Aim of this research work was to develop mouth dissolving tablet that disintegrates rapidly in mouth by using tasteless complex of Levocetirizine and Tulsion-335. Effect of different parameters such as swelling time, resin activation, drug resin ratio as well as stirring time was optimized by taste and percentage drug loading. Formulated DRC (Drug Resin Complex) was characterized by infrared spectroscopy, thermal analysis and X-ray diffraction pattern. Tablets were formulated by wet granulation with PVP as binder, Sodium Starch Glycolate (SSG) and Crospovidone as super disintegrants. In these batches optimum hardness was achieved but disintegration time was found to be very high as ≥ 70 second, so further trials were planned by using different superdisintegrants such as Croscarmellose sodium, Sodium Starch Glycolate (SSG) as well as Crospovidone by wet granulation method. Tablets formulated with 7.5% crospovidone showed comparatively low disintegration time (25 sec), wetting time (20 sec) and friability (0.60 %) than the other batches. In present study we optimized the conditions required for maximum drug loading of Levocetirizine with Tulsion-335. Among different superdisintergants, crospovidone was found suitable with drug-resin complex to get the low disintegration time, wetting time and friability of tablets. Among all route of administration, oral route is most important and preferable route of administration for solid dosage forms . TabletsLevocetirizine hydrochloride was obtained from Yegna Manojavam Drug and Chemicals ltd. (A.P.). Croscarmellose Sodium was obtained from DMV International, Crospovidone from FMC Biopolymer and Sodium Starch Glycolate from Rama production company. Polacrilin Potassium was purchased from Corel Pharma Pvt. Ltd. and MCC from Gujarat Microwax Pvt. Ltd.Formulation of drug resin complexFormulation of DRC was done by the batch process; 100 mg of resin Tulsion-335 was placed in a beaker containing 25 mL of deionized water and allowed to swell for a definite period of time. Accurately weighed amount of Levocetirizine hydrochloride (as per 1 : 3 and 1 : 5 drug resin ratio) was added and stirred for desired period of time. The mixture was filtered and residue was washed with deionized water. Filtrate was analyzed by U.V. spectrophotometer at 231 nm for the unbound drug and percentage drug loading was calculated.Optimization of drug resin ratio and stirring timeSeparate batches of drug-resin complex were prepared by altering the ratio of drug and resin as 1 : 3 and 1 : 5. Firstly the resin was soaked into 25 mL of deionized water contained in a beaker for 30 min and then the drug was added and stirred for different time period as 30, 60, 120 and 240 min. The complexation in batch process was performed and percentage drug loading and taste were determined.Optimization of resin activationAccurately weighed resin (25 mg), was placed on a filter paper in a funnel and then it was washed with double distilled water and subsequently with 1 N HCl (100 mL) for acid activation. The resin was rewashed with water until neutral pH was reached. Similarly, alkali activation of resin was performed, by replacing 1 N HCl with 1N KOH. For Acid- alkali activation, resin was treated with 1 N HCl and 1N KOH (1 N HCl: 1N KOH = 50:50). This activated resin was used for complexation process. The drug resin ratio, swelling time and stirring time kept constant for the DRC formulation by batch process and percentage drug loading was determined. Optimization of swelling and stirring timeOptimization of swelling and stirring time was done by changing swelling time as well as stirring time. For different batches Tulsion-335 (200 mg) was soaked in 250 mL of deionized water in a beaker for 40, 60, 90 and 120 min respectively. The complexation by batch process adopted for the formation of DRC by stirring for 240 and 300 min for different batches and percentage drug loading and taste were determined.Optimization of temperature and pH on complex formation0C, 400C, 600C and 800C using temperature controlled magnetic stirring for 30 min. The volume of filtrate was made up to 50 mL with aqueous washing of DRC. The amount of bound drug was estimated spectrometrically (231 nm) from the unbound drug in filtrate. The complexation of 50 mg of drug with 300mg of resin, slurred in 25 mL of deionized water in a beaker, was performed at 250C. The drug loading efficiency was estimated.Accurately weighed 50 mg of drug powder was added to 300 mg of resin slurred in 25 mL of different pH  solution prepared from standard solution of HCl and NaOH in a 100 mL beaker and maintained at 25Characterization of DRC-1. All three spectra were completely analyzed. FTIR spectra was obtained by Jasco FTIR 6100 type A, Japan spectrometer, sample was prepared in KBr disks, and spectra was recorded over the wave number 4000- 400 cm-3 cycles / sec was used. The powder X-Ray diffraction pattern of Levocetirizine, Tulsion 335 and DRC were taken by Philips PW 1729 X–ray differectometer Legroup interconnexion, Scient Jurie, Cadlada. Radiation generated from Copper source with wavelength of 30mA at 40 kV and the range of 5x10oC/min. nitrogen were purged at 50 mL/min and 100 mL/min through cooling unit.A mettler Toledo differential scanning colorimeter 821  equipped with an in cooler and refrigerated cooling system was used to analyze the thermal behavior of Levocetirizine and DRC. Sample (5-10 mg) was heated in hermetically sealed aluminum pans at temperature 20Taste characterizationTaste evaluation was done by a panel of six volunteer using time intensity method. One tablet was in mouth for 10 sec, bitterness level was recorded, written consent was prepared volunteer as per protocol previously prepared.Formulation of MDTs1 to B3 by taking DRC equivalent to 5 mg of Levocetirizine HCl. MCC was used as diluent, PVP K 30 as dry binder, mannitol as soothing agent, talc as an antiadherent and magnesium stearate as a lubricant. All the ingredients were accurately weighed and passed through 100 # sieve and mixed with complex. The above powder blend was compressed using rotary tablet machine using 13 mm concave punches. First of all formulation of MDTs was done by direct compression technique for batch B4 to B9 was formulated with PVP K-30 while batches B10 to B21 were formulated with different superdisintegrants having different concentrations. All the ingredients were passed through 100# sieve. Binder solution was added to the powder blend. Wet mass was then passed through 18 # sieve and dried at 60.C for 30 min. the dried granules were passed through 20 # and retained on 40 # sieve. Talc and Magnesium stearate were added extra granularly to the granules (before compression process). Then, the granules were compressed using rotary tablet machine.Next trial was performed with wet granulation technique. In this process batches BCharacterization of formulated MDTsin-vitro disintegration time, wetting time, in-vitro dispersion time and by in-vitro dissolution study. Characterization was done by different parameters such as weight variation, hardness, friability, thickness, In-vitro release study from drug resin complex and MDTDrug release study from drug resin complex was carried out at pH 6.8 using USP dissolution apparatus type II. Weighed quantity of drug-resin complex equivalent to normal dose was suspended in dissolution media and quantity of drug release was determined periodically. Drug release study from formulated MDTs was carried out in 900 mL of HCl buffer pH 1.2 using USP dissolution apparatus type II. Rotation speed of paddles was fixed at 50 rpm. Sample is taken from the medium and drug content was determined by taking absorbance in Shimadzu U.V. spectrophotometer (UV-1601).1 to F6. As in batches F1 and F2 drug resin ratio kept constant at 1:3 and stirring time varied from 30 min to 60 min in batch F1 to batch F2, taste was found to be slightly masked but percentage drug loading increases from 30.15% ± 1.04% for 60 min. But in batches F3 to F6 drug : resin ratio increases to 1:5, swelling time remain constant at 30 min and stirring time varied for 30, 60, 120 and 240 min respectively and percentage drug loading was found to be 49.97% ± 1.16%, 50.41 ± 1%, 52.17% ± 0.41%and 70.32% ± 1.2% wt/wt. respectively and taste was masked. Highest taste masking and percentage drug loading was achieved with drug resin ratio 1:5 and when stirring time was maximum upto 240 min.Optimization of swelling and drug: resin ratio was done by taking inactivated resin in batches FThe percentage drug loading was also determined with acid treated resin, alkali treated and resin treated with both acid and alkali was found to be 61.40% ± 1.32%, 45.80% ± 0.32% and 28.69% ± 0.62%. wt/wt respectively. Highest percentage drug loading was found for acid activated resin but as compared to inactivated resin no major effect was found on percentage drug loading. Thus further trials were made with inactivated resin.- groups of resin and compete with drug for binding.Complexation process in between drug and resin depends on pKa value of both, involves exchange of ions. Results indicate increase in pH from 2 to 5 also increases percentage drug loading. A maximum drug loading 97.69% wt/wt was found at pH 5. As pH increases above 5, the percentage drug loading decreases. This was due to that pH of solution affects both solubility and degree of ionization of drug and resin. Drug loading is low at lower pH due to -COOPercentage drug loading was determined at temperature 27°C, 40°C, 60°C and 80°C. Result indicate 97.52%, 96.81%, 95.49% and 93.43% drug loading at 27, 40, 60 and 80°C respectively.10 to F14, here drug: resin ratio kept constant for 1:5, swelling time and stirring time was changed from batch F10 to F14. In batch F11, complete taste masking was achieved as well as percentage drug loading was found to be 97.33% when swelling time kept as 60 min and stirring time kept 240 min.Effect of swelling time and stirring time was optimized in batches FInfrared spectra of Tulsion-335, Levocetirizine and DRC were shown in Several sharp peaks in XRD spectra of pure drug represent the crystalline nature of drug while a diffused peak in XRD spectra of Tulsion-335 represents amorphous nature of resin. But on the other hand XRD pattern of DRC shows disappearance of characteristic peaks of drug and also found to be broadened, these finding suggest the formation of drug resin complex.By DSC curves, the thermal behavior of Pure Drug Levocetirizine HCl shows peak endotherm at 213.55°C corresponding to loss of water of crystallization and melting of pure drug. Thermal behavior of Resin (Tulsion 335) shows peak endotherm at 152.28°C while thermal behavior of Drug Resin Complex shows peak endotherm at 70.29°C the reduction of height and sharpness of endotherm is due to loading of drug in resin. That shown, there was no interaction was observed between drug and complex. It concludes that, resin was not affecting the characteristic of drug due to complexation process and indicates the amorphous nature of DRC.In-vitro drug release studyIn-vitro release of drug from drug-resin complex at pH 6.8 was found to be less than 4.8% within 1 minute, indicating the presence of exchangeable ions of ionizable electrolytes in dissolution fluid of pH 6.8. Hence DRC is stable at salivary pH and poor released amount of drug from DRC do not impart bitter taste at the time of administration and while pass to GI tract from mouth. Further drug release profile of optimized formulation (B-20), plain tablet (do not have crospovidone) and marketed brand (Vozet 5 mg tablet) were analyzed in HCl buffer pH 1.2.10 to B21) showed all parameters within limit as well as good physicochemical properties. Drug release rate of formulated MDTs was also found to higher as compared to conventional tablet. Crospovidone 7.5% Batch B20 having hardness (3.0 kg/cm2), friability (0.60%), wetting time (20 sec) and assay 99.37%, hence tablets formulated with crospovidone not only increases rate of dispersion but also increases rate of drug release.In present study we optimized the conditions required for maximum drug loading of Levocetirizine with Tulsion-335. All the optimized tablet (MDT) formulations of Levocetirizine (B


