Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear.Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species T. canis larvae in vitro. Using a western blot (WB) assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm’s E/S antigens in different infected hosts including mice, rabbits and human, were examined.Excreted/secreted (E/S) antigens were prepared by culture of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band.In a mouse model of toxocariasis, intraperitoneal injection of 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation. Toxocara canis and Toxocara cati whose definitive hosts belong to the Canidae and Felidae respectively. In these hosts, the nematodes complete their development. In human and others animals, the development cycle of the nematodes is limited to the second larval stage which remains in the tissues for a long period of time  antigens in the circulation has been suggested as a mean to detect current toxocariasis  (20–25gr in weight) and rabbits (2kg in weight) were purchased from the Pasteur Institute in Ho Chi Minh City, Vietnam and then kept in our laboratory  during experiments. Pups and kittens (2–3 months old) were purchased in Ninh Thuan Province and Ho Chi Minh City, respectively, Vietnam.All procedures performed in studies involving animals were in accordance with the ethical standards of the Local Science and Ethical Committee at the Institute of Tropical Biology.Toxocariasis human sera were a gift from Prof. Tran Thi Kim Dung . Cord blood sera from volunteer women were used as negative control.Toxocara spp. eggs by phase contrast microscopy. Infected animals were anesthetized with ketamine (100 mg/kg of body weight). Toxocara spp. adult worms found in the small intestine were collected in pre-warmed PBS buffer. The worms were then washed several times in 1% formalin-PBS buffer. For egg production, all male and female worms were cultured together in PBS supplemented with 1% human serum plus penicillin (100U/ml) and streptomycin (100ug/ml) at 37 °C in a 5% CO2 atmosphere for up to 7 days. The eggs were collected by centrifugation at 200-x g for 10 min and then counted.Pup and kitten feces were sampled to test for the presence of 2HPO4, 0.4mM KH2PO4, 1.3mM CaCl2, 0.5mM MgCl2.6H2O, 0.6mM MgSO4.7H2O, and 137mM NaCl for 30 min at room temperature. The pH was adjusted to 7.4 using NaHCO3 and then to pH 2.0 with 5N HCl. The treated eggs were washed with sterile HBSS pH 7.4, concentrated by centrifugation at 200-x g for 10min and incubated in serum free DMEM medium supplemented with penicillin (100U/ml) and streptomycin (100ug/ml) at 37 °C in a 5% CO2 atmosphere. Three days later, >95% of fertilized eggs had hatched. Larvae were concentrated by centrifugation and living larvae were selected by letting them pass through a 40um cell strainer in DMEM medium overnight. All dead and weak larvae and unfertilized eggs remained in the upper side of the cell strainer. Our improved filtration method helped to obtain only strong and fast moving larvae. These were then used to produce E/S protein.Worm eggs were incubated in 1% formalin– PBS for 30 d at room temperature in a sterile flask. The fertilized eggs and embryo development were followed using an inverted microscope. Hatching was induced using a modified version of a published protocol . Briefly5T. canis larvae were cultured at a density of 1000 larvae/ml in DMEM medium in the presence of penicillin (100U/ml) and streptomycin (100ug/ml) at 37 °C in a 5% CO2 atmosphere. Culture medium was harvested every four weeks for at least one year. 1ml of culture medium was dialyzed in PBS and the protein concentration was determined by Bradford assay. E/S proteins in the culture medium were concentrated by centrifugation with the Amicon centrifugal filter units (10 kDa cut off). A cocktail of protease inhibitors   was added to E/S proteins which were then stored at −20 °C until use.3.7 × 10T. canis or T. cati by intraperitoneal injection (ip)  were used as negative controls.Mice (N =5–10) and rabbits (N=6) were infected with 1000 living larvae of ion (ip) –25. The Four weeks after infection in mice, organs including the liver, the lungs and the brain were harvested. Half of the brain was used to recover migratory larvae by using the Baermann funnel method. For histological analysis, the half of the brain and other tissues were embedded in paraffin. 