It manifested even before hemodialysis in 28 patients, and only 4 patients out of 81 showed significant improvement after hemodialysis. Pruritus was found to be severe in diabetic patients (21/37). Xerosis was observed in 52% of the study population. Diffuse blackish hyperpigmentation was seen in 40% and was predominant in sun-exposed areas. 53% of patients had skin infections, of these fungal, bacterial and viral infections were 27.2%, 14.6%, and 11.2%, respectively. Among the fungal infections onychomycosis, tinea versicolor and tinea cruris  were theNail changes were observed in 46 patients of whom 27 patients had onychomycosis . Other cPruritus was the most common skin manifestation observed in our patients as compared to other studies . PrurituDiffuse blackish hyperpigmentation was seen in 40%, which was relatively high when compared to 20–22% in other studies . This miNail changes observed in our study were onychomycosis, discolouration, onycholysis, and splinter hemorrhages. Hair changes were observed with a sparse distribution over body which included dryness and hair discoloration. Dryness was possibly due to decreased sebum secretion. This was high compared to 10–30% in other studies . The repIn our study, pruritus, xerosis, and pigmentation were higher among skin changes. Recognition and management of some of these dermatological manifestations vastly reduce the morbidity and improve the quality of life.


Escherichia coli (VTEC), with undercooked ground beef and raw milk being the major vehicles of food borne outbreaks. VTEC has been implicated as an etiological agent of individual cases and outbreaks in developed countries. This study was designed to determine the prevalence of VETEC in diarrheic dairy calves up to 20 days of age in Najaf, Iraq.Dairy cattle have been implicated as principal reservoir of Verotoxin-Producing Escherichia coli O157:H7 and non-O157 VTEC isolates. Non-sorbitol fermentation, enterohemolysin phenotype, and slide agglutination with antisera were used for screening and detection of these serotypes.326 fecal samples from diarrheic calves were collected for isolation of E. coli were obtained. Only 9 were agglutinated with available antisera and none of them belonged to the O157:H7 serotype. Three were found to be verotoxin positive on Vero cell monolayers. These included serotype O111 (2 isolates) and serotype O128 (1 isolate). All three VTEC isolates were resistant to ampicillin and streptomycin. Two exhibited adherence phenotype on HEp-2 cells.Nineteen (5.8%) non-sorbitol fermenting and 3 (0.9%) enterohemolysin-producing E. coli O157:H7 serotype is not prevalent in diarrheic dairy calves, and VTEC is not a frequent cause of diarrhea in calves in Najaf/ Iraq. Escherichia coli (VTEC), including O157:H7, was identified in 1982 as an important human pathogen causing bloody diarrhea or hemorrhagic colitis (HC) which can lead to life-threating sequelae such as hemolytic-uremic syndrome (HUS) and has been reported with increased frequency during the past decade as a cause of human illness  and incubated at 37 °C for 18–24 h  for detection of enterohemolysin-producing E. coli , -glucuronidase (-), and KCN (-), were serotyped by slide agglutination technique using E. coli O157, and H7 antisera . All other E. coli isolates were serotyped by slide agglutination technique using monovalent 2, 3, and 4 antisera . Positive isolates were then identified by using API 20E .Preparation of bacterial lysate. Bacterial lysates were prepared as described by O‘Brien and LaVaeck  at 180 rpm. The bacteria was harvested by centrifugation at 10,000 rpm (4°C) for 20 min, washed twice with saline solution, and re-suspended in buffer . The cells were disrupted by 3 min. of intermittent sonic oscillation . The sonic extracts were clarified by centrifugation at 12,000 rpm at 4°C for 1 h, and sterilized with 0.22 µm Millipore filter . LaVaeck . A 100 mDetermination of verotoxin production. Verotoxin was determined in microtiter plates  containing 96 wells with 0.1 ml of cell culture  supplemented with 10% fetal calf serum (Flow Lab), 100 units/ml penicillin G and 100 µg/ml streptomycin. Vero cells  monolayer were obtained by seeding 1.6 X 104 cells per well, 1 to 2 days at 36°C in 5% CO2 incubator  before use. Four-fold dilutions of bacterial lysates in cell culture medium were added in 0.1 ml quantities into wells, and the plate were incubated at 36°C in 5% CO2 incubator. Wells containing cells not exposed to bacterial lysates were negative controls. Vero cells were examined daily for 7 days and morphological effects were recorded  in culture were assessed as previously described  calves in group 1, (8.7%) in group 2, and (2.7%) in group 3. However, no significant difference was found among the cattle of different age groups (P > 0.05). On the other hand, enterohemolysin positive E. coli isolates were detected in age groups 2 and 3 in very low percentages. No enterohemolysin positive E. coli isolates were detected in age group 1 of the tested calves. The isolation rate of enterohemolysin-producing E. coli showed no significant association among groups of calves tested (P > 0.05).All case calves excreted watery manure of grayish or yellow color and in 20 samples blood flecks and/or mucous (6.1%) were presented.E. coliphenotyping and serotyping. Biochemical characteristics of the non-sorbitol fermenting (19 isolates) or enterohemolysin-producing E. coli (3 isolates) showed that they behave as typical E. coli when they grow on the classic screening medium sorbitol MacConkey agar. These isolates were further screened serologically. Only 9 isolates were agglutinated with available antisera , the bacteria were bound to localized areas of HEp-2 cells in which they form very clearcut microcolonies. This pattern is called localized adherence , but only 1 isolate was documented of 662 adult cows (0.15%). Surveys of United States dairy and beef cattle have found E. coli O157:H7 in 0 to 2.8% of animals, with the highest isolation rates reported from younger rather than older animals  of the 61 samples from diarrheic cattle were positive for VTEC and all the positive samples were from calves below 6 months of age (et al. (et al. (Results of this study revealed that out of three VTEC isolates, two were from calves with 3 to 5 months of age. This result agreed with the finding obtained by Dutta s of age . Other ss of age , 7. Djor (et al.  showed t(et al.  found noet al. reported that E. coli strains isolated from diarrheic dairy calves showed low resistance to cephalosporins and quinolones antibiotics (The high sensitivity of VTEC isolates in the present study to most of the antibiotics tested may be due to low number of the VTEC isolates obtained. However, all the isolates were resistant to ampicillin and streptomycin. Orden ibiotics . It is sibiotics .et al. (et al. (E. coli O157:H7 serotype is not prevalent. This study also showed that VTEC is not a frequent cause of diarrhea in calves in Najaf.High adherence capacity has been considered as an important factor for the maintenance of bacteria on the mucosal surface of the host organism. Different studies from various parts of the world , 31 incret al.  found th