Reduced ejection fraction (EF) has previously been shown to be a risk factor for adverse outcomes in patients undergoing percutaneous coronary intervention (PCI). However, with the advent of stents, procedural complications and restenosis rates have reduced dramatically. The aim of this study was to assess the association between left ventricular (LV ) ejection fraction and in-hospital and longterm outcomes using a prospective registry.n = 293), group 2: EF = 41−49% (n = 268) and group 3: EF ≥ 50% (n = 1 469). The frequency of in-hospital and follow-up outcomes between groups was compared using appropriate statistical methods.After exclusion of patients with acute myocardial infarction (MI) and those with missing data on left ventricular ejection fraction, 2 030 patients undergoing PCI between March 2002 and 2004 remained in our prospective registry. Patients were divided into three categories: group 1: EF ≤ 40% (p = 0.16); and 91.1, 90.3 and 92.9%, (p = 0.09), respectively. The respective cumulative major adverse cardiac events (MACE) and cardiac death rates at follow-up were 5.8, 2.2 and 3.3% (p = 0.04) and 2, 0.4 and 0.3% (p = 0.02), respectively. The hazards ratio (95% CI) for MACE and cardiac death in the lowest versus highest EF groups were 2.07 (1.03−4.16) and 5.49 (1.29−23.3).Stents were used for over 85% of the patients in each group. The mean EF ± SD in the lowest to highest EF groups was 35.8 ± 5.4%, 45.5 ± 1.6% and 57 ± 5.7%, respectively. The angiographic and procedural success rates were 91.8, 92.1 and 94.1%, (Patients with significant left ventricular dysfunction had higher long-term major adverse cardiac events and cardiac death rates. Even the use of newer techniques such as stenting did not compensate for this. Although mortality from coronary artery disease is declining, the incidence of associated heart failure is rising.13It is unclear whether coronary artery bypass grafting (CABG) provides a more appropriate choice for revascularisation than does PCI in patients with LV dysfunction. Previous studies have shown that patients with left ventricular ejection fraction (LVEF) ≤ 25% showed improved results with surgery.15Recent technical advances in the field of interventional cardiology and an increase in operator experience have resulted in substantial increases in the complexity and volume of the procedures, and improvement of acute and long-term success rates. With the advent of stents, outcomes in terms of periprocedural complications and restenosis have been much better than previously reported for PTCA alone.17In the present study, we aimed to assess the association between LVEF and in-hospital and long-term outcomes in the current catheter-based practice, using a prospective registry of 2 591 patients who underwent PCI between March 2002 and 2004. Also, as a subgroup study, we analysed these outcomes in a group of patients who had been treated with only stenting techniques.All patients treated with PCI at our centre between March 2002 and 2004 were entered into a prospective registry. The first wave of this registry consisted of 2 591 patients. We excluded 73 patients in whom the primary reason for revascularisation was acute myocardial infarction within the preceding 48 hours. Quantitative data on LV function were available for 2 030 (80.6%) of the patients, using contrast ventriculography, LV angiography and echocardiography. These patients were divided into three groups according to LVEF: group 1: EF ≤ 40%; group 2: EF 5 41−49%; and group 3: EF ≥ 50%.Baseline demographic and clinical details, angiographic and procedural characteristics, and in-hospital outcomes were obtained by research physicians. Follow-up outcomes were obtained by cardiologists in clinics at one and six months, and once a year thereafter, or by formal telephone interviews in patients who had given consent to participate in follow-up programmes after discharge from hospital. Computer operators entered all the data into a computerised database. Consent to collect post-discharge follow-up data was not obtained for 214 (10.5%) of the patients who survived hospitalisation. Of these, 33 (11.3%) were in EF group 1, 33 (12.3 %) were in EF group 2, and 148 (10.1%) were in EF group 3.Patients received 325 mg of aspirin before the procedure, and it was continued indefinitely. Coronary stenting was performed by standard methods. After stent placement, ticlopidine (250 mg twice daily) or clopidogrel (75 mg once daily) was given routinely for four weeks to patients with bare metal stents, and six to 12 months with drug-eluting stents. Angiographic findings such as vessel dimensions, pre- and post-procedural stenoses, lesion length and thrombolysis in myocardial infarction (TIMI) flow grade were determined by visual estimation using the guiding catheter as a reference object for calibration. The angiographic characteristics were also further analysed by an independent interventional cardiologist not involved in the procedure.Angina symptoms were defined according to the classification of the Canadian Cardiovascular Society.20Angiographic success was defined as residual stenosis below 20% for stenting and below 50% for balloon angioplasty plus normal TIMI 3 flow. Procedural success was defined as angiographic success without major complications  during hospitalisation. Major adverse cardiac events (MACE) were defined as the presence of cardiac death, non-fatal MI, or target vessel revascularisation (TVR) during the follow-up period. TVR was defined as ischaemia-driven repeat percutaneous intervention or bypass surgery of the target vessel. Target lesion revascularisation (TLR) was defined as ischaemia-driven repeat percutaneous intervention of the target lesion or bypass surgery of the target vessel.21This study was approved by the Tehran Heart Centre Ethics Committee. Informed consent was obtained from all patients before enrolment into this study.The trends for differences in baseline characteristics and inhospital outcomes across the groups were compared by the Mantel-Haenszel and Kendall’s tau rank correlation tests. Multivariate Cox proportional hazards models were developed for comparison of the rates of MACE and its constructing components  in followup, identification of variables associated with each outcome, and calculation of adjusted risk ratios. Event-free survival curves were drawn using the Kaplan-Meier method. The log rank was used to test for differences between survivals.A multivariate logistic regression model was constructed to compare the in-hospital outcomes, to determine the independent predictors, and to calculate the adjusted risk ratios. The risk ratios and 95% confidence intervals for events were calculated for each of the first EF groups (EF ≤ 40% and EF 5 41−49%) compared to the third group (EF ≥ 50%). A 0.15 level of significance for baseline and procedural characteristics in univariate analysis was considered suitable as a cut-off point for entering into multivariate models. We also analysed EF as a continuous variable. Univariate analyses were performed with SPSS software version 13. Multivariate analyses were conducted with SAS software version 9.1.p < 0.001) and to have a history of MI (p < 0.001). Patients in the highest EF value were less likely to be smokers (p = 0.03) and more likely to have a history of hypertension (p < 0.001).Among the 293 patients (14.4%) with an EF ≤ 40% (group 1), the mean EF was 35.8 ± 5.4% . Two hundred and sixty-eight patents (13.2%) had an EF between 41 and 49%, with a mean of 45.5 ± 1.6%. In the remaining 1 469 patients (72.4%) with EF ≥ 50% (group 3), mean EF was 57 ± 5.7% . The three groups had significant differences in baseline characteristics p < 0.001). Total occlusions in one or more coronary arteries were more frequent in the two lowest EF groups compared to the highest one . Moreover, the lower the EF value, the more likely lesions were to be longer. Stents were used for over 85% of patients in each group and the most frequently used technique was stenting after predilation. Drug-eluting stents were less frequently used for patients with the lowest EF (p = 0.03). However, there was no significant difference across the groups in lengths and diameters of stents  p < 0.001, and OR 5 0.4, 95% CI 5 0.23−0.68, p < 0.001, respectively). The same factors plus diffuse lesions were independent predictors of procedural success rate .The Mantel Haenszel test showed no statistically significant trend across the three groups for the angiographic and procedural success rates (p = 0.006). No patient underwent emergent CABG in the hospital. The rates of Q- and non-Q-wave MI were also similar for all groups Cardiac death rate was higher in patients who had EF between 41 and 49% (1.1%). The corresponding values were 0.3% in group 1 and 0.1% in group 3 (p = 0.004). No such trend was present for cumulative MACE  or for TVR, TLR and CABG. Table 4 shows the Kaplan Meier estimates for adverse clinical outcomes in follow-up.Finally, multivariate Cox regression models were used to determine the independent predictors of long-term outcomes including cardiac death and MACE. After adjusting for potential confounding factors with p-values ≤ 0.15, patients in the lowest versus highest EF group were at increased risk of cardiac death and cumulative MACE Use of drug-eluting stents tended to be associated with a lower risk of MACE . PCI in the left anterior descending artery territory increased the probability for MACE  and TVR . By analysing EF as a continuous variable, a lower EF was shown to be associated with a higher risk of MACE and cardiac death at long-term followup .p = 0.02).In the subgroup study of patients treated with stents, patients with the lowest versus highest EF category had significantly higher rates of MACE and cardiac death . Survival rates were 94.1, 97.4 and 96.1% in the lowest, intermediate and highest EF groups, respectively (The finding that emerged from this study is that patients with significant LV dysfunction (EF ≤ 40%) had increased rates of cardiac death and cumulative MACE in long-term follow-up compared to those with EF ≥ 50%.Previous studies have shown that outcomes of PCI in patients with LV dysfunction were poorer compared to those who had normal EF.In our study, however, there was no statistically significant trend across the three groups for in-hospital cardiac death and MI, nor were there any trends across the groups for acute success rates. It seems that with the advent of stents, the in-hospital outcomes have improved dramatically. For example, while in the early 1990s, the in-hospital cardiac mortality rate in patients with severe LV dysfunction has been reported to be as high as 7%;The other aim of our study was to compare the rate of MACE in these three groups. In multivariate analysis, the rate of both cardiac death and MACE differed between groups 1 and 3. Similar to our results, the authors of another study have shown that the single and composite endpoints of death/MI and CABG were significantly higher in the lowest versus highest EF groups. In this study, the death rate in the lowest EF group was 11% and the actuarial survival rates were 79.1 to 88.2% in the lowest to highest EF groups.11Prior to these studies, the rate of one-year death was reported between 13 and 21%.p = 0.02). The EF category 1 versus 2 was again the independent predictor for MACE and cardiac death. The rates of cardiac death and MACE in this population were 2.3 and 6%, respectively.The outcomes of stenting in patients with LV dysfunction have not been widely reported. To assess these effects in relation to the use of stents, we further conducted a study on a subgroup of patients treated with only stents. This time, the analysis showed that even MACE-free survival rates were significantly lower in patients treated with stents (94.1 vs 97.4% and 96.1%, The authors of two studies reported cardiac death rates of 6 and 12%Ultimately, while the study has shown fewer target-vessel revascularisation and cardiac event rates with CABG compared to PCI in long-term follow-up,Patients with lower EF have similar in-hospital outcomes and higher long-term major adverse cardiac events and cardiac death rates after percutaneous coronary intervention. Even the use of newer techniques such as stenting may not compensate for this. In fact, the use of new stenting strategies has reduced the overall rate of these outcomes in the total population.One of the limitations of this study was that 10.5% of patients were missed in follow-up. Although our study provided long-term evaluation of outcomes in patients, controlled analysis of these patients for a longer duration is required.