3um thick sections were then prepared and stained with Giemsa staining solution . Images were obtained using an OPTIKA microscope equipped with an OPTICAM B5 camera .E/S proteins (0.2μg to 2.4μg/well) were electrophoresed by SDS-PAGE under reducing condition on a 10% gel. Page Ruler broad range unstained protein ladder  was used as molecular weight standards. For protein detection, silver staining was used. Protein molecular weight was estimated by using Quantity One software ver. 4.6.3 .T. canis infected mouse or rabbit or human sera diluted 1:1000 in TBST and then washed with TBST buffer. The membrane was incubated with a 1:5000 diluted goat anti-mouse or anti-rabbit or anti-human IgG antibodies conjugated with HRP  for 1h and then washed with TBST buffer. For detection of antigen-antibody immune complexes, the membrane was incubated in enhanced chemiluminescence reagent for 5min and then scanned on C-digit blot scanner  to obtain images which were analyzed by Image Studio Lite programme.After SDS-PAGE, E/S proteins were transferred to a nitrocellulose membrane (0.45um of pore size) using a semi-dry blotter system . For optimization of the protein transfer, blotting times of 60min, 90min, or 120min were tested. The membrane was then blocked with 0.1% casein in TBST buffer  for 1hr and washed three times in TBST buffer for 10min. The membrane was incubated for 1h with In some experiments, western blot membrane strips were prepared as described elsewhere  but withThe mean values of protein MW were calculated using GraphPad Prism 5.0 .T. canis L2 larvae in vitro efficiently produced high amount of E/S proteins. After every four weeks of culture, the concentration of E/S protein (Mean ± SD) was approximately 23.9 ± 9.0μg/ml of culture medium . The protein components in the E/S products were then determined by SDS-PAGE.Our modified culture procedure of When 200ng of E/S product was used, we found 15 bands ranging from 19.5 kDa to 111.1 kDa in which the most dominant protein had a MW of 33.1 kDa . SeveralT. canis larvae and the humoral immune response of infected mice in order to clarify which E/S proteins result in the generation of an IgG response. Mice were injected ip with 1000 live or dead larvae of T. canis. Four weeks later sera and organs  were harvested for histochemical analysis and for the recovery of migrating larvae. Organs from control untreated mice and heat-treated larvae injected mice had no sign of tissue damage, whereas mice treated with live larvae showed an apparent abnormality in the lungs but not in the livers or in the brain . However, the 33.1 kDa protein started to be lost after 120min of blotting (Data not shown) and we, therefore, applied 120min of blotting for all following WB experiments.T. canis larvae was determined and the results are shown in To analyze the IgG response in mice, WB strips were prepared from one gel to be sure that each strip contains the same amount of bound antigens. At least 10 bands were detected on the strips using the sera of animals infected with live larvae (N=10) . The strT. canis larvae strongly cross-react with serum from animals infected with T. cati larvae using an ELISA assay . WB band patterns from toxocariasis patients were more similar to those obtained from Toxocara spp. infected rabbits than those obtained from Toxocara spp. infected mice  show >96% specificity  defined in original studies , 26, 29.al model . If thisT. canis 33.1 kDa E/S protein is the most reactive constituent recognized by specific IgG in animals and human infected with T. canis and/or T. cati indicating that this protein may be considered as a critical common biomarker for future applications in diagnosis both veterinary and human medicine. However, further investigation is required to prove its specificity.


Streptococcus pneumoniae is the leading cause of bacterial pneumonia among children worldwide, and nasopharyngeal colonization precedes infection.Measles vaccine (MV) may have non-specific beneficial effects for child health and particularly seems to prevent respiratory infections. We investigated whether providing early MV at 18 weeks of age reduced pneumococcal colonization and/or density up to 9 months of age.6.5) and age 9 months (PR9) were estimated using Poisson regression with robust variance estimates while the pneumococcal geometric mean ratio (GMR6.5 and GMR9) was obtained using OLS regression.The study was conducted in 2013–2014 in Guinea-Bissau. Pneumococcal vaccine was not part of the vaccination program. Infants aged 18 weeks were block-randomized 2:1 to early or no early MV; at age 9 months, all children were offered MV as per current policy. Nasopharyngeal swabs were taken at baseline, age 6.5 months, and age 9 months. Pneumococcal density was determined by q-PCR. Prevalence ratios of pneumococcal colonization and recent antibiotic treatment (yes/no) by age 6.5 months (PR6.5 was 1.02 (95%CI = 