Parmelia sulcata, Flavoparmelia caperata, Evernia prunastri, Hypogymnia physodes and Cladonia foliacea were evaluated. The total phenolic content of the tested extracts varied from 78.12 to 141.59 mg of gallic acid equivalent (GA)/g of extract and the total flavonoid content from 20.14 to 44.43 mg of rutin equivalent (Ru)/g of extract. The antioxidant capacities of the lichen extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging. Hypogymnia physodes with the highest phenolic content showed the strongest DPPH radical scavenging effect. Further, the antimicrobial potential of the lichen extracts was determined by a microdilution method on 29 microorganisms, including 15 strains of bacteria, 10 species of filamentous fungi and 4 yeast species. A high antimicrobial activity of all the tested extracts was observed with more potent inhibitory effects on the growth of Gram (+) bacteria. The highest antimicrobial activity among lichens was demonstrated by Hypogymnia physodes and Cladonia foliacea. Finally, the antiproliferative activity of the lichen extracts was explored on the colon cancer adenocarcinoma cell line HCT-116 by MTT  viability assay and acridine orange/ethidium bromide staining. The methanol extracts of Hypogymnia physodes and Cladonia foliacea showed a better cytotoxic activity than the other extracts. All lichen species showed the ability to induce apoptosis of HCT-116 cells.The antioxidative, antimicrobial and antiproliferative potentials of the methanol extracts of the lichen species  They are considered to be the earliest colonizers of terrestrial habitats on the earth . Nowadayper se, while others are exclusively produced by synergistic action of both partners in lichens. Secondary metabolites are usually insoluble in water and can be extracted into organic solvents. Their amount ranges from 0.1 to 10% of the dry weight of tallus and sometimes reaches 30% -2,5-diphenyltetrazolium bromide and dimethyl sulfoxide were purchased Sigma, St. Louis, USA. All other solvents and chemicals were of analytical grade.3.2.Hypogymnia physodes (L.) Nyl.,  Ach., Parmelia oregana Gyelnik; common names: Monk's-hood lichen, Hooded tube lichen, Puffed lichen), Evernia prunastri (L.) Ach. (common name: oakmoss), Flavoparmelia caperata (L.) Hale (syn: Parmelia caperata (L.) Ach.; common name: greenshield lichen), Parmelia sulcata Taylor (common name: shield lichen), growing on the Prunus domestica and Salix sp., were collected in the southeast of Serbia in April 2009. The collection site is Bojanine vode near Niš at 860 m. Terricolous lichen species Cladonia foliacea  Fr.) was collected at Jelašnička klisura at 330 m. To determinate lichens we used several standard keys [Corticolous lichens species: ard keys –51. Lich3.3.Air-dried lichen thalli were ground . Extractions were performed with 250 mL of methanol at room temperature for a period of 24 h. The extracts were filtered using Whatman No.1 filter paper and then concentrated in rotary vacuum evaporator at 40 °C.3.4.3.4.1.The total phenolic content of the lichen extracts was determined spectrophotometrically by Folin-Ciocalteu method . Briefly3.4.2.The total flavonoid content was evaluated using aluminum chloride . The sam3.4.3.50values  were estimated from % inhibition versus the concentration sigmoidal curve using non-linear regression analysis. The data were presented as mean values ± standard deviation (n = 3).Chlorogenic acid was used as standard .The antioxidant activity of lichen extract was evaluated according to scavenging activity of stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) . DPPH assay was performed by a modified method of ,36. Seri3.5.3.5.1.Lichen extracts were dissolved in DMSO and then diluted into nutrient liquid medium to achieve a concentration of 5% DMSO. An antibiotic doxycycline  was dissolved in nutrient liquid medium, a Mueller–Hinton broth , while an antimycotic fluconazole  was dissolved in Sabouraud dextrose broth .3.5.2.Parmelia sulcata, Flavoparmelia caperata, Evernia prunastri, Hypogymnia physodes and Cladonia foliacea) was tested against 29 microorganisms including the 15 strains of bacteria ; 10 species of filamentous fungi: Aspergillus niger ATCC 16404, Aspergillus fumigatus PMFKG-F23, Aspergillus flavus PMFKG-F24, Aspergillus restrictus PMFKG-F25, Aspergillus niger PMFKG-F26, Penicillium italicum PMFKG-F29, Penicillium digitatum PMFKG-F30, Penicillium chrysogenum PMFKG-F31, Trichothecium roseum PMFKG-F32, Botrytis cinerea PMFKG-F33 and 4 yeast species Candida albicans ATCC 10231, Candida albicans ; Rhodotorula sp. PMFKG-F27 and Saccharomyces boulardii PMFKG-P34. All clinical isolates were a generous gift from the Institute of Public Health, Kragujevac. The other microorganisms were provided from the collection of the Laboratory of Microbiology, Faculty of Science, University of Kragujevac.The antimicrobial activity of methanol extracts of five lichens (3.5.3.2 × 2H2O + 99.5 mL 1% w/v H2SO4). When adjusted to the turbidity of the 0.5 McFarland’s standard, the bacteria suspension contains about 108 colony forming unites (CFU)/mL while the suspension of yeast contains 106 CFU/mL. 1:100 dilutions of the initial suspension were additionally prepared into sterile 0.85% saline. The suspensions of fungal spores were prepared by a gentle stripping of the spore from the slopes with growing aspergilli. The resulting suspensions were 1:1000 diluted in sterile 0.85% saline.The bacterial suspensions and the yeast suspension were prepared by the direct colony method . The col3.5.4.5 CFU/mL for bacteria and 5 × 103 CFU/mL for fungi and yeast. Finally, a 10 μL resazurin solution was added to each well inoculated with bacteria and yeast. Resazurin is an oxidation–reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. The inoculated plates were incubated at 37 °C for 24 h for bacteria, 28 °C for 48 h for the yeast and 28 °C for 72 h for filamentous fungi. MIC was defined as the lowest concentration of tested substance that prevented resazurin color change from blue to pink. For filamentous fungi, MIC values of the tested substance were determined as the lowest concentration that visibly inhibited mycelia growth.The antimicrobial activity was tested by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) using a microdilution method with resazurin . The 96-Doxycycline and fluconazole were used as a positive control. A solvent control test was performed to study an effect of 5% DMSO on the growth of a microorganism. It was observed that 5% DMSO did not inhibit the growth of a microorganism. Also, in the experiment, the concentration of DMSO was additionally decreased because of the twofold serial dilution assay (the working concentration was 2.5% and lower). Each test included growth control and sterility control. All tests were performed in duplicate and MICs were constant.The minimum bactericidal and fungicidal concentration was determined by plating 10 μL of samples from the wells, where no indicator color change was recorded, on the nutrient agar medium. At the end of the incubation period the lowest concentration with no growth (no colony) was defined as minimum microbicidal concentration.3.6.3.6.1.2 flasks  and after a few passages the cells were seeded in 96-well plate. Cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C. The cell numbers were determined by trypan blue exclusion.The colon cancer adenocarcinoma cell line HCT-116 was obtained from the American Tissue Culture Collection . These cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin. The cells were grown in 75 cm3.6.2.4 cells per well) and cultivated for 24 h. After that the cells were treated with 100 μL of diluted lichen extracts (concentration ranged from 50 to 1000 μg/mL) 24 and 72 h. The untreated cells served as a control. At the end of the treatment period, MTT   was added to each well, which was then incubated at 37 °C in 5% CO2 for 2 h. The colored crystals of the produced formazan were dissolved in DMSO (dimethyl sulfoxide) . The absorbance was measured at 550 nm on Microplate Reader. Cell proliferation (% viability cells) was calculated as a ratio of the absorbance of the treated group divided by the absorbance of the control group, multiplied by 100 to give percentage of the proliferation.After 24 and 72 h of treatment, the cell viability was determined by the MTT  reduction assay . MTT ass50(inhibitory dose which inhibits 50% of cell growth) and by the magnitude of the maximal effect in exposed cells. The IC50values were calculated from calibration curve by a CalcuSyn computer program.The antiproliferative effect of each extract was expressed by IC3.6.3.For the analysis of cell death, we used fluorescent assays of the acridine orange/ethidium bromide (AO/EB) double staining. Acridine orange is taken up by both viable and nonviable cells which emit green fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA). Ethidium bromide is taken up only by nonviable cells which emit red fluorescence by intercalation into DNA .4 cells per well) for 24 h. After that, 2 mL (250 μg/mL) of each lichen’s methanol extracts were added and the cells were cultivated for 24 and 72 h. The untreated cells served as a control. The incubation was performed at 37 °C in an atmosphere of 5% CO2 and 95% of relative humidity. After 24 and 72 h of treatment, 200 μL of dye mixture (100 μL of 100 mg/mL AO and 100 μL of 100 mg/mL EB in distilled water) was added to each well. The suspension was immediately (fast uptake) examined by fluorescence microscopy (NICON Eclipse Ti) at 400× magnification. A minimum of 300 cells was counted in every sample.HCT-116 cells were grown in a 6-well plate . In all cases p values <0.05 were considered statistically significant.The data were expressed as the means ± standard deviation (SD). All statistical analyses were performed using SPSS package  . Mean differences were established by Student’s 4.Parmeliacea  were demonstrated. The best results were obtained from Hypogymnia physodes and Cladonia foliacea. Further work will be done on the isolation and purification of active components in these species.The antioxidant, antimicrobial and antiproliferative activities of the five lichen species from Familia


Health beliefs are an important factor in the maintenance of an adherent behaviour. However, specific interventions based on the modification of health beliefs to promote adherence have not been applied in naïve HIV-infected subjects.Prospective randomized 48-week study to evaluate the efficacy of a psychoeducative intervention based on health beliefs to promote adherence in a sample of naïve HIV-1-infected men who started antiretroviral therapy. Participants were randomized to follow three intervention visits to promote adherence with the use of projective drawing techniques, Life-steps and Motivational interview  or to continue with the routine care . Adherence was assessed through self-report and drug plasma levels. Mann-Whitney nonparametric test, w2 or Fisher exact test were used to compare variables.3 and HIV-RNA VL of 65.000 (22.500–250.000) copies. The infection route had been mainly MSM (90%). QD and BID ARV therapy was prescribed in 29 (72.5%) and 11 (27.5%) subjects. Seven patients  were lost to follow-up. At week 48, 100% of subjects in GI and 60% in GC had 100% adherence (p=0.01). In GC, 26% and 14% of subjects had ≥95% and <95% adherence, respectively. No differences were found in adherence regarding QD or BID therapy. All subjects except for 3 had VL <25 copies at week 48.Participants were 40 men with a median (IQ) age of 35.2 (30.2–44.8) years, CD4 cell count at the study entry of 316 (229–539) cells/mmHigh adherence was observed in the majority of this group of naïve HIV-infected men who initiated their first antiretroviral therapy. However, all subjects following the intervention had 100% adherence after one year of follow-up. A psychoeducative intervention based on the modification of health beliefs may be a useful strategy to promote adherence in naïve HIV-infected patients.