To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components.Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months.Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures . With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes . With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal—temporal width by 13% . In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT . With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% .The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection. MYOC), optineurin (OPTN), and WD repeat domain 36 (WDR36) =11.86 + 0.12* ). Likewise, WT littermate B6 animals had mean IOP=13.0±3.6 mmHg, which rose similarly and significantly with age . There was no difference in IOP between Aca23 and WT mice . Baseline IOP measurements for animals in the bead injection experiment showed no significant differences between genotypes or treatment groups (Aca23 homozygotes had mean IOP=13.0±3.9 mmHg (n=122 mice from 4.0 to 20.6 months of age). In a regression model, IOP was significantly higher in older animals . In a similar model, nasal—temporal eye width was significantly larger in older mice, in Aca23 mice, after glaucoma, and after inflation testing and perfusion fixation . Likewise, superior—inferior width was also significantly longer by age, in Aca23 mice, and after glaucoma , but was not related to whether inflation testing had been performed nor to the fixation method employed (both 0.4).Puk et al.  demonstrt-test). From 4 months to 17 months of age, Aca23 eyes became 9.7% longer and 3.9% wider . WT mice grew 5.7% longer and 8.6% wider during the same age span. Both length and width remained significantly larger in older Aca23 mice than WT mice .The Aca23 mice at 4 months of age were 12.2% longer and 7.3% wider than WT, and at an average of 17 months of age they were 16.4% longer and 2.6% wider than the wild type . In the analysis, use of either one eye per mouse or both eyes when both were tested did not alter the conclusions as presented.In the more posterior (R2–3) scleral region, strain rose significantly in both circumferential and meridional directions with increasing IOP . Aca23 animals had less strain than younger WT animals at all of the pressure steps analyzed from 10 to 30 mmHg in both circumferential and meridional directions , but there were no significant differences among the 3 groups in stress resultant values .In the more anterior (R4) scleral region, strain rose significantly circumferentially and meridionally with increasing IOP , but did not differ significantly between Aca23 and older WT, Aca23 and younger WT, nor between younger and older WT.Chronic glaucoma was induced and its effects measured in 37 Aca23 and 30 WT mice beginning at 3.5 to 4.4 months of age, for a period of 6 weeks from injection of beads to sacrifice. Aca23 control eyes were significantly longer than they were wide . In 8 older Aca23 mice, aged 16–18 months, axon number was 51,285±16,106 .After 6 weeks of glaucoma, the Aca23 mice lost 0.57±17% of axons, while WT lost 21±31% (median loss: 1% (Aca23) versus 10% (WT), n=37, 29, respectively; t-test). The median decrease in RGC cell bodies labeled by β-tubulin was 17% in Aca23 eyes and 50% in WT retina . The corresponding median axon loss for the eyes whose retinas were analyzed was 17% in Aca23 and 41% in WT nerves, showing excellent agreement between the RGC cell body loss and axon loss .Stiffening of the ONH and sclera has been reported in animal glaucoma models and in living and post-mortem human glaucoma eyes. Zeimer et al.  reportedThe decreased susceptibility to glaucoma damage conferred by the Aca23 mutation could be related to features other than those studied as yet. In development, it is hypothesized that collagen 8α2 is a factor in capillary development. Potentially, the mutation could lead to altered nutritional support to RGC and their axons in the retina and ONH that protects these neurons from damage. The expression of collagen 8α2 in astrocytes of the optic nerve has been suggested ,37. ThisClearly, the Aca23 mouse exhibits dramatic structural changes in ocular connective tissues. The larger eye and thinner cornea are not an apparent result of exposure to a different IOP level, as these eyes are insignificantly different from control in this parameter. Nor is the phenotype simply a difference in the cornea alone, as the widths of the globe at the equator are significantly different from controls. There are no detectable differences in the number of RGC at baseline, either judged by axon counts or retinal anatomy. This mouse is only one of several strains that we have identified, including those with genetic alterations in elastin and fibromodulin, that lead to larger than normal eyes. It will be interesting to compare how these various models respond to chronic experimental glaucoma.We have confirmed past studies of age effects in mice  regardinWe recognize that several aspects of the present research deserve further study. The inflation studies conducted here did not take into account the local scleral thickness. We have recently developed methods to measure this for incorporation into a more sophisticated, constitutive model of inflation behavior. There may be differences in susceptibility to experimental glaucoma by age. Further studies of Aca23 mice at different ages could be informative.In summary, a mutation induced in the collagen 8α2 gene produced a larger eye and altered biomechanical behavior on inflation testing. These mice were significantly less susceptible to loss of RGC in an experimental glaucoma model. Study of the role of ocular connective tissues in this and other genetically-altered mouse models could lead to insights into pathogenesis and new therapeutic directions.


Diagnosis of pulmonary tuberculosis (TB) among human immunodeficiency virus (HIV) patients remains complex and demands easy to perform and accurate tests. Xpert®MTB/RIF (MTB/RIF) is a molecular TB diagnostic test which is rapid and convenient; the test requires minimal human resources and reports results within two hours. The majority of performance studies of MTB/RIF have been performed in high HIV burden settings, thus TB diagnostic studies among HIV patients in low HIV prevalence settings such as Peru are still needed.3 and 45 (34.4%) had TB. For TB detection among HIV patients, sensitivity of MTB/RIF was 97.8% (95% CI 88.4–99.6) (44/45); specificity was 97.7% (95% CI 91.9–99.4) (84/86); the positive predictive value was 95.7% (95% CI 85.5–98.8) (44/46); and the negative predictive value, 98.8% (95% CI 93.6–99.8) (84/85). MTB/RIF detected 13/14 smear-negative TB cases, outperforming smear microscopy . For rifampicin resistance detection, sensitivity of MTB/RIF was 100% (95% CI 61.0–100.0) (6/6); specificity was 91.0% (95% CI 76.4–96.9) (30/33); the positive predictive value was 66.7% (95% CI 35.4–87.9) (6/9); and the negative predictive value was 100% (95% CI 88.7 –100.0) (30/30).From April 2010 to May 2011, HIV-positive patients with high clinical suspicion of TB were enrolled from two tertiary hospitals in Lima, Peru. Detection of TB by MTB/RIF was compared to a composite reference standard Löwenstein-Jensen (LJ) and liquid culture. Detection of rifampicin resistance was compared to the LJ proportion method. We included 131 patients, the median CD4 cell count was 154.5 cells/mmIn HIV patients in our population with a high clinical suspicion of TB, MTB/RIF performed well for TB diagnosis and outperformed smear microscopy. Tuberculosis (TB) is the leading cause of death in human immunodeficiency virus (HIV) infected patients Achieving accurate diagnosis of TB disease is more complex in HIV patients than in subjects with normal immunity Mycobacterium tuberculosis (Mtb).Smear microscopy is the cornerstone of TB diagnosis and case detection in the vast majority of TB control programs Culture does allow for drug resistance testing; however the test requires proper laboratory infrastructure and trained personnel and the time required for culture growth is long B gen. This test works with the GeneXpert® System device  that fully automates a real-time polymerase chain reaction (rt-PCR) and provides results within two hours. It has minimal biosafety requirements and reduced technical manipulation Xpert®MTB/RIF  is a semi-quantitative molecular test for simultaneous detection of TB and rifampicin resistance through detection of the rpoTo date, most studies have evaluated the performance of Xpert®MTB/RIF test (from now on referred as MTB/RIF) in pulmonary and extrapulmonary specimens mostly in HIV-endemic countries in Africa where up to 80% of TB patients are HIV co-infected We conducted a cross-sectional study to evaluate the diagnostic test accuracy of MTB/RIF in identifying pulmonary TB disease in HIV patients in two tertiary hospitals: Hospital Nacional Hipólito Unánue (HNHU) in Eastern Lima and Instituto de Medicina Tropical Alexander von Humboldt (IMTAvH) in Northern Lima. In 2010, the incidence for all TB cases in Peru was 110 per 100,000 population and 2.6% were co-infected with HIV We included patients 18 years of age or older with an HIV diagnosis confirmed by Western Blot, a high clinical suspicion of TB and who had not received more than two doses of TB treatment. A high clinical suspicion of TB was defined as cough for ten or more days with concurrent abnormal chest x-ray  and at least one of the following symptoms: fever, fatigue, night sweats, hemoptysis, chest pain or weight loss. We included those who agreed to participate and completed the written informed consent. Patients who did not provide a second sputum sample with the required volume were subsequently excluded.Trained study health personnel interviewed and enrolled study patients using a structured questionnaire for demographic, clinical and epidemiological data. Interviews were conducted prior to obtaining the first sputum sample. Clinical records were reviewed in case of discrepancies between the reference standard and the MTB/RIF.The microbiology laboratory at IMTAvH conducted diagnostic tests requested by the Peruvian National TB Program  and Mycobacteria Growth Indicator Tube (MGIT). Routine tests done on the first sputum sample, usually an on-the-spot sample, were not included in the primary analysis. The following day, the second sample, usually a morning sample, was collected and used to perform direct MTB/RIF and to repeat all the tests done to the first sample. Sputum samples that could not be processed on the same day were stored at 4°C and processed the following morning or on Monday if it was collected on a Saturday.The study staff transported all the samples by car on a daily basis at 4°C from HNHU to the microbiology laboratory at IMTAvH (distance of ∼20 minutes). All tests were performed according to standard protocols and established guidelines Three trained laboratory technicians performed routine tests and the MTB/RIF test was performed by a single technician with experience in handling the GeneXpert® System. All technicians remained blinded to results of the tests they did not perform.Due to test manufacturer modifications to MTB/RIF software during study performance, we worked with two different MTB/RIF software versions: 2.1 and 4.0. The new version (4.0) had higher cutoff values for rifampicin detection and did not include changes for Mtb