0.94–1.10), and the PR9 was 1.04 (0.96–1.12). The GMR6.5 was 1.02 (0.55–1.89), and the GMR9 was 0.69 (0.39–1.21).Analyses included 512 children; 346 early MV-children and 166 controls. At enrolment, the pneumococcal colonization prevalence was 80% (411/512). Comparing early MV-children with controls, the PR6.5 0.60 (0.34–1.05) and PR9 0.87 (0.50–1.53)). Antibiotic treatment was associated with considerably lower colonization rates, PR6.5 0.85 (0.71–1.01) and PR9 0.66 (0.52–0.84), as well as lower pneumococcal density, GMR6.5 0.32 (0.12–0.86) and GMR9 0.52 (0.18–1.52).Early MV-children tended to be less frequently treated with antibiotics prior to follow up (PREarly MV at age 18 weeks had no measurable effect on pneumococcal colonization prevalence or density. Higher consumption of antibiotics among controls may have blurred an effect of early MV.NCT01486355clinicaltrials.gov  In a recent WHO commissioned review, the livStreptococcus Pneumoniae (pneumococci) by 75% (OR 0.25 (0.07–0.90)) when comparing carriage status 4 weeks after vaccination with before.A recent observational study from ThePneumonia is the single largest killer of children <5 years worldwide with more than 95% of cases occurring in low-income countries. The mosGlobally, pneumococci are estimated to cause 11% of all deaths among children <5 years with 90% of fatalities due to pneumonia, 11 and S. Pneumoniae through an effect on the nasopharyngeal colonization. Thus, within an ongoing randomized controlled trial (RCT) of early MV among infants in Guinea-Bissau, we investigated whether MV reduced pneumococcal colonization and/or -density.We hypothesized that MV decreases respiratory infections with Since 1978, the Bandim Health Project (BHP) has maintained a health and demographic surveillance system (HDSS) currently covering more than 100,000 individuals in 6 suburbs in the capital of Bissau, Guinea-Bissau. Through monthly home visits newborns are identified and followed with trimonthly home visits until 3 years of age to register growth, morbidity, vaccinations and vital status. At the initial registration, each child is given a unique ID number to facilitate linkage between all BHP registers.The present pneumococcal study (MVPneumo) was nested within a RCT conducted within the BHP urban study area from 2011 to 2015, aiming at investigating the effect of early vs. no early MV at 18 weeks of age on overall mortality until age 5 years (MVUrban). All children were offered the recommended MV at age 9 months. Pneumococcal conjugate vaccine (PCV) had not been implemented in Guinea-Bissau when MVPneumo was conducted. There were no reported measles cases throughout the duration of the study.Inclusion criteria in the MVUrban trial were receipt of the third dose of the pentavalent vaccine (DTP-HepB-Hib-3) at least 4 weeks before enrolment, and age below 7 months. Through home visits, eligible children were invited for inclusion at one of the three health centers in the HDSS study area. Here, children underwent a clinical examination by a physician and had anthropometric values measured. Severely sick children in need of hospitalization, malnourished children (defined as a mid-upper-arm circumference less than 115 mm) and children with severe malformations were excluded.Provided informed oral and written consent from a parent/guardian was obtained, children were block-randomized 2:1 stratified by sex to early or no early MV at age 18 weeks. Parents picked a folded lot from a sex-specific envelope containing 24 lots, 2/3 of the lots assigned the child to the intervention group (early MV), 1/3 of the lots assigned the child to the control group (no early MV). Twins of the same sex were allocated to the same treatment to avoid confusion. Children randomized to early MV received a single dose of 0.5 ml MV  as a subcutaneous injection in the shoulder region. No placebo was given, since mothers could get the false impression that their child was vaccinated against measles and therefore abstain from the recommended 9 months MV if they travelled or moved from the BHP study area.From August 2013 to January 2014, children enrolled in MVUrban were invited to participate in MVPneumo. Provided informed consent, three nasopharyngeal swabs (NPS) were scheduled to be taken: At enrolment, 2 months later (approximately age 6.5 months), and at 9 months of age. Children older than 6 months at enrolment were not followed up before age 9 months. The last child was followed up by June 2014.Children and their caretakers were visited at home by field assistants and invited to the health center for NPS.At the health center a questionnaire was completed comprising health