In developing nations, the spread of STIs/HIV infection continues to affect millions of young and productive population. In Ethiopia youths including university/college students are at greater risk of STIs including HIV infection often due to many risky sexual behaviors. Although there are some anecdotal evidences suggesting widespread unsafe sexual practices among university students, the paucity of research finding, especially in newly established public universities are the major bottle necks to commence feasible interventions. Therefore, this study was designed to assess the magnitudes and factors associated with risks for STIs/HIV infections among Madawalabu university students in Southeast Ethiopia.An institution based cross sectional study was conducted from May-June 2012. A total of 390 students were selected using stratified then simple random sampling method. Descriptive statistics, binary logistic and multivariable logistic regression analyses were employed to identify factors associated with risks for STIs/HIV infection.Combined risk measure showed that 51.4% of students were at risk of having STIs and/or HIV infection. Practicing casual sex/sex for benefits with first sexual partner (OR = 3.9[95%C.I: 1.86-8.03]), life time multiple sexual partner  (OR = 2.7[95%C.I: 1.13-6.28]), and number of sexual partners in the last 12 months (four and above) (OR = 4.8[95%C.I: 1.77-13.53]) showed statistically significant association with risks for STIs and/or HIV infection. Practicing casual sex/ sex for any benefit with their first sexual partner (AOR = 3.9 [95%CI: 1.80-8.50]) and multiple sexual partners in the last 12 months (four and above) (AOR = 3.7 [95%C.I: 1.15-11.80]) were found to be the independent predictors of risks for STIs and/or HIV infection.This study has identified risks and risk sexual behaviors for STIs and/or HIV infection on university students. The knowledge of the students towards STIs and/or HIV is unsatisfactory. More than half of the students were at risk for STIs and/or HIV infection. Casual/benefit based sexual relationship with first sexual partner and having multiple sexual partners  in the last 12 months were independent predictors of STIs and/or HIV infections. Therefore, university based, risk reduction and behavior change focused interventions are recommended. The HIV pandemic remains the most serious infectious disease and a challenge to public health intervention. Globally, the epidemic has regional, population and gender disparity. Sub-Saharan Africa remains the most affected region in the global AIDS epidemic in which Ethiopia is one of the hardest hit sub-Saharan countries. Most of the deaths attributed to HIV/AIDS were because of inadequate access to HIV prevention and treatment services. It disproportionately affects youth generation which is almost half of the global population and leaves countries, communities, and households without productive people. Therefore its impact is not limited only to health problem -7.Not only from HIV infection, adolescents are being affected and infected from STIs or both. The prevalence of STIs is highest in developing nations. Because of young people’s risky behaviors and low use of preventive behaviors and/or services, the epidemic has fastest and growing effect on youth population. It is estimated that in developing countries, one in 20 youth contracts an STI each year. Young people especially who are unlikely to have access to quality health care services such as university students have higher rates of STIs but they have largely been ignored ,8. UniveIn universities- in countries with high HIV/AIDS prevalence-significant proportion of their students and staffs might have been infected with HIV . In EthiA study conducted in China showed that 17.6% of males and 8.6% of females were sexually active. The mean age at first sexual debut was 19.4 and 19.7 for males and females, respectively. But the proportion of male students who had experienced sex before joining university was significantly higher than female students . MultiplIn Ethiopia, significant proportion of the population, particularly the youths have been identified at risk of HIV infection despite high level of knowledge about HIV/AIDS . In JimmA study conducted in Bahir Dar University, has reported that 69.1% of students were sexually active; of which 25.3% started sexual intercourse before the age of 18 years. The study also indicated that 27.8% of students have had multiple sexual partners and 34.4% had practiced unprotected sex. About eight percent (7.8%) of students had sex with commercial sex workers .In Haromaya University, 41.2% of students were sexually active, and 27.8% of students had multiple sexual partners (2–5 partners on average). In this study, (39.9%) of the students had reported sexual contact with commercial sex workers (CSW) while 23.51% of the students reported to have sex with casual friend ). Similarly, the number of life time sexual partner  was found to be statistically significant with the outcome variable (OR = 2.7[95%C.I: 1.13-6.28]). Number of sexual partners in the last 12 months (four and above) was statistically associated with risks for STIs/HIV infection (OR = 4.8[95%C.I: 1.77-13.53]) (Table 3.9[95%CI: 1.80-8.50]) and having four and above sexual partners in the last 12 months (AOR = 3.7[95%C.I: 1.15-11.80]) were independent risk predictor of STIs/HIV infection ; practicing sex for any kind of benefit (9.7%), wide range of age difference (10 years or more) with first sexual partner 10.4%), life time multiple sexual partners (47.4%) , three sexual friends (13.1%) and 23.1% of students had more than three sexual partners in their life time. The proportion of life time multiple sexual partner is almost similar with the finding of the study conducted in Haromaya University (35.4%). But the figure is by far greater than the findings in Jimma University (28.9%) and Bahir Dar University (27.8%). The figurative difference could be due to the difference in sample size and comprehensive university based behavioral change interventions in the above mentioned universities. But a study in Wolaita Sodo University has reported life time multiple sexual partner rate of 70.6%. The study conducted in Nigerian University, students have reported an average of 3.5 sexual partners. A study conducted in Benin, Togolese university, showed that 63% of students had more than one sexual partner ,15,20-23In this study, 30.4% of students have had multiple sexual partners in the last 12 months. A similar study in Malawi University showed that 40.4% of the students reported multiple sex partners in the last 12 months. In our study, 44.0% of the students practiced unprotected sexual intercourse in the last 12 months. This rate of unprotected sexual practice is nearly similar with the finding in Malawi University (37.4%) and Bahir Dar University (34.4%). But the rate of unprotected sex with casual partners in Jimma University looks somehow greater (56.3%) ,18,19,22In this study, 24.0% of sexually active male students reported ever had history of sexual contact with commercial sex workers (CSW). Similarly, in Gonder University, 23% of university students had reported sexual contact with prostitutes while a study in Haromaya University reported that 39.9% of sexually active male student had sexual contact with commercial sex workers. But in Bahir Dar University, 7.8% of students reported sexual contact with commercial sex workers ,22,23. CThe current study has indicated that having sex with casual sexual friend/ sex for any benefit has a statistically significant association with risks of STIs and/or HIV infection. In line with its theoretical/clinical significance, those students who have had casual/benefit based sexual intercourse with their first sexual partner were about 4 times more likely to have risks for STIs and/or HIV infection when compared to those who have permanent sexual friend/spouse (OR = 3.9[95%C.I: 1.86-8.03]). Similarly, the number of life time multiple sexual partners has statistical association with the risks for STIs and/or HIV infections. Therefore, students who have had more than three sexual partners in their life time were about 3 times more likely to have risks for STIs/HIV infection when compared to their counter parts who have had one sexual partner (OR = 2.7[95%C.I: 1.13-6.28]).The number of sexual partners in the last 12 months (≥ 4) showed statistical associated with risks for STIs/HIV infection with an increased odds ratio as compared to life time multiple sexual partners (OR = 4.8[95%C.I: 1.77-13.53]). Students those who have had four or more sexual partners in the last 12 months were about 5 times more likely to have STI/HIV infection risks when compared with their counter parts who have one sexual partner.The multivariable logistic regression analyses showed that practicing casual/benefit based sexual activity with first time sexual partner and having four and above sexual partners in the last 12 months were statistically significant predictors of STIs/HIV infection risks.3.9(95%CI: 1.80-8.50). Similarly, those students who had multiple sexual partners in the last 12 months  were 3.7 times more likely to have risks for STIs/HIV infections when compared to their counter parts who have had only one sexual partner in the last 12 months (AOR = 3.7[95%C.I: 1.15-11.80]).Given that, life time multiple sexual partner is in the model, those who practiced casual sex/ sex for any benefit with their first sexual partner were 4 times more likely to have risks for STIs/HIV infection when compared to those students who practiced their first sex with their permanent sexual friends/spouse  , casual sexual intercourse/sex to get any benefit, sex with risk groups(CSW). Unprotected first time sexual act was reported-which might be practiced after students has joined university- which could be evidenced by one third of the students had practiced their first sexual intercourse after joining university.Generally this study has identified that more than half of the students were at risk for STIs and/or HIV infection and casual/benefit based sexual relationship with first sexual partner and having multiple sexual partners in the last 12 months  were independent risk predictors of STIs and/or HIV infections. Therefore, strengthening student clubs, designing and implementing awareness rising and risk reduction activities and promoting peer education are recommended interventions.The authors declare that they have no competing interests.TS conceived and designed the study, performed analysis and interpretation of data and drafted the manuscript. AT assisted with the design conception, analysis, and interpretation of data. ND assisted the study design, data interpretation and critically reviewed the manuscript. BT assisted data collection, entry and reviewed the manuscript. All authors read and approved the final manuscript.