detection A head-to-head per-sample analysis of MTB/RIF was the primary analysis for Mtb detection: the second sputum sample was examined using the MTB/RIF as the index test and the reference standard was a composite culture (LJ/MGIT) of the second sputum sample. The reference standard was positive if there was Mtb growth in at least one slope of LJ or in a MGIT culture tube, and negative if the results of both cultures - LJ and MGIT- were negative. In addition, the reference test was considered contaminated if both LJ and MGIT were contaminated. Contaminated reference standard tests or a MTB/RIF result reported as invalid were excluded from the analysis. We compared the performance of MTB/RIF with that of sputum smear microscopy. Finally, considering the LJ PM as the reference standard, we assessed the performance of MTB/RIF for the detection of rifampicin resistance and MDR-TB. Accordingly, a rifampicin resistance case was defined as rifampicin resistance detected by the LJ PM and rifampicin sensitive case was defined if the results of the LJ PM showed patterns of full drug sensitivity or drug resistance excluding rifampicin.As a secondary analysis we conducted a head-to-head per-patient assessment for Mtb detection. A subject with a positive reference standard test in at least one of the two sputum samples was considered a PTB case, and one with a negative reference standard test in both sputum samples was not considered a PTB case. Unless we refer to a “PTB case”, all other mentions to culture-positive patients or patients with tuberculosis refers to the primary analysis, thus to the second sputum sample.2.p(1-p)/e2Study sample size was calculated using sample size formula for estimating a proportion with a normal approximation n = zAll data from questionnaires and laboratory results was entered into Microsoft Access database . Sensitivity, specificity, likelihood ratios (LLR), kappa coefficient and predictive values of the tests were calculated using 2×2 tables and OpenEpi v 2.3.1 The study protocol was approved by the Institutional Ethics Committee at Universidad Peruana Cayetano Heredia and by the Institutional Ethics Committee at HNHU. Written informed consent was received from all participants and all data was processed anonymously. MTB/RIF results were not used for treatment management; only routine tests results were given to the treating physicians. TB cases were then referred to the National TB Program center at each site where free treatment under directly observed treatment short course (DOTS) was provided.3 (IQR 51.3–341.5). A prior TB episode was reported by 25% of patients and 32% were receiving highly active antiretroviral therapy (HAART) at enrollment. Other demographic data is listed in From April 2010 to May 2011, 158 patients were screened in the two sites, of which 136 were eligible for the study and 131 were included in the analysis, as shown in Out of the 131 patients included, 45 (34.4%) had TB and among these 14 (31.1%) were smear negative. The proportion of TB per site was 45.1% (95% CI 31.4–58.6) at HNHU and 27.5% (95% CI 18.6 –38.0) at IMTAvH.Overall, MTB/RIF sensitivity for detection of Mtb was 97.8% (95% CI 88.4–99.6) (44/45); the specificity, 97.7% (95% CI 91.9–99.4) (84/86); the positive predictive value, 95.7% (95% CI 85.5–98.8) (44/46); and the negative predictive value, 98.8% (95% CI 93.6–99.8) (84/85). The positive likelihood ratio (+LLR) of MTB/RIF was 42.0 (95% CI 15.8–112.1), and the negative LLR 0.0 (95% CI 0.0–0.2). The kappa coefficient value was 0.9 (95% CI 0.8–1.1).MTB/RIF outperformed smear microscopy  and detected 13 out of 14 (92.2%) smear negative, culture-positive versus 31 out of 31 (100%) smear-positive, culture-positive patients. Six MTB/RIF cartridges with improved rifampicin resistance software (v 4.0) were used but they were all Mtb negative for both reference standard and MTB/RIF tests. Rifampicin resistance was assessed in 39 (86.7%) out of 45 patients with TB. In five cases the results of the LJ PM were not available .Rifampicin resistance was found in six out of 39 patients, all of these were also detected by MTB/RIF: five had MDR-TB and one was sensitive to isoniazid. Furthermore, MTB/RIF detected rifampicin resistance in three additional patients that were not detected by the reference standard. Overall, MTB/RIF sensitivity for rifampicin detection was 100% (95% CI 61.0–100.0) (6/6); the specificity was 91.0% (95% CI 76.4–96.9) (30/33); the positive predictive value was 66.7% (95% CI 35.4–87.9) (6/9); and the negative predictive value 100% (95% CI 88.7 –100.0) (30/30). The +LLR was 11 (95% CI 5.7–21.1), and the kappa coefficient value was 0.8 (95% CI 0.5–1.1).In terms of Mtb detection by MTB/RIF, three (2.3%) patients had discordant results with the reference standard. A false negative MTB/RIF test was observed in a patient with a negative smear. The patient showed a positive response to treatment  and was reported as cured. Two patients had false positive MTB/RIF tests, one of them was also smear-positive, responded well to treatment and was reported as cured; the other did not start treatment and died one month after study inclusion with no defined cause of death.Finally, there were two cases with positive smears and negative results in the reference standard and MTB/RIF tests. Both of them completed TB treatment and were reported as cured.Three patients were MTB/RIF rifampicin resistant and LJ PM sensitive; all three started TB treatment for sensitive cases . Two of them finished treatment and were reported to be cured and the other one was lost to follow-up.3 had a sensitivity of 100% (95%CI 83.9–100.0) (20/20) while patients with CD4 counts above 200 cells/mm3 had a sensitivity of 95.5% (95%IC 78.2–99.1) (21/22); p = 0.5. Patients with CD4 counts below 200 cells/mm3 had a specificity of 96.1% (95%CI 86.8–98.9) (49/51), while patients with CD4 counts above 200 cells/mm3 had a specificity of 100% (95%IC 89.6–100.0) (33/33); p = 0.4.Performance of MTB/RIF by immunological status of the patients was evaluated for 96.2% (126/131) patients with available CD4 count at study inclusion. MTB/RIF performance was not affected by immunological status. Patients with CD4 counts below 200 cells/mmOne patient had a contaminated reference standard test  with a negative MTB/RIF result. Two patients  had invalid results with MTB/RIF (which translates into the sample not properly processed or rt-PCR inhibited). As sufficient sample remained, MTB/RIF test was repeated in these two patients, without detection of Mtb in concordance with the results of their reference standard.We assessed performance of the index test for Mtb detection, done only on the second sputum sample, with a reference standard defined as any positive result in either the first or the second sputum sample to have a per-patient analysis.MTB/RIF sensitivity for detection of Mtb was 86.3% (95% CI 74.3–93.2) (44/51); the specificity, 97.5% (95% CI 91.3–99.3) (78/80); the positive predictive value, 95.7% (95% CI 85.5–98.8) (44/46); and the negative predictive value, 91.8% (95% CI 84.0–96.0) (78/85). The +LLR of MTB/RIF was 34.6 (95% CI 12.9–92.6), and the negative LLR was 0.1 (95% CI 0.1–0.2). The kappa coefficient value was 0.9 (95% CI 0.7–1.0).A comparison of the performance of MTB/RIF for Mtb detection between per-sample and per-patient analysis is described in We report that MTB/RIF had a high specificity (97.7%) in detecting Mtb, confirming the findings of other studies A lower sensitivity (73%) of MTB/RIF was found among patients with or without TB symptoms in an antiretroviral therapy clinic in South Africa Three previous studies - two in South Africa and one in Tanzania- reported sensitivities of 70%, 84% and 88% respectively, while in our setting the sensitivity to detect TB was 98% Despite all the advances of HAART scale up worldwide, much of the preventable burden of TB related mortality is concentrated in populations with advanced immunosuppression and without HAART, as that of this study. Only 32% of HIV patients had initiated HAART at the time of enrolment in our study.When we analyzed the performance of MTB/RIF compared to a reference standard including results from both sputum samples, the sensitivity was considerably reduced, yet this could be related to the fact that MTB/RIF was only done in one sample.The performance of MTB/RIF to detect rifampicin resistance and thus its contribution for MDR-TB detection were not equally convincing. The index test did detect all the rifampicin resistant cases but also reported three false positives. Previous studies have addressed this issue Our study has some limitations. WHO new guidelines recommend that TB should be suspected in any HIV-positive individual with any of the following symptoms: cough, weight loss or fever. Our study was designed before these guidelines were set, and it aimed to evaluate performance of MTB/RIF in a group of HIV-positive individuals with at least two of these symptoms, thus a more selected population. We decided to study a selected population of HIV-positive patients to narrow the risk of tuberculosis to a higher one. MTB/RIF performance could decrease among a less selected population of HIV-positive individuals as compared to our results. Currently, MTB/RIF is still costly and targeting its use in patients with the highest risk of TB could be a strategy for resource-limited settings.Due to resource limitations, we only evaluated MTB/RIF performance on a single sputum sample; we could not genotype the strains of three cases with false positive rifampicin resistance results. However this reflects the commonly available resources in settings with high prevalence of TB. Also, the three false positive tests were performed with MTB/RIF software v 2.1 and not with the improved software v 4.0. Nonetheless, false positive rifampicin results have been previously reported with the latest version In our study, MTB/RIF showed an excellent performance in detecting TB among patients with advanced immunosuppression and a high clinical suspicion of TB. A positive MTB/RIF result was almost 40 times more likely to occur in a subject with TB than in a subject without TB, and a negative MTB/RIF was also much more commonly seen in patients without TB.We conclude that MTB/RIF can be an important diagnostic tool for TB disease amongst HIV-positive patients, particularly in patients with a high pre-test probability of TB. Many studies of new rapid TB diagnostic tests have been conducted in Africa where high HIV rates place a different perspective on TB programs and health systems. Further evaluation of MTB/RIF in Latin America is needed. Operational research should evaluate the yield of scaling up diagnostic algorithms of such strategies in order to evaluate the cost-effectiveness of rapid treatment initiation, improvement of individual prognosis and reduced disease transmission, within well established tuberculosis programs in TB endemic settings