status of the child, anthropometric values were measured, and the nasopharynx was swabbed. Information on recent medication of the child (within last three days) and the mother (within last week) was registered  of medicine taken was registered).At 9 months of age, the NPS was taken immediately before measles vaccination.MVPneumo staff were blinded to the randomization status of children since randomization took place in a separate room after the first NPS was obtained. Moreover, randomization status was not registered on the health card of the child so the staff remained blinded at follow up visits.All procedures of MVUrban and MVPneumo were carried out by employees of the BHP, including doctors, nurses and trained assistants.NPS procedures followed a protocol based on WHO recommendations. A flexiThe swab was then rotated in 0.5 ml 0.9% saline and a 0.25 ml aliquot of this specimen solution was transferred by a 1 ml single-use Pasteur-pipette to a cryotube containing 100 μl DNA stabilizing transport medium  and shaken. Samples were temporarily contained in a cold box until frozen at minimum -20°C within a maximum of 7 hours. All equipment was disinfected twice with 70% ethanol between swabbing procedures and the staff wore disposable gloves within the process.™ spin kit for soil  with elution into 100 μl. The number of pneumococci detected from 5 μl DNA-extraction was determined using a quantitative real-time PCR (q-PCR) detecting the autolysin gene (lytA) as described by the US CDC[S. Pneumoniae DNA in TE buffer containing calf-thymus DNA as stabilizer/carrier DNA[Genomic DNA was extracted from 125 μl of the NPS specimen solution using the FastDNAhe US CDC. Furtherhe US CDC. PCR wasrrier DNA. ResultsAll analyses were based on intention-to-treat. One mother agreed to participate in MVUrban but refused to let her child be vaccinated when randomized to early MV; however, estimates from per-protocol analyses were similar since reclassification of one observation did not alter the findings.Categorical variables describing pneumococcal colonization status (No/Yes), were defined based on the presence of pneumococcal DNA detected by q-PCR. In addition, a categorical variable describing recent antibiotic treatment of the child (No/Yes) was defined. The analysis of the effect of antibiotics on pneumococcal colonization and density was not originally planned but was conducted after discovering that early MV children had a markedly lower consumption of antibiotics than controls.6.5 and PR9), were estimated using Poisson regression with robust variance estimation[6.5 and GMR9) of pneumococcal density according to MV status, were computed for colonized children. We applied linear ordinary least square (OLS) regression, using the logarithmic transformed density as variable.The primary outcomes, pneumococcal prevalence ratios at 6.5 and 9 months of age  during campaigns prior to early MV is associated with a less beneficial effect of early MV, hence, Moreover, in a Guinean study investigStratification by receipt of OPV and season were not pre-specified in the protocol, but based on the above mentioned findings.Lower pneumococcal density values are related to increased measurement uncertainty, and only single assessments were done. Nonetheless, exploring cut off values of density ranging from 100 through 2,000 genome equivalents did not modify the estimate of the MV effect on colonization or density. Thus, a cut off was not implemented in the analyses.All estimates are reported with 95% confidence intervals. Analyses were performed in Stata version 12.0 .We intended to include 420 early MV-children and 210 controls based on detecting a 10% reduction of pneumococcal colonization among MV-children, from 85% to 75%, with a power of 80% and significance level α = 0.05.The early MV trial and the pneumococcal sub-study were approved by the Ethical Committee in Guinea-Bissau (CNES-2013-031). The Developing-Country Committee of The National Committee on Health Research Ethics, Denmark, gave its consultative approval. All participants gave their informed consent. MVUrban is registered at clinicaltrials.gov NCT01486355Flowchart of the study). Specimens from 512 children having a successful swab available from both inclusion and 9 months follow up were analyzed (17 samples were lost). Of these, 410 children (80%) also had the 6.5 months swab available and analyzed. Children lost to follow-up differed on a number of factors, e.g. they were older and came from more deprived groups (data not shown). Losses to follow-up were comparable in the early MV group  and the no early MV group .Within the pneumococcal study period, 