Soap Lake is a small saline lake in central eastern Washington that is sharply stratified into two layers. In addition to being highly alkaline (~pH 10), Soap Lake also contains high concentrations of sulfide. Here, we report the community profile of the prokaryotic microbiota associated with Soap Lake surface water. Cyanobacteria  using a sterile 50-ml sampling tube. The water temperature and pH were 20°C and 10, respectively. DNA was extracted using the FastDNA SPIN kit for soil from MP Biomedicals, according to the manufacturer’s protocol. The V6 to V8 region of the bacterial 16S rRNA gene was amplified using the primer set 926f/1392r and sequenced using the Roche 454 FLX+ platform . The raw pyrosequence reads were quality filtered and analyzed using QIIME version 1.7.0 (Firmicutes (45.4%), Proteobacteria (15.4%), Tenericutes (10.5%), Actinobacteria (10.2%), and Bacteroidetes (5.1%). Cyanobacteria contributed 3.2% of the sequence reads. Interestingly, 80.3% of the phylogenomic data were recruited by 18 OTUs, and 41.8% were by a single OTU assigned to an uncultured microorganism classified as a member of the Firmicutes. This data set reveals a relatively low diversity of microorganisms capable of coping with the extreme conditions of Soap Lake.A total of 2,702 high-quality sequences, representing 172 distinct OTUs and 13 phyla, were obtained, and a Good’s coverage of 0.98 was calculated. The five most abundant phyla were SRP033727.The DNA sequences from this metagenome project have been deposited in the NCBI Short Read Archive under the accession no.


AA amyloidosis is the most severe complication of hereditary autoinflammatory diseases. In TRAPS it has been reported to occur in approximately 25% of patients in the absence of an effective treatment. However, susceptibility to AA is difficult to predict. Identification of key risk factors affecting the development of AA would improve the clinical management of TRAPS patients, by allowing a more tailored treatment approach.To evaluate the relative contribution of clinical and genetic factors to the risk of AA amyloidosis in TRAPS.TNFRSF1A mutation and written informed consent. DNA was available for patients recruited into the EUROTRAPS research project and SAA1 was genotyped by direct sequencing of exon 3.Clinical data were obtained from the EUROFEVER/EUROTRAPS web-based registry. Inclusion criteria for this study were: age ≥ 18 years at last follow-up, identification of a SAA1 genotype was established in 89/104 patients and 31 (35%) were homozygous for the SAA1.1 allele. 77 patients (74%) had a clearly pathogenic TNFRSF1A variant. 27 had either P46L or R92Q.104 patients  with TRAPS (39 different mutations) were included in the study. Median age was 41 years (range 18-88), median age at TRAPS onset was 6 years (range 0.5-63), and median age at diagnosis was 37 years (10-81). 21 patients had AA amyloidosis, with a median age at AA onset of 38 years (range 18-76). Family history for AA amyloidosis was observed in 33 patients (32%). SAA1.1 homozygosity, disease course, age at TRAPS onset and the type of mutation were significantly associated with AA amyloidosis. At multivariate analysis homozygosity for SAA1.1 and age at TRAPS onset independently predicted development of renal amyloidosis. SAA1.1/1.1 genotype was the variable with the strongest influence on AA development, with a 5.3 fold increased risk whereas older age at TRAPS onset was associated with a reduced risk of AA amyloidosis. Survival according to SAA1 genotype  was estimated by Kaplan-Meier analysis. Median amyloid free survival from birth was 47 years vs. not reached (p=0.01).At univariate analysis, family history for amyloidosis, SAA1.1 allele is the strongest predictor of AA risk in TRAPS. This result is extremely relevant for the clinical management of TRAPS patients and supports SAA1 genotyping on a routine basis to guide treatment approach.Homozygosity for the None declared.


S.aureus bacteraemia (SAB).The recent decline in MRSA infection is well recognised but the contribution of hospital-acquired (HA) versus community-acquired (CA) is not always clear, nor if MRSA has substituted for MSSA or is an additional burden. We examined our experience with S. aureus was meticillin-sensitive or resistant, and if infection was CA or HA. SAB within 2 days of admission was defined as CA, 2 days or more after admission as HA and a new episode if more than 2 weeks between 2 positive SAB.From April 2008 to March 2010, at Imperial College Healthcare NHS Trust (ICHNT), electronic microbiological records of patients with SAB were linked with patient administration data to determine if the Of 345 SAB detected, 60% were CA and 40% HA; 250 (72%) were MSSA and 95 (28%) MRSA. Of MRSA bacteraemias, 49% were HA, but 37% of MSSA bacteraemias were HA. The rate of SAB per 100,000 bed-days had a downward trend due a decrease in both MRSA and MSSA bacteraemia. Since April 2009, the number of HA MRSA and MSSA decreased simultaneously. During the period April 2008 to March 2009, the rate of HA SAB was 19.6. In the period April 2009 to March 2010 this declined by to 11.7. Between these two periods the rates of MRSA and MSSA bacteraemia rates decreased by 1.7 (from 19.6 to 11.7), 2.1 (from 7.2 to 3.4) and 1.5 times (from 12.4-8.3).S. aureus as a cause of bacteraemia at ICHNT is decreasing, particularly HA cases. MRSA and MSSA bacteraemia are decreasing simultaneously, suggesting either that MRSA substituted MSSA rather than reflecting additional cases or that MRSA control measures may also have had an effect on MSSA.None declared.


Prevalence surveys serve as a rapid and simple means of obtaining healthcare associated infection (HAI) data. There is variability in following either the point or the period prevalence strategy, and whether the four major HAI categories  should be reported rather than all HAI.The aim of the study was to analyse the difference in outcome depending on the used survey strategy.The University of Geneva Hospital (HUG) is a primary and tertiary care centre at 8 hospital sites with 1908 beds, 48’112 admissions and 671’709 patient-days in 2011. Since 1994 yearly period prevalence surveys are performed. Surveillance includes all HAI as defined by the Centers for Disease Control and Prevention. The HUG surveillance allows to distinguish between period and point prevalence outcome from the same dataset. This study focused on the data between 2007 and 2012. Differences were analysed by stratifying the data by clinical setting , and the group of HAI .The HAI prevalence for point and period methodology was 7.45% and 9.87% (+32%), respectively. The additional 33% (p<0.01) HAI were due to UTI (+52%), LRI (+31%), and other HAI (+40%). The difference for BSI (+15) and SSI (+19) was less important. HAI prevalence for point and period methodology in ICU, acute care, subacute care, and longterm care were 19.1%/24.1% (+25%), 6.3%/7.9% (+25%), 9.5%/13.4% (+41%), and 5.8%/8.9% (+53%), respectively. The differences were stable over time. Additional 43% LRI (p=0.05) and 79% (p=0.01) other HAI were detected by the period prevalence in longterm care, where the difference was most pronounced. The point and period prevalence the four major HAI categories were 5.88% and 7.65% (p<0.001), respectively.More HAI are identified by the period prevalence method. There is a tendency for period prevalence surveys to overestimate HAI of short duration  and in subacute and longterm care settings. Point prevalence data cannot be benchmarked with period prevalence data.None declared