Manihot esculenta Crantz) can produce cyanide, a toxic compound, without self-injury. That ability was called the cyanogenic potential (CN). This project aimed to identify quantitative trait loci (QTL) associated with the CN in an outbred population derived from 'Hanatee' × 'Huay Bong 60', two contrasting cultivars. CN was evaluated in 2008 and in 2009 at Rayong province, and in 2009 at Lop Buri province, Thailand. CN was measured using a picrate paper kit. QTL analysis affecting CN was performed with 303 SSR markers.Cassava  of 26%.The phenotypic values showed continuous variation with transgressive segregation events with more (115 ppm) and less CN (15 ppm) than either parent ('Hanatee' had 33 ppm and 'Huay Bong 60' had 95 ppm). The linkage map consisted of 303 SSR markers, on 27 linkage groups with a map that encompassed 1,328 cM. The average marker interval was 5.8 cM. Five QTL underlying CN were detected. Five new QTL affecting CN were successfully identified from 4 linkage groups. Discovery of these QTL can provide useful markers to assist in cassava breeding and studying genes affecting the trait. Manihot esculenta Crantz) has been cultivated as an important food source and industrial feedstock since agriculture was developed. Accordingly, it ranked fourth among all crops in worldwide production. Thailand was the world's leading exporter [Throughout the world, cassava  , 425 priThe linkage map Figures , 4 and 5The cassava genome database contained a draft sequence in late 2010 , in whicCN09R1 was located on linkage group 10 associated with marker locus CA141. In addition, CN09R1 also explained the largest portion of variation at 26.Based on multiple-QTL models (MQM) analysis, only 5 QTL were detected across 4 linkage groups, 1-2 from each environment Table . Each QTThe LOD scores of some QTL were lower than genome wide significant threshold as expected for sparse maps (maps with marker density > 1 cM) ,23. HoweCN08R1, all identified QTL showed significance by KW analyses  analysis was used for single marker analysis to avoid type 2 errors. Except for s Figure . Even th1 errors ,24. No cCN09L1. However, SSRY242 was 34.4 cM far from the position of CN09L1. It should be kept in mind that QTL analysis is based on statistical analysis and there is a strong environmental effect on CN [In a previous study by Kizito et al. (2007)  two QTL CN08R1, CN09R1, CN09R2, CN09L1 and CN09L2, respectively were identified  unit from a sugar nucleotide derivative to a sugar or amino acid acceptor (EC2.4.-) . CyanogeThere is no report to link Nbp35 and Adenosine/Guanosine diphosphatase directly to the cyanogenesis pathway, however these two enzymes were involved in common biogenesis. In addition, sequences of linamarase (EC 3.2.1.21) and hydroxynitrile lyase (4.1.2.11) which are key enzymes involved in the cyanogenesis pathway were found on scaffold09743 and scaffold01206, respectively. However, these scaffolds were not anchored by any of the markers in this study linkage map. Thus, it would be useful to identify additional markers to link these enzymes to the map.1 population of a cross between 'Hanatee' and 'Huay Bong 60'. The map was used for analysis of QTL underlying CN, and five potential QTL were detected. Among all the QTL, CN09R1, which was located on linkage group 10 was the strongest with the LOD value of 5.75 and it explained 26.0% of the variation in CN. In addition, all loci on the genetic map were compared with the data from cassava genome sequence. The anchor markers common to both could help the organizing and completion of chromosome sized scaffolds. The loci found in this study will be useful for identification of genes controlling the traits as well as establishing MAS of cassava in the future.In this study, an SSR based genetic linkage map of cassava was constructed using an FCassava variety 'Hanatee' , exhibited low CN and 'Huay Bong 60'  , displayAll samples were planted in May during years 2007-2008. In 2007, the population and its parents were separately cultivated at Rayong province, Thailand. Ten cutting stems per genotype were planted with ten rows at a space of 1 × 1.5 m. In 2008, they were cultivated at Rayong and Lop Buri provinces, Thailand. Each genotype was replicated two times in 10 × 10 simple lattice designs at a space of 0.8 × 1 m. Fertilizer , 312.5 kg/Hectare and chicken manure, 3,100 kg/Hectare were applied at one month after planting. Pest management was applied as necessary. Roots were harvested for CN evaluation at one year after planting. The climate at both locations was warm and humid all year round with an average temperature of 28°C. The average rainfall at Rayong and Lop Buri was 1,339.4 and 1,211.9 mm per year , respect-1 fresh weight (ppm) using picrate paper kit as described by Bradbury et al. (1999) [The CN was evaluated in roots as mg HCN equivalents kg. (1999) . The roo1 population. The PCR reactions were carried out in 20 μl final volume containing 50 ng of genomic DNA, 1 × PCR buffer  with 1.5 mM MgCl2, 0.2 μM of each PCR primer, 200 mM of each dNTP and one unit of DNA-polymerase (Promega). The PCR program for SSR amplification consisted of the following steps: 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 1 min, then a final step of 72°C for 5 min  [Genomic DNA of the population and its parents was extracted from young leaves based on CTAB selective precipitation of DNA, modified according to Fulton et al. (1995) . A total. (2008) ). Produc. (2008) . The amp. (2008) .® 3.0 program software [2) test. Statistically, significant markers at P < 0.05 were excluded from further analysis. The similarity of loci was then tested and markers showing 100% similarity were also excluded. The remaining markers were mapped into linkage groups based on the LOD threshold of 6.0 and maximum recombination threshold of 0.4. The genetic distance in a unit of recombinant frequency or centimorgan (cM) was calculated using Kosambi mapping function.For linkage map construction, all genotypic data were loaded into JoinMapsoftware . The pro® 3.0) were loaded into MapQTL® 4.0 program [P < 0.05 from the 1,000 permutations were selected as the cofactor. The automatic cofactor selection tool was used for selection of cofactors. They were selected by user before performed MQM mapping analysis. The markers that showed LOD score higher than the chromosome wide threshold with P < 0.05 from the 1,000 permutations were identified as QTL. KW analysis, a single marker analysis, was also analyzed based on one way ANOVA.As a first step, the data file containing the marker observations, the mean trait values and the genetic linkage map (output from JoinMap program . IM analOn-line BLAST against the cassava genome with primer sequences located on linkage map was performed on Phytozome . FunctioSW and KT conceived of the study together with the other authors, carried out the major part of the experiments, analyzed the results and prepared the manuscript. SP and SK participated in the genotypic analysis. ST involved in the data analysis. OB prepared plant materials. DAL was involved in drafting the manuscript and revising it critically. All authors read and approved the final manuscript.List of annotated genes within 2-LOD support regions of the QTL.Click here for file


Little data exists to detail the effect of hypotensive epidural anesthesia on differential tissue oxygenation changes above and below the level of neuraxial blockade. This study was designed to investigate tissue oxygenation in a clinical setting, using non-invasive near-infrared spectroscopy.Patients aged 18 to 85 years scheduled to undergo primary total hip arthroplasty were enrolled. Muscle oxygenation saturation was measured above and below the level of neuraxial blockade . Other continuously recorded parameters included cardiac output, stroke volume, heart rate, invasive mean arterial blood pressure and arterial oxygen saturation. Recordings of muscle oxygenation were compared over time separately for upper and lower extremity.nd quintile: 71.0% vs. 63.3%, p<0.0001). This decline was followed by a return to baseline towards the end of the surgery . Mean muscle oxygenation saturation did not change for the same period of time in the deltoid muscle. When adjusting for covariates, the changes in muscle tissue oxygenation remained significant.10 patients were enrolled. We found an intermittent and significant unadjusted decline of mean muscle oxygenation saturation in the vastus lateralis muscle during first part of the surgery (nadir 2These results indicate that muscle oxygenation saturation, a surrogate parameter for tissue perfusion, is decreased by hypotensive epidural anesthesia, but only within the functional limits of the neuraxial blockade. The etiology of these findings remains to be elucidated. Total hip arthroplasties (THA) are performed more than 280,000 times per year in the United States with predictions for this number to more than triple within 20 years ,2. IncreIn an attempt to enhance these beneficial effects and based on the theory of reduction of blood pressure while maintaining flow via stabilization of cardiac output (CO), the technique of hypotensive epidural anesthesia (HEA) was developed decades ago and since used with great success –11. This2) as a marker of tissue perfusion above and below the level of sympatholysis under conditions of HEA in THA patients  and 65.5 (SD ± 14.2) vs. 66.1 mmHg , respectively,); mean HR showed a modest but significant increase over the same period (66.2 (SD ± 9.1) vs. 71.5 [(SD ± 7.6). p=0.005] and 66.2 (SD ± 9.1) vs. 74.6 bpm [(SD ± 7.6) p=0.0001], respectively). No significant change was seen for mean CO over time (3.51 (SD ± 0.66) vs. 3.78 [(SD ± 0.82) p=0.2565] and 3.51 (SD ± 0.66) vs. 4.05 L/min [(SD ± 1.20) p=0.0515], respectively). Mean values for each quintile of parameters obtained are shown in From the unadjusted regression analyses we found an intermittent and significant decline of mean SmOd muscle . When an2 with respect to baseline were identified for the first part of the surgical procedure  which cannot directly be extrapolated to our setting ,16. Neve2 in the deltoid muscle remained unaffected. Except changes in MAP, no other studied covariates impacted significantly on outcomes. Reasons for the former finding have to remain speculative. While correlation with actual clinical outcomes is warranted, the maintenance of SmO2 in the deltoid is encouraging and suggests adequate tissue perfusion to the level above the sympathetic block. Therefore, further research with a larger number of patients is needed to determine if these findings are of clinical significance.In conclusion, this study showed a significant unadjusted decrease of muscle tissue oxygenation saturation of the vastus lateralis muscle in patients undergoing THA using HEA. The SmO