703 children  were enrolled in the MVUrban trial. Of these, 651 children (93% of inclusions) were enrolled in the MVPneumo study. At age 6.5 and 9 months, 515 (79%) and 529 (81%) children were followed up with a NPS, respectively  were randomized to early MV and 166 (32%) to no early MV. Enrolment in rainy season (Jun-Nov) comprised 65% of children, and 52% of children received OPV during national campaigns before enrolment ; no chilAt enrolment, 80% of children were colonized with pneumococci 411/512, . At age Three health professionals obtained NPSs; controlling for investigator did not impact the assessment of early MV’s effect on colonization status or density (data not shown).6.5 1.02 (0.94–1.10) and PR9 1.04 (0.96–1.12), respectively . At age 9 months, the overall GMR9 was 0.69 (0.39–1.21)  . Again, 6.5 being 0.60 (0.34–1.05) and PR9 0.87 (0.50–1.53).At both follow up visits  early MV6.5 0.85 (0.71–1.01) and PR9 0.66 (0.52–0.84), and decreased density, GMR6.5 0.32 (0.12–0.86) and GMR9 0.52 (0.18–1.52)  . Predomi6.5 NO OPV 0.50 (0.21–1.23), whereas the opposite tendency was seen among early MV-children who did receive campaign OPV before enrolment, (GMR6.5 OPV 1.83 (0.78–4.28). Hence, there was a significant interaction between early MV and campaign OPV before enrolment (p = 0.04). This association was not maintained at age 9 months (GMR9 NO OPV = 0.58 (0.26–1.30), GMR9 OPV = 0.83 (0.38–1.82), test for interaction p = 0.53), possibly due to the effect of later OPV campaigns, as subsequent OPV campaigns tended to blur the difference between those who had and had not received campaign OPV before enrolment (data not shown).Stratification by receipt vs. no receipt of OPV during campaigns before enrolment showed no effect regarding colonization prevalence . At age The analysis by season of enrolment did not show any significant differences between children enrolled in the rainy and dry season, respectively .We found no overall effect of early MV on pneumococcal colonization or pneumococcal density at age 6.5 or 9 months.The randomization ensured that background variables were balanced between randomization groups .In post hoc analyses we found that early MV-children were less frequently treated with antibiotics at follow up. This is in line with the findings of a morbidity subgroup study within the main trial, which found that early MV was associated with a reduced risk of morbidity from 4 to 9 months of age. Thus, ainconsistent mediation[However, we chose not to adjust for antibiotic treatment in the analyses, because bias would be introduced when adjusting or stratifying for a post randomization variable associated with a prior intervention .Unfortunately, antibiotic treatment of children was only registered if taken within three days prior to examination so we are unsure if this observation reflects differences in the overall consumption of antibiotics in each group. Also, no objective measure of antibiotic activity in a child was assessed. Information on antibiotic intake relied solely on reporting by mothers. They do not necessarily know whether a given medication prescribed by the doctor is an antibiotic and they might not remember the exact name of the medicine. At the 6.5 months visit, however, the information was also based on seeing the medicine packaging by the home visiting nurse, increasing the reliability of the information at this visit.The sample size was intended to include minimum 420 early MV-children and 210 controls. However, only samples from 346 and 166 children (early MV/no early MV) were analyzed resulting in a statistical power to show a significant difference of 74% in contrast to the anticipated power of 80%.Additionally, 21% and 19% of enrolled children were lost to follow up at 6.5 and 9 months of age, respectively, and comprised a predominance of the less advantaged children (data not shown), which may reduce the generalizability of the findings. However, since loss to follow-up was evenly distributed between randomization groups we do not believe it affected the estimated early MV effect.Pneumococcal carriage rates among infants in Guinea-Bissau have not previously been investigated, but the observed rates are consistent with reports from other West African countries: The Gambia Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S1 ProtocolEffect of early measles vaccine on pneumococcal colonization: A randomized trial from Guinea-Bissau.(DOC)Click here for additional data file.S2 ProtocolEstudo dos Efeitos não Específicos da Vacina do Sarampo na presença de S.pneumoniae nas vias aéreas nasais das Crianças Guineenses.(DOCX)Click here for additional data file.