Baseline thyroid-stimulating hormone (TSH), FT4, FT3, and TT3 were measured for potential relationships to treatment response across groups. In males only, there was a significant inverse correlation between baseline free T4 and time to response . In both males and females across all treatment conditions, as measured by Kaplan–Meier (K–M) maintenance failure time, baseline TSH below the mean (1.5 ng/dL) was associated with a shorter time to response  . Patients with baseline TSH above the mean were less likely to reach full remission (MADRS ≤ 7) . No significant differences between groups emerged in the mean response time. Baseline thyroid function, as measured by serum free T4 and TSH, may predict a patient's response time to antidepressant treatment with citalopram.The lack of reliable outcome predictors and the delayed onset of therapeutic response to antidepressants are among the clinical challenges in the treatment of depression. Identifying clinical correlates associated with antidepressant response would reduce symptom severity and morbidity for patients with depression. Twenty-three subjects with major depression were treated with citalopram 20 mg/day in a 6-week open trial and were also simultaneously randomized to either adjunctive triiodothyronine (T3) 25  Major depression is a serious medical illness that affects more than 13% of adults in the U.S. during their lifetime . The vasP = 0.002). The response was greater in women than men. Along the same lines, P < 0.05). These gender differences at baseline; or during acceleration or treatment responses have not been prospectively confirmed in larger studies.Another clinical challenge stems from the fact that less than half of treatment-seeking depressed patients reach the expected therapeutic benefits after several weeks of adequate antidepressant treatment . This deP = 0.03).Another accelerating agent is pindolol, a β-blocker with activity at the 5-HT1A receptor , which hThe aim of this pilot study was to explore the relationship between pretreatment thyroid function measures and response to treatment in subjects enrolled in a study to compare the efficacy of T3, pindolol, and placebo in accelerating the antidepressant effect of citalopram in patients with unipolar major depressive disorder. We hypothesized that within normal range, lower baseline TSH levels will be associated with better antidepressant response outcome.All study procedures were approved by UCLA IRB. Twenty-three subjects  with first episode unipolar major depressive disorder (DSM-IV-TR) signed an informed consent and were recruited in the study through local advertisement at UCLA campus. All 23 subjects were referred to the study either by self or by their primary care physician or psychiatrist. Diagnosis was confirmed by structured clinical interview (SCID) . SubjectDepressive symptoms were rated over eight visits  using the following rating scales: Montgomery and Asberg Depression Rating Scale (MADRS) , Beck Deμg BID (n = 7), pindolol 5 mg BID (n = 8), or placebo (n = 8) at the start of citalopram treatment.All subjects received open label citalopram (20 mg) for 6 weeks and were randomized in a blinded fashion to receive additionally triiodothyronine (T3) 25 Serum TFTs were checked at baseline and at the end visit. TSH and total triiodothyronine (TT3) were assessed by immunoassay , free triiodothyronine (FT3), and free thyroxine (FT4) by Enzyme Immunoassay Assay Diagnostic System Laboratory (EIA-DSL).Primary outcome measure was the time to 50% reduction in baseline MADRS scores.Collected data were screened for distributional properties and determined to be appropriate for parametric analysis. Simple correlation analysis and proportional hazard regression (Cox Model) and accelerated failure time survival regression analysis were used to predict time to response (i.e. 50% improvement in MADRS scores) and remission (MADRS score ≤ 7) with baseline and delta TSH, FT4, FT3, and TT3 as independent variables. Analysis was done using SPSS software .Of the randomized 23 subjects, 19 completed the study. Two subjects in the placebo group dropped out  and two subjects missed follow-up. The mean age of the cohort was 38.34 (±12.6) years and the mean length of the index episode was 8.9 (±5.9) months with an age of onset of 32.9 (±13.5) years. All patients were required to not be receiving antidepressants for at least one month prior to starting. All, but five, patients were antidepressant naive at the time of the study. The mean baseline MADRS score was 29.7 (±5.85), BDI score was 23.4 (±7.3), and a mean CGI severity score was 4.1 (±0.3). Six patients met DSM-IV criteria of life comorbid generalized anxiety disorder, four with posttraumatic stress disorder, and one patient with social phobia. All comorbid conditions were clinically stable and none of the patients receives treatment or therapy for comorbid conditions during the study.The mean baseline TSH was 1.5 (±0.7) mIU/L, FT4 was 1.1 (±1.2) ng/dL, FT3 was 3.0 (±1.5) ng/mL, and TT3 was 120.2 (±20.9) ng/dL. No significant gender differences were observed in any of the baseline thyroid indices .2 = 4.53, df = 1, P = 0.03). Furthermore, patients with baseline TSH above the mean were less likely to reach full remission .Across genders, low baseline mean TSH was associated with shorter time to response as measured by K–M maintenance failure time : 16 ± 7.8, pindolol (n = 8): 12.2 ± 10.6, and placebo (n = 8): 16.4 ± 9 days. One-way ANOVA F  = 0.9, P = 0.4. Males (n = 9) reached response faster than females (12.4 ± 7.6 vs. 16.8 ± 9.9 days); however, this difference was not statistically significant .The mean time to response, defined as a 50% reduction in MARDS score, 14.9 (±9.1) days, was not significantly different between the three groups. T3 (The two major limitations of our study are (1) the open label design; and (2) the small number of subjects in each group, which may have precluded finding an accelerating effect of either medication. We estimated to require 20 patients per treatment arm based on a priori power analysis to detect a 15% difference in the primary outcome (MADRS) scores from the mean with 80% power and an alpha of 0.05. However, due to the stringent exclusion criteria , the enrollment was slow and was terminated before a minimum number of intended-to-treat (ITT) subjects were enrolled. However, this pilot study with treatment group, combined allowed us to evaluate the relationship of thyroid status at baseline to treatment outcome. The study suggests that optimal thyroid function may be associated with faster response to citalopram and perhaps more so in men than in women. Low baseline TSH was associated with shorter time to response, while patients with baseline TSH above the mean were less likely to reach full remission. Moreover, higher baseline free T4 was inversely correlated with the time to response in males. We  and otheThe significant inverse correlation between baseline free T4 and response time only in males is in agreement with our previous report . HoweverPerhaps due to the small sample size, coinitiating T3 or pindolol with citalopram in patients with major depression did not show a significant difference from placebo in reducing the time to response. However, P = 0.03). One explanation for this disparity is that T3 may shorten the response time to TCAs and not SSRIs. Of course, the small sample size of our study may have resulted in a Type II error in evaluating the accelerating response of both T3 and pindolol.This observation is in contrast with the significant effect of T3 in accelerating the antidepressant effect of TCAs , and theThe mean time to response in our sample was only two weeks despite the relatively high baseline MADRS scores (29.7 ± 5.8). This interesting observation is in line with other published meta-analyses of double-blind randomized clinical trials reporting statistically significantly greater response to fluoxetine  and bothDespite the negative acceleration results in this study, the main finding suggests that hyperactive hypothalamic pituitary thyroid axis may be associated with better antidepressant response. Further research is needed to further explore this potential marker.