Mortality from bacterial meningitis in African adults is significantly higher than those in better resourced settings and adjunctive therapeutic interventions such as dexamethasone and glycerol have been shown to be ineffective. We conducted a study analysing data from clinical trials of bacterial meningitis in Blantyre, Malawi to investigate the clinical parameters associated with this high mortality.3 (>50% neutrophils) in HIV negative participants and >5 cells/mm3 in HIV positive participants. Outcome was measured by mortality at discharge from hospital (after 10 days of antibiotic therapy) and community follow up (day 40).We searched for all clinical trials undertaken in Blantyre investigating bacterial meningitis from 1990 to the current time and combined the data from all included trial datasets into one database. We used logistic regression to relate individual clinical parameters to mortality. Adults with community acquired bacterial meningitis were included if the CSF culture isolate was consistent with meningitis or if the CSF white cell count was >100 cells/mmS.pneumoniae (84% of positive CSF isolates) and N.meningitidis (4%). 607/694 (87%) participants tested were HIV antibody positive. Treatment delays within the hospital system were marked. The median presenting GCS was 12/15, 17% had GCS<8 and 44.9% had a seizure during the illness. Coma, seizures, tachycardia and anaemia were all significantly associated with mortality on multivariate analysis. HIV status and pneumococcal culture positivity in the CSF were not associated with mortality. Adults with community acquired bacterial meningitis in Malawi present with a severe clinical phenotype. Predictors of high mortality are different to those seen in Western settings. Optimising in-hospital care and minimising treatment delays presents an opportunity to improve outcomes considerably.Seven hundred and fifteen episodes of bacterial meningitis were evaluated. The mortality rate was 45% at day 10 and 54% at day 40. The most common pathogens were  Since the beginning of the HIV epidemic, many high prevalence Sub-Saharan African countries have reported an increase in patients presenting to health care facilities with acute bacterial meningitis (ABM) Interventions such as adjunctive dexamethasone and glycerol have failed to impact on this high mortality in large randomised controlled trials in Malawi and thus ABM remains a considerable clinical challenge http://www.mlw.medcol.mw) has provided a quality controlled cerebral spinal fluid (CSF) and blood culture service for QECH for more than 10 years.Queen Elizabeth Central Hospital (QECH), Blantyre is a 1250-bed government funded hospital with free care at the point of delivery that admits 50,000 patients per year. QECH serves a population of approximately 1 million including the city of Blantyre. The diagnostic laboratory at the Malawi Liverpool Wellcome Trust Clinical Research Programme  undertaken at the Department of Medicine, College of Medicine, QECH between 1990-to the current time, where adults with ABM were recruited prospectively and data were freely available. Database access for each study was obtained from the principal investigator.3 in HIV negative or 5 cells/mm3 in HIV positive. The latter was selected because the CSF inflammatory response in ABM may be attenuated in HIV infected African adults Cryptococcus neoformans or Mycobacterium tuberculosis, if the CSF white cells were >50% lymphocytes, or on study specific criteria, such as type two diabetes for the glycerol trial Included participants were over 13 years of age (adult ward admission age) and enrolled in a clinical study of ABM at QECH with either CSF proven microbiological evidence of ABM, or a high clinical index of suspicion of ABM plus a CSF white cell count that was >50% neutrophils and >100 cells/mmThe outcome measure for included subjects was mortality which was measured at day 10 and at day 40. Day 40 was prospectively agreed as the last follow up time point in included studies. Day 0 was recorded as the date and time of entry into the hospital; day 1 was 24 hours from this point.Variables were standardised and a single database was created. The first recorded clinical observations on admission were used. Eighteen clinical and demographic parameters were selected on the basis of previous published associations and suspected risk factors, and subjected to analysis for associations with death All constituent studies had been approved by the College of Medicine Research and Ethics Committee in Malawi, and by the Liverpool School of Tropical Medicine Research Ethics Committee and conformed to institutional guidelines. All participants in the included clinical trials and studies gave written informed consent, or this was given by a named guardian if the participant was under 18 years of age.Data were analysed using SPSS/PASW version 20. Univariate analysis was performed for each variable. For continuous variables, parametric (Student t) and non-parametric (Mann-Whitney U) tests were used to compare survivors and non-survivors; Fisher exact/chi-square tests were used to compare categorical variables. A multivariate analysis model was generated using backwards stepwise logistic regression methods to estimate the influence of each variable on clinical outcome. Each model was compared at each step with the previous model and only variables with on-going significance remained in the analysis. Data from the final model retaining all significant variables were reported. The strength of relationships was expressed using odds ratios with 95% confidence intervals. All statistical tests were two-tailed, and a p value of <0.05 was used to denote statistical significance. Due to the heterogeneous nature of the studies included, some significant variables had missing data; if >50% of the data were missing, these variables were excluded from the multivariate analysis. The multivariate analyses were repeated using multiple imputation methods to enable subjects with missing data to be included; this did not alter the results and these analyses are not presented.3 (n = 38). A further sixty one patients were excluded because no mortality data were available at day 10 yielding 715 cases for the analyses. We excluded a relatively high proportion of participants from the cohort study (118/161) compared to 26 from the glycerol trial and 32 from the dexamethasone trial. This was due to a high number of patients in this database either not meeting the white cell count inclusion criteria for this analysis or having mortality data available. Free access to HIV treatment become routinely available during the study period; co-trimoxazole prophylaxis in 2002 and anti-retroviral therapy (ART) from 2004 Five studies were identified. One observational study was excluded as it was conducted prior to the introduction of ceftriaxone in 2001 under different admission conditions The median age was 31 years (inter-quartile range IQR 25–38) and half were female (50.3%). 20 participants (2.8%) were adolescents. 608/695 (87%) of those tested were HIV antibody positive, of which 36 (5.9%) were on ART . CD4 celThe overall mortality rate was 45.3% in hospital by day 10, and 54.3% by day 40, with no significant difference across the three studies . AlteredStreptococcus pneumoniae was the most common isolate  followed by Neisseria meningitidis  . CSF opening pressure was not routinely recorded. Blood cultures were positive in 185/664 (28%) of individuals; the most commonly isolated organisms being S.pneumoniae , enteric gram negative organisms  and N.meningitidis . Of 352 pneumococcal CSF isolates where paired blood culture data were available, 139 (40%) were positive. Comprehensive data on antimicrobial resistance were not available. Pneumococcal serotype was determined in 128 of 352 isolates; 56 (43.8%) were serotype 1. Other serogroups were 6 (4.7%), 7 (3.1%), 9 (4.7%), 12 (3.9%), 14 (4.7%), and 19 (3.1%). Mortality was 59% (33/56) for the participants infected with serotype 1 and 50.1% (36/71) for the other serogroups. Available data were too few to analyse this trend further.CSF culture was positive in 419 of 712 specimens (59%). 419, 4%) . Other iPresentation out of routine working hours comprised 318 (44.5%) of admissions. For a subset of patients with culture proven ABM ‘the observational cohort’, (n = 26) data were collected prospectively relating to the patient pathway on a standard form to investigate delays in presentation, investigation and initiation of therapy. Nine of 26 patients (35%) died before day 10. Median self-reported journey time to QECH was 0.8 hours (IQR 0.5–1.6) in non-survivors, and 1.5 hours (IQR 0.7–2.0) in survivors. Median time from presentation to medical review was 2.8 hours (IQR 0.8–5.8) in non-survivors and 1 hour (IQR 0.6–3.0) in survivors. The median time from presentation at the hospital with meningitis to receipt of intravenous antibiotics was 5.0 hours (IQR 2.5–8.9) in non-survivors and 2.7 hours (IQR 1.7–6.0) in survivors. No time difference reached statistical significance.Eighteen parameters were subjected to univariate analysis for associations with survival at day 10 (n = 715) and day 40 (n = 668). There was no statistically significant difference between these two time points for any of the variables except pneumococcal culture and therefore the day 40 follow up data are presented .S.pneumoniae isolated from the CSF, 181 (54%) died and 158 (46%) were alive at day 40 OR 0.923 (0.68∶ 1.25) p = 0.61. As culture of S.pneumoniae in the CSF and HIV status have been associated with mortality in other studies of bacterial meningitis The following clinical parameters were significantly associated with poor outcome on univariate analysis: age, clinical presentation with altered mental status, acute and post discharge seizures, hypoxaemia, tachycardia, hyponatraemia, anaemia and treatment with glycerol. Neither gender, out of hours admission, HIV antibody status, blood glucose, mean arterial blood pressure, anuria, respiratory rate, nor serum creatinine were associated with poor outcome . Of the Presentation with coma was the strongest independent predictor of outcome after multivariate analysis; OR for death with a GCS<8/15 was 5.9 (95% CI 3.31∶ 10.86) p<0.001, GCS of 8–11 was 1.665 (1.08∶ 2.5 ) p = 0.007. Acute seizures were associated with death, OR 1.55 (1.08∶ 2.24) p = 0.019. The severity of anaemia was associated with a corresponding increasing risk of mortality, OR death of haemoglobin (Hb) 5–8 g/dL was 3.41 (1.61∶ 7.23) p = 0.001; at Hb<5 g/dL the OR was 6.34 (1.86∶ 21.62) p = 0.003. Every 1 g/dL increase in haemoglobin was associated with a 12% reduction in mortality, OR 0·88 (0.82–0.94). Tachycardia >120 bpm was also associated with death, OR 1.80 (1.08∶ 2.99) p = 0.023. Glycerol receipt was independently associated with mortality 1.88 (1.09∶ 3.25) p = 0.023. Age >40 years was not significant, OR 1.33 (0.86∶ 2.06) p = 0.20.Neither CSF pneumococcal culture positivity nor HIV status were significantly associated with mortality at day 40. The OR of death were 0.70 (0.49∶ 1.02) p = 0.06 for pneumococcal infection, and 1.18 (0.70∶ 2.00) p = 0.52 for HIV infection. No parameter reaching non-significance at the univariate level was shown to have significant association at the multivariate level.Mortality was not seasonal; 279 (36%) of cases presented in the cold winter season (May-September), with 145 (52%) deaths and 388 (64%) cases presented during the remaining hot months of the year with 217 (56%) deaths p = 0.34 .Streptococcus pneumoniae is more common in HIV infected than HIV uninfected individuals in sub-Saharan Africa S.pneumoniae infection via altered mucosal and cell-mediated immunity Adults and adolescents presenting to hospital in Malawi with bacterial meningitis are young, largely HIV positive and have a very high mortality rate, contrasting significantly with published data from high income countries A striking finding from this very large ABM dataset was the excessive mortality which is much higher than in series from industrialised countries and has remained unchanged for 10 years Early access to resuscitation and antibiotic treatment has been instrumental in reducing mortality rates from paediatric meningitis Similarly to the Dutch cohort, we show that coma