The 6-minute walk test (6MWT) in a regular hallway is commonly used to assess functional exercise capacity in patients with chronic obstructive pulmonary disease (COPD). However, treadmill walking might provide additional advantages over overground walking, especially if virtual reality and self-paced treadmill walking are combined. Therefore, this study aimed to assess the reproducibility and validity of the 6MWT using the Gait Real-time Analysis Interactive Lab (GRAIL) in patients with COPD and healthy elderly.Sixty-one patients with COPD and 48 healthy elderly performed two 6MWTs on the GRAIL. Patients performed two overground 6MWTs and healthy elderly performed one overground test. Differences between consecutive 6MWTs and the test conditions (GRAIL vs. overground) were analysed. Patients walked further in the second overground test  and in the second GRAIL test . Healthy elderly improved their second GRAIL test . The GRAIL 6MWT was reproducible (intra-class coefficients = 0.65–0.80). The best GRAIL 6-minute walk distance (6MWD) in patients was shorter than the best overground 6MWD . Healthy elderly walked further on the GRAIL than in the overground condition . Validity of the GRAIL 6MWT was assessed and intra-class coefficient values ranging from 0.74–0.77 were found.The GRAIL is a promising system to assess the 6MWD in patients with COPD and healthy elderly. The GRAIL 6MWD seems to be more comparable to the 6MWDs assessed overground than previous studies on treadmills have reported. Furthermore, good construct validity and reproducibility were established in assessing the 6MWD using the GRAIL in patients with COPD and healthy elderly. Chronic obstructive pulmonary disease (COPD) is a highly prevalent chronic disease affecting about 10% of adults above the age of 40 , 3. ExerAccording to the European Respiratory Society/American Thoracic Society (ERS/ATS) guidelines, a flat corridor of at least 30 meters is required to perform a 6MWT , 5. HoweTo date, two studies have been conducted in assessing treadmill-based 6MWDs in patients with COPD. Both studies established a significantly greater mean overground 6MWD compared to a regular treadmill-based 6MWT  , 8. HoweBoth COPD studies with the 6MWT have used regular fixed-paced treadmills. Conversely, the use of a self-paced treadmill, a feedback controlled function that adapts treadmill speed to its user, could be beneficial to adjust the walking speed more naturally and resulting in a more natural gait pattern compared to fixed speed treadmill walking . In addi1/FVC <0.7) were recruited at pre-rehabilitation assessment between February 2014 and June 2015 [A cross-sectional observational study was conducted in CIRO, a centre of expertise for chronic organ failure located in Horn, the Netherlands. Sixty-one patients with COPD  was used to assess the self-paced treadmill 6MWDs. The GRAIL comprises of a 3D motion analysis system with a dual-belt, instrumented treadmill and a virtual reality 180 degrees projection screen . Four reSample size calculation was based on the results of Stevens et al.. Patientt-tests or two related samples tests. Differences between groups were identified by independent t-tests or two independent samples tests. For consistency with previous studies, mean values of non-normally distributed variables are reported. Predicted values of the 6MWDs for patients and healthy elderly were calculated using the formula of Troosters et al. [p-value <0.05.The assumption of normally distributed data was checked with the Shapiro-Wilk test. If data were not normally distributed, non-parametric tests were used. Differences between overground 6MWTs in patients, differences between GRAIL 6MWTs in each group and differences between the best GRAIL 6MWT and best overground 6MWT in both groups were identified by paired sample s et al. . The Bla1/FVC and FEV1% predicted differed between patients and healthy elderly . The FEV elderly .2nadir ≤ 88% [In the overground condition, patients walked 481.4 m (95% CI 465.7–497.0 m) in the first 6MWT and 506.2 m (95% CI 488.3–524.0 m) in the second 6MWT, with an average increase of 24.8 m . Eighty percent of patients improved during the second overground 6MWT. The best 6MWD in patients was 511.0 m (95% CI 494.5–527.6 m), which was 79% (95% CI 76–82%) of the predicted value. At the end of the best overground 6MWT, patients had Borg dyspnoea and fatigue scores of 5.4 (95% CI 4.8–6.0) and 5.3 (95% CI 4.7–5.9), respectively. Forty-nine percent of patients desaturated (SpOir ≤ 88% ) during Healthy elderly covered 668.8 m (95% CI 647.4–690.1 m), which was 103% (95% CI 100–106%) of the predicted value. At the overground 6MWT, healthy elderly had Borg dyspnoea and fatigue scores of 1.2 (95% CI 0.9–1.5) and 1.2 (95% CI 0.9–1.5), respectively. Oxygen saturation decreased by 1.2% (95% CI 0.2–2.2%) and heart rate increased by 47.3 bpm (95% CI 42.8–51.9 bpm). Difference in best overground 6MWD of patients and healthy