Helicobacter pylori (H. pylori) presents a challenge. We aimed at investigating the presence of H. pylori, susceptibility profile, and associated mutations in an effort to validate the effectiveness of GenoType HelicoDR assay in H. pylori typing in our environment. Two hundred and fifty-four biopsy specimens were cultured and DNA extracted from seventy-eight positive cultures using the Qiagen DNA extraction kit. The GenoType Helico DR which employs reverse hybridisation was used to confirm the presence of H. pylori, determination of its susceptibility to antimicrobials, and detection of mutations conferring resistance to clarithromycin and fluoroquinolones. The organism was isolated from 168/254 (66.1 %) of the specimens by culture. Of the 78 strains used for further investigation, 12/78 (15.38%) were resistant to clarithromycin while 66/78 (84.61%) were susceptible. For fluoroquinolone, 70/78 (89.74%) strains were susceptible while 8 (10.26%) were resistant. Mutations were observed in 17 strains with A2147G being the most prevalent; A2146C and D91N were the least. The reverse hybridisation assay is an easy and fast technique in confirming the presence of H. pylori, its antimicrobial profile, and associated mutations. Analysis regarding the suitability of this assay for H. pylori typing is warranted in other regions.Rapid diagnosis and treatment of  Helicobacter pylori (H. pylori) presents a tremendous challenge therapeutically [H. pylori infection seems tenacious because the organism lives in an environment not easily accessible to many medications, the overwhelming presence of antibiotic resistance and poor patient compliance [The burden of utically , 2. Clinmpliance . Eradicampliance . Regimenmpliance .H. pylori with very low minimum inhibitory concentration compared to other molecules [H. pylori can become resistant to these compounds, which jeopardize the success of treatments [H. pylori isolates is regarded as a main cause of treatment failure in developing countries [H. pylori [Clarithromycin currently remains the available most powerful antibiotic against olecules . Fluoroqolecules , 7. Howeeatments , 8. Partountries . The orgountries , and henountries , 10. Extountries . These mountries , 11, 12.ountries . Besidesountries , 7. Quin. pylori . Resista. pylori , 13, 14.H. pylori to antibiotics is currently widely determined in clinical bacteriology laboratories by standard methods, such as disk diffusion, microbroth dilution assay or Etest [H. pylori needs about 3–7 days to grow) and do not give insight to the type of mutation present, which could be of epidemiological and clinical significance. Conventionally, mutations are detected using molecular typing schemes such as PCR restriction fragment length polymorphism (PCR-RFLP) and sequencing [H. pylori and mutations at codons 2146-2147  in 23S rRNA gene in clarithromycin resistant isolates and mutations at codon 87 and 91  which are associated with resistance to fluoroquinolone were employed and validated in a study by Cambau et al. [H. pylori, its antimicrobial profile, and mutations associated to clarithromycin and fluoroquinolone. We sought therefore to employ the use of this assay to investigate the presence of H. pylori and associated mutations to these antibiotics in an effort to validate its effectiveness in H. pylori related studies in the environment of the Eastern Cape province of South Africa due to its high prevalence reported in our previous studies [Resistance of or Etest , 16. Thequencing , 12, 17.quencing . A PCR-bu et al.  in FrancIn this study, 168/254 (66.1%) of the specimens collected from patients who underwent endoscopic examination for upper gastrointestinal problems with no history of treatment with macrolide and fluoroquinolone antibiotics were found positive for culture. Seventy-eight of the positive strains isolated from 50 males and 28 females were used for further investigations. Antral and corpus gastric mucosal biopsy specimens were taken from each dyspeptic patient. The biopsies were immediately placed in sterile bijou bottles containing 0.2 g/L of cysteine and 20% glycerol in brain heart infusion (BHI) broth and transported in ice to the laboratory within 2 h of collection for culture [2, 10% CO2, 80%–85% N2) . Isolates were identified based on colony morphology and positive oxidase, urease, catalase tests, and confirmation by amplification of the glmM gene as previously reported [H. pylori reference strain NCTC 11638 was included in all experiments. Approval for this study was obtained from the Research Ethics Committee of the University of Fort Hare and the Eastern Cape Department of Health (protocol number EcDoH-Res 0002).Biopsies were homogenised under aseptic conditions in 0.2 g/L of cysteine and 20% glycerol in BHI broth and a loop full plated primarily on freshly prepared Columbia agar base  supplemented with 7% sheep's blood  and Skirrow's supplement ; trimethoprim (2.5 mg), vancomycin (5 mg), cefsulodin (2.5 mg), and amphotericin (2.5 mg) were also added to the medium. All plates were incubated at 37°C for 3–5 days under microaerophilic conditions (5%-6% Oμg/mL for ciprofloxacin and 0.125–256 μg/mL for clarithromycin.Minimum inhibitory concentration (MIC) was determined for clarithromycin and ciprofloxacin as previously described . MIC valμL containing 35 μL primer/nucleotide mix (PNM), 5 μL 10x polymerase incubation buffer, 2 μL of 1.5 mM MgCl2, 3 μL of nuclease free water  0.2 μL Thermo-Start Taq DNA polymerase (1-2 units were added to each tube), and 5 μL DNA template. PCR was performed with a thermal cycler . The amplification cycles consisted of an initial hot start of 95°C for 15 min, initial denaturation of target DNA at 95°C for 5 min, denaturation at 95°C for 30 sec and 58°C for 2 min, primer annealing at 95°C for 25 sec, 53°C for 40 sec, and 70°C for 40 sec, and extension at 70°C for 8 min. All reactions were performed through 32 cycles . DNA was extracted from 78 strains using QIAamp tissue kit  following the manufacturer's recommendation closely. Amplification of the bacterial DNA was done using hot-start DNA polymerase . Biotinylated primers were used for this study and were provided in the amplification kit. Primers were designed using the gene sequence from GenBank accession number NC_009151. Polymerase chain reaction for a single mixture had a final volume of 50 H. pylori, antimicrobial susceptibility, and mutational analysis to clarithromycin and fluoroquinolone was performed using the GenoType HelicoDR kit . The kit employs the use of reverse hybridisation performed using hybridisation trays and TwinCubator  according to the manufacturer's instructions. Briefly, 20 μL of amplified DNA was denatured and added to biotinylated probes on the strip and the hybrids formed were detected by enzyme-linked immunosorbent assay (ELISA) upon addition of substrate conjugate and substrate. Four gyr87 wild type probes (gyr87WT1–gyr87WT4) and one mutant probe (gyr87MUT), one wild type probe (gyr91WT1), and three mutant probes (gyr91MUT1–gyr91MUT3) were used for detecting fluoroquinolone resistance at position 87 and 91, respectively. For clarithromycin, one wild type probe (23SWT) and three mutant probes (23SMUT1–23SMUT3) were used for detecting resistance. On the strip, were designated conjugate control (CC), amplification control (AC) and H. pylori (HP). When one of the WT probes stained positive together with the gyr91WT as well as 23SWT and no mutation band formed, the results were interpreted as susceptible to the respective antibiotic. The presence of a band at CC and AC meant that the conjugate control and amplification control were in the right frame while at HP implied presence of H. pylori according to the manufacturer's instruction . Confirmation of isolates as P value <0.05. The sensitivity and specificity of the GenoType HelicoDR kit for detection of resistance to clarithromycin and fluoroquinolone, respectively, were calculated as previously described [Epi Info version 2000  was used for statistical analysis. Chi square or Fischer exact test was applied to test whether differences in susceptibility/resistance between values of males and females were significant at escribed . H. pylori by culture and confirmed using polymerase chain reaction/reverse hybridisation assay (GenoType HelicoDR). Seventy-eight of the 168 strains were used for further investigations. In this study, 168/254 (66.1%) specimens were positive for Seventy (89.7%) of the 78 strains were susceptible to fluoroquinolone while eight (10.3%) were resistant. For clarithromycin, 66/78 (84.6%) were susceptible and 12/78 (15.4%) were resistant. The sensitivity and specificity of detecting resistance were 98% and 100% for clarithromycin and 89% and 93% for fluoroquinolone, respectively. P = 0.006), although not statistically significant for fluoroquinolone (P = 0.127).Of the 78 strains employed for further analysis, 28 (35.9%) were from females and 50 (64.1%) from males. Prevalence of clarithromycin resistance in females and males was 32.1% (9/28) and 6% (3/50), respectively, while for fluoroquinolone it was 17.9% and 6%, respectively. A higher prevalence of resistant isolates was observed in female compared with male subjects in this investigation. There was statistically significant difference with the use of clarithromycin for both sexes (Some mutations designed in line with the kit to detect resistance to clarithromycin and fluoroquinolone were delineated. Mutations observed in 17 strains are summarised in Three strains had 2 or more mutations with the highest number of mutations (4) occurring in 252C. A2147G mutation associated with resistance to clarithromycin was the most prevalent mutation type in this study while A2146C and D91N were the least. Twelve of the 17 strains studied possessed A2147G mutation . μg/mL. The frequency of A2146C mutation was very low occurring in only one (252C) of 17 strains (5.8%). The MIC of clarithromycin for these mutants (A2147G and A2146C) ranged from 32 to 256 μg/mL to 32 μg/mL. Strains with mutations for both clarithromycin and fluoroquinolone (252A and 252C) had MIC 32 μg/mL while 247A had 256 μg/mL.Of the 8 strains found to be resistant to fluoroquinolone, all (100%) possessed N87K mutation associated with fluoroquinolone. No designated mutation was found in five strains  using this assay as there was the complete absence of the gyrase 87 wild type indicating a mutation (designated as N87K mutation). Also, D91N associated with resistance to fluoroquinolone was detected in 1 (5.8%) of the 17 strains whilst D91Y and other designated mutation associated with fluoroquinolone resistance were not found. Minimum inhibitory concentration for fluoroquinolone  , ranged H. pylori infection [H. pylori infection, and its associated disease conditions coupled with the difficulties of rapid diagnosis and management of patients [H. pylori strains isolated from the Eastern Cape province known to have a high prevalence of H. pylori-related morbidities [Clarithromycin and fluoroquinolone are presently the drugs of choice employed for triple combination therapy in the treatment of nfection . Resistanfection , 13, 19.patients  necessitbidities , 16 usinH. pylori was detected in 168/254 (66.1%) of the specimens studied. Clarithromycin is used worldwide as one of the potent antibiotics in the eradication therapy of H. pylori [. pylori . However. pylori , 17. Thi. pylori . The pre. pylori . Finding. pylori  who repo. pylori . TherefoThe moderate rate of resistance of 10.26% to fluoroquinolone reported in this study ties with the findings of Wang et al.  who repoP = 0.006), but not for fluoroquinolone. Importantly, more males were recruited for this study than females and this could account for the difference observed. Noteworthy is the fact that the number of strains resistant to clarithromycin (12) and fluoroquinolone (8) is almost the same, suggesting that these antibiotics could be close in their suitability as drugs of choice for H. pylori treatment in our environment corroborating our previous finding [The prevalence of antibiotic resistance was higher in males than in females in this study reaching statistical significant difference for clarithromycin  of the 17 strains reported to be resistant in this study had A2147G mutation (Tables H. pylori is thus relevant in this area to guide empiric treatment. In conclusion, this study revealed a moderate rate of resistance to fluoroquinolone and clarithromycin, with A2147G and N87K being the main mutations associated with clarithromycin and fluoroquinolones, respectively. However, continuous surveillance of fluoroquinolone and clarithromycin resistance in