and tachycardia are independently associated with death on multivariate analysis S.pneumoniae was the most common organism isolated from CSF. Only 4% of cases were due to N. meningitidis, all of whom survived. Malawi is south of the African meningitis belt, and meningococcal epidemics occurring during the study period did not affect Malawi. Our results therefore are not generalizable to that region, but are applicable to other African countries in sub-Saharan Africa with high rates of HIV infection and invasive pneumococcal disease. Mortality was highest in those with S.pneumoniae serotype 1 S.pneumoniae culture positivity and poor neurological outcome or seizures in survivors In contrast to Western data, a sub-analysis (not presented) did not show an association between . Our study recorded a significant number of participants who had a negative CSF culture. All data were from clinical studies with strict inclusion criteria including expert clinical evaluation. S.pneumoniae is a fastidious organism and culture has a low sensitivity, PCR and antigen detection tests were not available. Although our CSF WCC inclusion criteria was >5 cells/mm3 in HIV positive participants, the median WCC was significantly higher than this (480 cells/mm3 IQR 170–1680). It is unlikely that any participants had a CSF inflammatory response due to seizures alone M.tuberculosis. Specific TB meningitis (TBM) data from our centre demonstrated a median CSF WCC of only 40 cells/mm3 (IQR 0–240) in TBM, 10 fold lower than our study of ABM All studies used ceftriaxone as the first line antibiotic. Only a very small proportion were taking antiretroviral therapy (ART) and we do not have data on co-trimoxazole use. We reported pneumococcal resistance between 2000–2009 to the following antibiotics: multi-drug resistance  in 30%, penicillin in 15%, ceftriaxone in 0%; co-trimoxazole resistance increased from 74% to 93% Some findings contrasted with other studies. Hyponatraemia was associated with mortality, although the numbers were small, contrasting with Dutch data, where no association was seen Data heterogeneity limited our analysis. All data were collected prospectively but the lack of uniform data acquisition across the studies and missing data limited our ability to analyse each trend fully. We used random imputation models to test the robustness of our findings, but where the proportion of missing data was higher than collected data those variables could not be included. We were not able to evaluate some of the other indicators that are prognostically significant in other settings. We were unable to stratify our results by CD4 count. A small proportion of adults screened by the included clinical trials died before recruitment, refused consent or were excluded due to study specific criteria, but overall the majority of adults with bacterial meningitis presenting to our centre were enrolled in these studies and the risk of selection bias is low. The consistency of the numbers recruited over each time period and mortality suggests that the confounding effect of time on the analysis is minimal. Despite the introduction of major public health interventions in Malawi over the time period studied the mortality rate from bacterial meningitis remains unchanged.This study is the largest clinical analysis of bacterial meningitis in adults and adolescents in Africa to date, and the first to investigate the role of clinical presentation and key physiological parameters in outcome. Our findings are likely to be generalisable to much of sub Saharan Africa. Coma, seizures, tachycardia and anaemia all independently predict a poor outcome. Disease severity at presentation is likely to be worsened by delays in presentation to hospital and further in-hospital delays. Early antibiotics and goal directed resuscitation have been shown to reduce mortality significantly in sepsis


Diabetic nephropathy is the most important cause of end stage renal disease (ESRD). Aldosterone is involved in renal damage through induction of fibrosis, inflammation and necrosis in the kidney tissue. Previous studies have demonstrated that the combination of angiotensin receptor blocker (ARB) and spironolactone  are efficient for albuminuria reduction.This study was designed to evaluate the effect of spironolactone alone on diabetic nephropathy.In this double blind randomized clinical trial, 60 type II diabetic patients with microalbuminuria were enrolled. They were divided into two groups: case group  and control . The treatment success rate  was compared between the two groups.After three months, successful treatment was seen in 70% (95% CI: 52 - 83) and 83.3% (CI 95%: 66 - 93) of case and control groups, respectively (P = 0.4). Mean ± SD of serum potassium levels after three months in case and control groups were 4.56 ± 0.38 and 4.39 ± 0.34 mEq/L, respectively (P = 0.08). Mean ± SD of systolic blood pressures in case and control groups were 129.67 ± 9.4 and 130.97 ± 9.4 mmHg, respectively (P = 0.6). Mean ± SD of serum creatinine levels at the end of the study were 0.95 ± 0.15 in case and 0.90 ± 0.22 mg/dL in control group (P = 0.4).Spironolactone alone is as effective as the combination of spironolactone and losartan on albuminuria reduction in type 2 diabetic patients and can be used alone as an effective drug for diabetic nephropathy. Diabetic nephropathy is the most significant cause of end-stage renal disease (ESRD) and the main cause of mortality and morbidity in diabetic patients. The prevalence of nephropathy in diabetes mellitus (DM) type 1 is more than DM type 2, but due to the greater number of patients presenting DM type 2, its nephropathy rate is as high as type 1 DM . DiabetiUrinary albumin excretion  is one of the important risk factors for the progression of renal disease to ESRD -4, 6. ThPresent study was performed to evaluate the effect of spironolactone alone compared to the combination of spironolactone and losartan on albuminuria reduction in type II diabetic patients.This study was a double-blinded randomized clinical trial, performed in the DM health care centers of Sari, Iran from 2008 to 2011. The study was registered in IRCT (Iran) with the following registration code: IRCT 138806211241N2. Sixty patients with type 2 diabetes mellitus, suffering from diabetic nephropathy, were enrolled in the study. The inclusion criteria were age range of 25 to 75 years, HbA1c < 9% and random urinary albumin to creatinine ratio of 20 - 200 mg/gr Cr in two random measurements with a month interval. If only one of the two microalbumiuria tests was positive, it would be repeated the next month. Exclusion criteria were: diastolic and systolic blood pressures more than 100 and 160 mmHg, respectively; serum potassium level > 5.5 meq/L; prior acute myocardial infarction (MI) or stroke during the preceding six-month period; taking proteinuria-affecting medications ; renovascular disease; collagen vascular disease; obstructive uropathy; alcohol and substance abuse; pregnancy or lactation.This study had two phases; screening and treatment. During the screening phase, patients were selected according to the inclusion criteria and then eligible patients were entered into the treatment phase. Five milliliters of fasting blood was taken for the serum creatinine, potassium, and glycosylated hemoglobin (HbA1C) assessments, and the tests were repeated 4 and 12 weeks after the intervention. Limitation of protein consumption (0.6-0.8 g/kg/d) was advised during the study. The samples were assigned to case or control group through the RANDBETWEEN function of Microsoft Excel software. Case group comprised 30 diabetic patients who took spironolactone 25 mg once daily plus half a tablet placebo twice a day. Control group took spironolactone 25 mg once daily plus losartan 12.5 mg twice a day. The intervention phase lasted for 12 weeks.Patients’ drugs consumption quality was evaluated by counting the remaining tablets at the end of each month. Albuminuria was measured in the beginning, after 4 weeks and at the end of the intervention. For these measurements, immunotorbidometric assay was performed by prestige 24i automated clinical chemistry analyzer , using the Pars Azmon kit (Iran). Patients were visited every 4 weeks and their serum potassium levels were measured. Serum potassium level was measured by Alfa Wassermann Starlyte III electrolyte analyzer and serum creatinine was assessed by Cobas Integra 400 , using the Creatinine Plus kit .Spironolactone dosage was reduced by half if the serum potassium level increased more than 5.5 mmol/L or systolic BP decreased less than 90 mmHg. The study was approved by the Mazandaran University of Medical Sciences Ethics Committee (Ethic code: 88-89) and informed consent was obtained from all the participants.The data were described as mean ± SD (standard deviation). Independent sample t-test was used to compare the baseline and after intervention levels of microalbuminuria between case and control groups. Repeated measures analysis was applied for evaluation of the treatment effect as well as the trend of treatment in each group. The treatment success rate was reported as 95% confidence level. P value < 0.05 was considered statistically significant.Twenty-three (76.7%) of the case and 22 (73.3%) of the control group were female (P = 0.8). One patient of the case group was dropped out from the study because of not using the medications regularly. Mean age of the patients was 51.78 ± 11.39 years. Fifty patients (83.3%) were under oral antidiabetic agents and 10 (16.7%) were taking insulin. Patients under treatment with insulin were 13.3% of the case and 20% of the control group (P = 0.5). The treatment success rate  1 month after the intervention was 66.7% and 56.7% in the case and control groups, respectively (P = 0.4). After 3 months, this rate was 70% in the case and 83.3% in control group (P = 0.4) . The treThere were no significant increases in the serum creatinine or potassium levels of any patient. Microalbuminuria, serum creatinine level, and systolic/diastolic blood pressures were not different between the two groups after the study . Using tIn this study, we found that in diabetic patients, treatment with spironolactone alone has the same effect as combination therapy with spironolactone and losartan on microalbuminuria reduction. ACEIs and ARBs effectively reduce proteinuria and postpone renal disease progression in diabetic and non-diabetic patients , 24. AntType IV collagen is a component of glomerular basement membrane and mesangial matrix, production of which can be induced by aldosterone. Its urinary appearance can be a reflection of its production level  and it cHyperkalemia is one of the complications of spironolactone. It is dependent on drug dosage; so low dose spironolactone can provide a safe margin . In our Our study had some limitations. Its population was pretty small and the level of microalbuminuria was different between the two groups before the intervention, but it was not statistically significant. Also, we just evaluated microalbuminuria in diabetic patients. According to our findings, we suggest that spironolactone alone can be effective in treatment of patients with diabetic nephropathy. More studies need to be done to establish the long-term beneficial clinical effects of spironolactone alone in different stages of diabetic nephropathy.