elderly was 157.8 m . All overground 6MWT outcomes are documented in Patients walked 449.7 m (95% CI 426.8–472.5 m) in the first GRAIL 6MWT and 476.4 m (95% CI 454.2–498.7 m) in the second test , with anHealthy elderly walked 639.8 m (95% CI 617.5–662.3 m) at first, and then 689.5 m (95% CI 671.4–707.6 m) , with anOn average, the best GRAIL-based 6MWD in patients was significantly shorter than the best overground 6MWD . Conversely, the GRAIL 6MWD in healthy elderly was significantly greater than the overground 6MWD  on the GRAIL compared to overground walking. This difference in 6MWD between the test conditions is smaller than previous studies using regular treadmills have established .5 m on t. Our finDespite the fact that the familiarisation session is only performed prior to the first GRAIL 6MWT, which could have led to less distance covered in the second GRAIL 6MWT, our results indicate that 75% of patients improved their walk distance during the second GRAIL test. This is comparable to the 80% of patients who improved during their second overground test. Therefore, we consider this effect to be minimal. Concerning the duration of a GRAIL 6MWT. One GRAIL session takes more time to perform compared to the overground 6MWT. In addition, the GRAIL is less accessible to all centres compared to regular treadmills. We do however not argue that the GRAIL should be implemented everywhere to assess the 6MWD only. The GRAIL is a unique method to conduct analysis of gait impairments in patients with COPD, as these patients have reported walking as one of the most problematic activity in daily life . Future A first limitation of this study is that healthy elderly performed one overground 6MWT, which might have led to a shorter best overground 6MWD. However, a previous study found a minimal difference of 5 meters between two overground 6MWTs in healthy elderly . TherefoIn conclusion, the GRAIL is a promising system to assess the 6MWD in patients with COPD and healthy elderly. The 6MWD assessed by the GRAIL seems to be more comparable to the 6MWDs assessed overground than previous studies on treadmills have reported. Furthermore, good construct validity and reproducibility were established in assessing the 6MWD using the GRAIL in patients with COPD and healthy elderly.S1 Dataset1 ≥ 80% predicted. GOLD II defined as FEV1 50% ≤ FEV1 < 80% predicted. GOLD III defined as 30% ≤ FEV1 < 50% predicted. GOLD IV defined as FEV1 < 30% predicted.GOLD I defined as FEV(XLSX)Click here for additional data file.


Purpose. To evaluate primary trabeculectomy with adjunctive mitomycin-C (MMC) in diabetic patients without retinopathy with primary angle-closure glaucoma (PACG). Design. This is a retrospective case series comparison. Participants. This retrospective trial compared outcomes of 88 eyes that underwent trabeculectomy in patients with diabetes mellitus (DM) without retinopathy and in 97 patients without DM. Methods. In this study, the intraocular pressure (IOP), visual acuity, visual field, and postoperative complications were compared between the two groups. Qualified surgical success is defined as an IOP between 6 and 18 mmHg with or without topical antiglaucoma medication. Results. After a follow-up of 5 years, the IOP decreased from a mean basal IOP of 27.8 ± 7.3 mmHg to 15.0 ± 5.6 mmHg in the DM group and from 27.3 ± 6.0 mmHg to 12.4 ± 5.3 mmHg in the control group. The mean number of antiglaucoma medications was 3.4 ± 1.3 and 3.3 ± 1.2 preoperatively (P = 0.587) whereas it was 1.7 ± 1.5 and 1.1 ± 1.4 at the 5-year follow-up (P = 0.049). The 5-year qualified surgical success rates were 42.9% and 65.4% for both groups . Encysted blebs were seen in 21 (23.9%) patients in the DM group and in 12 (12.4%) patients in the control group (P = 0.041). Conclusion. PACG patients with DM without retinopathy undergoing primary trabeculectomy with MMC may have a lower long-term surgical survival rate compared with patients without DM. According to current literature, diabetes mellitus (DM) is a risk factor for the failure of trabeculectomy –3. HowevThe purpose of this study is to evaluate the long-term outcomes of primary trabeculectomy with MMC in diabetic patients without retinopathy with PACG.This is a retrospective case series comparison of the outcomes of primary trabeculectomy with MMC between PACG patients with DM (DM group) and PACG patients without DM (control group). A total of 88 eyes from 88 patients with DM who had undergone primary trabeculectomy with MMC were compared with 97 eyes from 97 patients without DM. The study was approved by The Second People's Hospital of Jinan and followed the tenets of the Declaration of Helsinki. Medical records of patients with PACG who had undergone trabeculectomy with MMC between July 2005 and December 2008 and had at least 1 year of follow-up were reviewed.PACG was defined as the structural alteration of the optic disc typical for glaucomatous optic neuropathy or visual field