Concentrations of PM3 increase in PM2.5–10 (single-pollutant model). The association with PM2.5–10 was stronger for November through May, when road dust is most important , compared with the rest of the year , although the difference was not statistically significant. When adjusted for other pollutants, particularly PM2.5, the effect estimates per 10 μg/m3 for PM2.5–10 decreased slightly but were still higher than corresponding effect estimates for PM2.5.Results: We estimated a 1.68% increase  in daily mortality per 10-μg/m2.5–10. Regulation of PM2.5–10 should be considered, along with actions to specifically reduce PM2.5–10 emissions, especially road dust suspension, in cities.Conclusions: Our analysis shows an increase in daily mortality associated with elevated urban background levels of PM Particle effects on mortality. Hundreds of epidemiological studies have shown that the ambient particulate air pollution is associated with daily mortality, generally studied using the concentration of particulate matter (PM) with an aerodynamic diameter ≤ 10 μm (PM10) or fine PM with aerodynamic diameter of ≤ 2.5 μm (PM2.5) (2.5–10) on mortality has been less studied. In their review article, 2.5–10 effects on mortality after adjusting for PM2.5. However, PM2.5–10 levels are expected to be more spatially heterogeneous than are PM2.5 levels, which increase exposure misclassification when one or a few monitors provide exposure data  . The effure data . Moreove Arizona , Coachel Arizona , and Mex Arizona . In arid2.5–10 and daily mortality , particularly among demographic subgroups of lower socioeconomic status. In their study, only those participants who resided close to an air pollution monitor were included in the study in order to reduce exposure misclassification. Adjusting for PM2.5 had no effect on the effect estimates for PM2.5–10, likely due to its low correlation with PM2.5–10 in these California counties. An even larger study of U.S. cities found an association between PM2.5–10 and daily mortality that persisted after adjusting for PM2.5  was based on daily counts of deaths excluding external causes , for the years 2000 through 2008 from the Cause of Death Register at the Swedish National Board of Health and Welfare .Environmental data. Data on daily urban background concentrations of PM10, PM2.5, ozone (O3), and carbon monoxide (CO) were obtained from the Environment and Health Administration of Stockholm (2011). PM10, PM2.5, and O3 were measured in central Stockholm at Torkel Knutssonsgatan, a monitoring station located at rooftop level (at a height of 25 m) not directly affected by nearby emissions . To account for losses of volatile material in the PM, all data were corrected following 3 was based on its absorption of ultraviolet light . The urban background CO concentrations were based on continuous measurements of two rooftop stations  in central Stockholm. The instruments were based on a nondispersive infrared technique . The coarse fraction of PM10 (PM2.5–10) is based on the difference between PM10 and PM2.5.The mass concentrations of PM2.5–10 levels therefore depends on the meteorological conditions, especially during the late winter and spring. Therefore, we adjusted for meterological data that was collected from the Swedish Meterological and Hydrological Institute. Daily temperature and relative humidity were measured at Bromma Airport, a city airport, 9 km from Stockholm city center.The contribution of road dust to the particle concentrations varies with the wetness of the road surfaces  and is nStatistical methods. We studied the association between daily mortality and PM2.5–10 concentrations averaged over the day of death and the day before death (lag01) with a time-series analysis. The exposure lag01 has been commonly used when effects of air pollution on mortality have been studied . Influenza episodes were controlled by modeling the daily influenza hospital admissions as a smooth function. All influenza hospital admissions in Sweden were obtained from the Patient Register at the Swedish National Board of Health and Welfare . Each of the smooth functions in the model was represented using penalized regression splines.2.5–10, PM2.5, and CO and the maximum of 8-hr moving-average (between 0600 hours and 2200 hours) concentrations of O3 on the same day and the previous day (lag01). Results are reported for single-pollutant models  and for multipollutant models . Results also are reported for a 10-μg/m3 increase as well as for an interquartile range (IQR) increase in each pollutant.We modeled 24-hr average concentrations of PM2.5–10) and June through October as the reference season.The analysis was stratified by period because the composition of particles varies seasonally. In Sweden, passenger cars, light-weight trucks, and light-weight buses are required to have winter tires from 1 December to 31 March. Heavy vehicles are not required to use winter tires. Winter tires can be studded or nonstudded, but studded winter tires were allowed from 1 October to 30 April during the study period 2000 through 2008 and were used by 70–75% of vehicles in Stockholm during those years. The share of studded winter tires usually increases from zero in September through October to its maximum in December through March and then falls back to zero in May , dependi2.5–10 may affect mortality with a longer lag than lag01, we also fitted a distributed lag model for up to 7 days (same day and up to 6 days earlier) for PM2.5–10.To test the hypothesis that PMp-values < 0.05 to define statistically significant effect estimates and p-values between 0.05 and 0.10 to define borderline significant effect estimates. Data were analyzed using the mgcv package in R .We used 2.5 (260 days) and PM10 data (106 days) that did not allow calculation of PM2.5–10. In total, 2,789 days were included in the analysis. A statistical summary of the air pollution measurements for the whole period (2000 through 2008) is presented in There were 93,398 deaths  during the 2000 through 2008 study period . On average, there were 28.4 deaths per day . The average number of deaths per day for the road dust period (November through May) was 29.6 , and for the reference season (June through October) was 26.8 . Days with missing values for environmental variables during the study period were not included, most often because of missing PM2.5–10 are highest during late winter and spring. For PM2.5–10, 148 days with a daily mean concentration of 20 μg/m3 (95th percentile) or higher were observed within the road dust period, but only 4 days outside that season  of the total PM10 concentration. PM2.5–10 contributes 44% on average in November through May and 40% in June through October  based on the single-pollutant model .The correlations between PMnt model . Adjusti3 increase in PM2.5–10  was higher than the effect estimate for a 10-μg/m3 increase in PM2.5  when both pollutants were included in the same model. In addition, the estimated percent change in daily mortality for an IQR increase was larger for PM2.5–10 (5.2 μg/m3) than for PM2.5 from the single-pollutant model, adjusted for the covariates listed above , suggests that the more precisely estimated part of the curve does not deviate from linearity.The smooth function of PMed above , suggestp = 0.025) in daily mortality per 10-μg/m3 increase in PM2.5–10 for the period November through May (p = 0.81).We estimated a 1.69% increase  compared with the road dust period  per 10-μg/m3 increase in PM10 in the European APHEA 2 study . The uncertainty of PM2.5–10 has not been determined, but most of the uncertainty in PM10 is likely due to the correction for volatile PM, which is mainly present in PM2.5 fraction and not in the PM2.5–10 fraction.We did not directly monitor PM methods ] in Stoc2.5–10 during the road dust period originates from the road network covering the whole city, not from a few point sources. Thus, in Stockholm, where the main contributor to PM2.5–10 is road traffic, the urban background daily mean concentrations in different parts of the city should fluctuate in a similar way.A strength of the present study is that PM10. Other recent studies also have estimated large effects on daily mortality of crustal PM2.5–10  and filtered air in a single-blind, crossover study, exposure produced a mild pulmonary inflammation  on mortality. Results regarding the effect modification of Saharan dust days on a PM10–mortality relationship are inconsistent, despite positive associations, with negative effects and no interaction effects also reported. These inconsistent findings could reflect differences in the composition of other PM10 fractions, but also differences in correlations with other pollutants.Road dust is an important traffic-related pollutant, because road wear particles contributes substantially to local particle emissions in cities. In cities where studded tires are used, road dust may cause violations of limit values for PMor PM2.5 . This haor PM2.5  have, li2.5–10 on daily mortality in studies of U.S. cities (2.5–10. One must keep in mind that a large proportion of PM2.5 in many cities is transported over long distances and is difficult to avoid, whereas PM2.5–10, as in Stockholm, may be largely of local origin. Therefore, it also may be easier to improve health by reducing exposures to PM2.5–10.Given our results on road dust and other recent findings showing an impact of PM. cities  and dese. cities , it seem