To investigate the association between EGFL6 D535N polymorphism and obesity in Chinese children.Case-control study.A total of 384 obese and 205 healthy children were enrolled as obese and control groups.The tagged SNP in EGFL6 (rs16979033) was measured by automated platform MassArray. Anthropometric estimates  and biochemistry marker measurements  were performed.2=0.119, p=0.730). The EGFL6 D535N polymorphism was not associated with obesity in either the dominant or recessive model test. The anthropometric estimates and biochemistry markers in patients with EGFL6 353D were not significantly different than those in patients with EGFL6 353NThe frequencies of the N allele were 13.9% in 589 children. Among the obese group, 329 were homozygous for the 535D allele, 22 girls were heterozygous and 33 were homozygous for the 535N allele. In the control group, 173 were homozygous for the 535D allele, 15 girls were heterozygous and 17 were homozygous for the 535N allele. The frequencies of the N allele were 13.5% in obese group and 14.5% in control group, having no significant difference (χThe polymorphism of EGFL6 D535N was very common in Chinese children. EGFL6 D535N polymorphism was not associated with obesity in Chinese children and it may not be a cause of childhood obesity.


An. gambiae mosquitoes sampled in Dielmo , in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010.The aim of this study was to evaluate the susceptibility to insecticides of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kdr mutations were assessed by PCR. Longitudinal study of kdr mutations was performed on adult mosquitoes sampled monthly by night landing catches from 2006 to 2010.WHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae were resistant to DDT, Lambda-cyhalothrin, Deltamethrin and Permethrin  but highly susceptible to Fenitrothion and Bendiocarb . There was significantly more RR genotype among An. gambiae surviving exposure to DDT or Pyrethroids. An. arabiensis represented 3.7% of the sampled mosquitoes (11/300) with no kdr resistance allele detected. An. gambiae molecular form M represented 29.7% of the mosquitoes with, among them, kdr genotypes SR (18%) and SS (82%). An. gambiae molecular form S represented 66% of the population with, among them, kdr genotype SS (33.3%), SR (55.6%) and RR (11.1%). Only 2 MS hybrid mosquitoes were sampled and presented SS kdr genotype.No specimen studied presented the L1014S mutation. During the longitudinal study, L1014F allelic frequency rose from 2.4% in year before the implementation of LLINs to 4.6% 0–12 months after and 18.7% 13–30 months after. In 2010, An. gambiae mosquitoes in Dielmo  within 24 months of community use of LLINs. Molecular identification of L1014F mutation indicated that target site resistance increased after the implementation of LLINs.Biological evidence of resistance to DDT and pyrethroids was detected among  Recently, huge progress has been made in the control of malaria in Sub Saharan African countries in vivo biological test and by the identification of resistance alleles in a vast number of sites across Africa. Especially, kdr mutation genotype has been recognized to be related to DDT and pyrethroid resistance An. gambiae s.l. and An. funestus are the two major malaria vectors in Dielmo  Mosquitoes' resistance to insecticide has been demonstrated by both Anopheles gambiae s.l., 2 years before and after the implementation of LLINs and the results of resistance tests to 6 frequently used insecticides performed in 2010, 2 years after the implementation of LLINs.Since 1990, an epidemiological study is ongoing in Dielmo  that involves long-term investigations on host-parasite relationships and mechanisms of protective immunity in residents of this Senegalese village This study is part of the Dielmo Project that has been described in detail elsewhere Anopheles gambiae sensus lato (s.l.) group were stored for following steps.Adult mosquitoes were collected by human landing catches (HLC) monthly from July 2006 to December 2010. Night captures (7:00 PM–7:00 AM) were conducted once or more times each month in two indoor and two outdoor sites. In each site, two trained collectors  worked alternatively for one hour and rested for one hour. Anopheline identification was performed following the Gillies and DeMeillon morphologic identification keys An. gambiae s.l. were collected from 10 breeding sites situated in and around the village . Larvae were pooled and fed with Tetramin® baby fish food locally until emergence. Unfed 2–3 days female An. gambiae s.l. mosquitoes were used for insecticide susceptibility tests.In August 2010, during the rainy season, immature stages of An. gambiae s.l., with four batches tested against each insecticide. Mosquitoes were exposed to insecticide-impregnated filter paper for 1 hour at 25–27°C and 80% relative humidity. The number of knockdown mosquitoes was recorded at 10, 15, 20, 30, 40, 50, 60 and 80 min. After exposure, mosquitoes were kept in observation tubes and supplied with a 10% sugar solution. Mortality was recorded after 24 hours. The mortality of a control stain of An. gambiae  and used for molecular tests.Bioassays were carried out using WHO test kits for adults mosquitoes In the subsample of mosquitoes used for bioassay and in adults sampled by HLC during the longitudinal study, detection of L1014F and L1014S kdr mutations (thereafter identified as kdr-w and kdr-e respectively) was performed by PCR 50 and KDT95) were computed with probit regression models. Rates were compared using Fisher exact and Pearson Chi2 tests. Statistical analyses were performed using Stata 10.1 software. A P value of 0.05 or less was considered as significant.WHO (1998) criteria were used to evaluate the resistance/susceptibility status of the tested mosquito populations  The Dielmo project was initially approved by the Ministry of Health of Senegal and the assembled village population. Approval was then renewed on a yearly basis. Audits were regularly conducted by the National Ethics Committee of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institute and the Institut de Recherche pour le Développement.From July 2006 to December 2010, no specimen with L1014S (kdr-e) mutation was identified.2 = 1.4, p = 0.24). This rate significantly increased to 4.6% within the first 12 months that followed the distribution of LLINs  and again 13–30 months after to 18.7%  (The repartition of kdr genotype during the study period is presented in  period) .Mortality data indicated that mosquitoes were highly resistant to 4 of the 6 insecticides tested including DDT and all Pyrethroids . Mortality rates ranged from 46 to 63%, far below the susceptibility limit of 80% . Mosquit50) was higher than 40 minutes for Lambda-cyhalothrin, Permethrin, DDT and Fentrothion and KDT95 exceeded the 80-min observation period (50 = 28.0 min) and even shorter for Bendiocarb (KDT50 = 17.5 and KDT95 = 39.0 min).Knockdown time 50  An. gambiae s.s. molecular form M, 2 (0.7%) MS hybrids and 198 (66.0%) form S. When comparing the species and molecular forms of An. gambiae s.l. among dead and surviving mosquitoes, no association could be identified for Deltamethrin, Lambda-cyhalothrin, Permethrin and DDT  were Among the 300 surviving and dead specimens selected for kdr-w identification, 152 (50.7%) were SS, 126 (42%) SR and 22 (7.3%) RR kdr-w genotype . No specAn. gambiae . Allelic form R was totally absent in An. arabiensis and in An. gambiae MS form. In An. gambiae M form, SR genotype was present (18%) and RR genotype was absent. Allele R frequency was 38.9% for molecular form S vs. 7.8% in the other groups (Fisher exact test p<0.001).The frequency of kdr-w mutation was significantly different according to the molecular form of An. gambiae s.l. display a high biological level of resistance to DDT and pyrethroids . Similar resistance has been observed all around Africa but little information was previously published about Senegal. Investigations on the biological susceptibility to DDT performed in sentinel sites of Senegal reported a resistance to DDT in 4/10 sites 50 is short (6–26 min) 50 is longer (more than the 80-min observation period) 50 were at an intermediate level.The results of this study demonstrated that field population of 50 was 49 min in our study, slightly longer than that observed in Dakar in 1995 when susceptibility was higher  While the resistance to pyrethroids was limited in 2008 in Senegal . Interestingly, kdr-w mutation was identified at a low rate (9%) in the M molecular form. Many studies reported the high frequency of kdr-w mutation in molecular form S in Western and Central Africa and its low frequency or absence in molecular S form  since we observed 100% mortality. Susceptibility of An. gambiae after the implementation of LLINs in Dielmo . This coincided with a cross-resistance to DDT and all pyrethroids observed in 2010. Resistance was associated with a higher kdr-w allele frequency in surviving specimens. Moreover, kdr-w mutation was detected in both M and S molecular forms of An. gambiae and significantly more frequently in molecular form S. On the other hand, mosquitoes were fully sensitive to Bendiocarb and Fenitrothion.In conclusion, this study demonstrated an increased frequency of kdr mutation in