Figures 3 and 4 are currently in reverse order. Their legends are in the correct order.Figure 3. Range of variation from year to year for outcome metrics.Each boxplot covers the values for a given outcome metric measured for each of the 25 years in a single run. We show US Model 1 here as demonstration; comparable plots for the other three country/model combinations are in the online technical supplement.doi:10.1371/journal.pone.0050522.g003Please see figure 3 at the following link:Figure 4. Sensitivity analyses.a) Changes in main partnership duration relative to base model (100%). b) Changes in main and casual UAI relative to base model (100%). c) Changes in testing frequency relative to base model (100%).doi:10.1371/journal.pone.0050522.g004Please see Figure 4 at the following link:


Hemoglobin was measured using the HemoCue technique. Descriptive and multivariate analyses were performed to determine prevalence and factors associated with anemia. The prevalence of childhood anemia was 38.8% . Mean hemoglobin was 11.2 ± 1.2 g/dL. Variables associated with child anemia were age less than 24 months , stunting , and mother's low hemoglobin level . Anemia among young children in Fond des Blancs and Villa is a public health problem. Predictors of child anemia in this region include child's age, stunting, and mother's anemia. Interventions and strategies aimed at addressing effectively anemia in this population must therefore target mothers and children under two years of age.Anemia has serious consequences on child growth, development, and survival. This study was conducted in Fond des Blancs and Villa, Haiti, to assess the prevalence of childhood anemia and its risk factors in order to inform program design. Children 6–59 months old ( Childhood anemia is a major public health problem worldwide. It is associated with serious consequences including growth retardation, impaired motor and cognitive development, and increased morbidity and mortality . Estimatn = 557) were randomly selected using a two-stage random sampling scheme in 30 of the 69 villages of the area. The list of all villages and 2009 population estimates were used. The first sampling stage represents the selection of clusters, and the second sampling stage is the selection of households within the clusters. Children 6–59 months old , height-for-age (HAZ), and weight-for-height (WHZ) for SPSS . UnderweDescriptive statistics were calculated. Means (±SD) were derived for hemoglobin, and random effects logistic regression modeling was used to determine the risk factors associated with childhood anemia. All analyses were performed using the Windows' SPSS version 20.0. The study was approved by the health department and Saint Boniface Hospital authorities. Consents were obtained from children's parents before inclusion in the study. Treatment for anemia was provided to anemic subjects free of charge.The boy : girl sex ratio was almost 1.0 (0.9). The prevalence of anemia among children was 38.8% . Mean hemoglobin concentration was 11.2 ± 1.2 g/dL. The prevalence of anemia was slightly higher among boys (42.1%) than girls (35.7%). Among non-pregnant and pregnant women, the prevalence of anemia was 23.9% and 29.2%, and mean hemoglobin levels were 12.9 ± 1.5 g/dL and 11.8 ± 1.3 g/dL, respectively. The prevalence of child wasting was 4.5% (95% CI: 3.2–6.2) with 1.1% (95% CI: 0.5–2.4) being severe; 19.8% (95% CI: 16.5–23.6) of children were stunted and 9.8% (95% CI: 7.6–12.5) were underweight.P = 0.000), stunting , and mother's low hemoglobin   .z score < −2, and mother's anemia predict childhood anemia in this population. The same factors were found in other studies [This is the first population-based study to document childhood anemia prevalence and its risk factors in the region of Fond des Blancs and Villa and one of the very few in Haiti on this subject. We found that almost 4 children out of 10 are anemic and that boys are more affected than girls. Similar results were found in another study conducted in urban Haitian children two to five years old . We also studies , 7. The The prevalence of anemia among children 6–59 months in Fond des Blancs, though high, is still lower than that in the same age group nationally (60.6%) and in the south (54.4%) and southeastern 43.8%) departments where Fond des Blancs and Villa are located [.8% deparOur findings showed that in this rural setting of Haiti, based on WHO criteria, anemia among young children is a moderate public health problem . Factors to consider for reducing the burden of this disease on children comprise child's age, stunted growth, and hemoglobin status of the mother.


Infection is one of the most frequent causes of medical admissions to the emergency department in the adult population. However not all infected patients present with fever (>38º Celsius). As early administration of antibiotic is related to improved prognosis, the aim of the study was to compare the “door-to-antibiotics” time in infected patients admitted to the medical emergency ward with- and without fever at arrival.Prospective observational cohort study of all patients admitted acutely to the medical emergency ward, Odense University Hospital (September 1st 2010 – August 31st 2011). At arrival all patients had their temperature measured . After discharge, all patient records were evaluated manually and patients with infection were identified due to the National Healthcare Safety Network criteria of infections in combination with clinical judgment where focus was clinically evident. Time of antibiotic treatment was extracted electronically from the electronic patient journal.We included all adult (≥15 years of age) patients with a first time admission of community-acquired infection within the inclusion period with a registration of antibiotics within the first 24 hours after admission (ATC: J01*).There were 8133 admissions in 6257 different patients. 1987 patients fulfilled the inclusion criteria. 1003/2003 (50.5%) presented with fever and 984 (49.5%) without fever. Median age for patients with fever was 69.9 years (range 15.1-99.3 years) and without fever 76.0 years . Patients with fever more often were male , but less often had severe comorbidity  than among patients without fever.In patients with fever, the median time to antibiotic administration was 4.1 hours (IQR 2.5 - 6.3 hours) compared with patients without fever 5.9 hours (IQR: 3.7 – 9.3 hours) p<0.0001. For patients with pulmonary focus the time to antibiotics were 4.0 vs. 5.8 hours, p<0.0001 in patients with and without fever, urinary focus 4.0 vs. 6.7 hours, p<0.0001 and abdominal focus 5.2 vs. 7.8, p=0.004.Infected patients with fever have antibiotics administered earlier than infected patients without fever. Infected patients with fever are younger and have less severe comorbidity than infected patients who present without fever.


Sir,38.5 and base excess of - 18.2 mEq/L. Serum electrolytes, electrocardiogram and chest radiograph were normal. Blood group was AB positive. Peripheral smear was positive for Heinz bodies. Urinary free haemoglobin was positive.A 29-year-old male with copper sulphate poisoning presented with severe haemolysis that made blood grouping and other laboratory parameters difficult to determine. The patient had earlier been admitted in another hospital with symptoms of nausea, vomiting, diarrhoea, melena and haematuria. Investigations there revealed haemoglobin (Hb) 8.8 g%, serum bilirubin 1.4 mg%, serum creatinine 6.8 mg%. There he was managed conservatively; as the patient’s condition continued to deteriorate he was referred to a tertiary care centre for further management. On arrival at our hospital, he was conscious but agitated with pulse rate of 132/min; blood pressure 120/86 mmHg; respiratory rate 42/min. The skin was ‘mauve lavender’ and pulse oximeter readings were 82-85%. The patient was put on mechanical ventilation. Patients Hb further fell down to 2.8 g%. Due to massive haemolysis arterial blood gas analysis (ABG), blood grouping and cross matching, renal and liver function tests could not be done as the machine did not accept the haemolysed sample. As a life saving measure, 3 U of O negative packed red blood cells were transfused. Investigations later in the day revealed Hb 4.5 g%; leucocyte count 24000 cells/cu. mm; platelet count 14200/cu mm; blood urea 92 mg%, serum creatinine 2.8 mg%; total bilirubin 8.3 mg% with 3.6 mg% conjugated; prothrombin time 18.8 s (control 13 s); partial thromboplastin time 42.9 s (control 30 s); international normalized ratio 1.9. ABG revealed pH of 7.12, HCOThe patient received two additional units of O negative packed red cells and 2 U of fresh frozen plasma. Sodium bicarbonate was administered to correct acidosis and adequate hydration was maintained. Methylene blue was infused; a total dose of 4 mg/kg was given over 6 h. Facilities for measurement of methaemoglobin levels and co-oximetry are not available at our institution.On day 2, the patient remained haemodynamically stable; there was no melena or haematuria. Weaning from mechanical ventilation was initiated on day 2 of hospital stay and the trachea was extubated on day 3. The patient was transferred to the medical ward on day 4 and was discharged home on day 12.Following copper ingestion, gastrointestinal symptoms generally develop within 15 min and may be severe enough to produce shock. Early deaths are due to shock, while hepatic and renal failure account for delayed mortality. Copper poisoning affects the erythrocytes, the liver, and the kidneys in the order named. Intravascular haemolysis appears 12-24 h following ingestion. Haemolytic anaemia is caused either by direct cell membrane damage or indirectly as a result of the inactivation of enzymes which protect against oxidative stress.1 The haemCopper poisoning is associated with high mortality compared to other metal toxicities.Treatment of copper sulphate poisoning is largely symptomatic. Though chelating agents have been used in the treatment of acute copper poisoning, there ar1