defect typical for glaucoma, in the presence of a closed anterior chamber angle on gonioscopy . Exclusion criteria included glaucoma subtypes or optic neuropathy other than PACG, diabetic or nondiabetic retinal pathology based on documented dilated fundus examination within 6 months, history of retinal photocoagulation, previous intraocular procedure other than cataract surgery, and concurrent procedures performed with trabeculectomy.Patients are grouped by the presence or absence of DM. The DM group was composed of controlled diabetic patients without retinopathy. The control group was selected from all nondiabetic PACG patients undergoing MMC trabeculectomy. If the patient had two eyes that would qualify, the first operated eye was elected. Biomicroscopic, gonioscopic, and fundoscopic examinations were performed in all patients before the surgery. Data collected included demographic data (age and gender); medical data  level whenever available); preoperative and postoperative ocular data (intraocular pressure (IOP), best-corrected visual acuity , glaucoma medication, slit-lamp examination of the anterior segment, fundus examination, lens status, and visual field result whenever available); and surgical data (intraoperative and postoperative complications and any surgeries or procedures performed after trabeculectomy). Data for visual acuity, IOP, and antiglaucoma medication were collected preoperatively and at intervals of 6 months (±2 weeks) and each year (±1 months) thereafter after trabeculectomy. IOP was defined as the average of three IOP measurements by Goldmann tonometry. Snellen visual acuity was converted to logMAR scale for comparison. LogMAR values of 1.40, 2.70, and 3.70 were assigned to counting fingers, hand motions, and light perception, respectively . Only reAll surgeries have been performed by the same single surgeon. After the local anesthesia, the bulbus was stabilized with an atraumatic 4-0 silk traction suture suspending the superior rectus muscle. Trabeculectomy was performed with a fornix-based conjunctival flap (approximately between 11 and 1 o'clock). Then, a scleral flap  was dissected until the entire corneoscleral limbus was exposed. A surgical sponge  measuring 6 mm × 6 mm which was soaked with 0.4 mg/ml MMC  was placed under the sclera flap for 2 min , 11. AftComplete success was defined as the IOP between 6 and 18 mmHg without medications. Qualified surgical success was defined as the IOP between 6 and 18 mmHg regardless of topical antiglaucoma medication. A failure is defined as the IOP ≥ 18 mmHg regardless of any medication. Hypotony was diagnosed in cases with IOP levels of <6 mmHg. Needling of encysted blebs was not considered a failure. The conjunctival leakage was observed under cobalt blue slit-lamp illumination after a moistened sterile fluorescein strip was applied on the bleb surface. A leak was diagnosed if there was any apparent aqueous stream from any leaking point.t-tests were used to evaluate the between-group and within-group differences, respectively. Categorical variables and proportions were analyzed using Pearson's χ2 tests. Success rates in both groups were compared using the Kaplan-Meier life table analysis and the log-rank test. Statistical significance was defined as a P value of <0.05.Data are presented as mean ± SD. All statistical analyses were performed using SPSS statistical software . Independent P < 0.001). Since patients with DM were either on diet control  or diabetic medical therapy , their preoperative blood serum sugar levels were not very high. All cases in the DM group had a preoperative HgbA1c level checked in the preoperative physical examination (mean: 6.8 ± 0.8%). Overall, 69 eyes (78.4%) in the DM group and 75 eyes (77.3%) in the control group had reliable preoperative visual field results for comparison. The differences in MD  on the visual field between the two groups were not statistically significant (P = 0.586).A total of 88 eyes (88 patients) with type 2 DM that had undergone primary trabeculectomy with MMC were compared with 97 eyes (97 patients) without DM. All recruited patients were Asians. Table P = 0.587) whereas it was 1.7 ± 1.5 and 1.1 ± 1.4 at 5-year follow-up (P = 0.049). Mean postoperative number of antiglaucoma medications in the control group was also lower than that in the DM group at all visit intervals 1 year after the trabeculectomy . BCVA differences between the groups were not statistically significant at any visit interval.In the DM group, the mean BCVA at baseline was 0.28 ± 0.31 logMAR and 0.51 ± 0.21 logMAR at the 5-year follow-up. Differences between the preoperative and postoperative BCVA were statistically significant . MD differences between the groups were not statistically significant at any visit interval  in the DM group and 41 eyes (42.3%) in the control group had reliable preoperative visual field results. At the 5-year follow-up, the MD was −15.2 ± 10.2 dB in the DM group and −14.9 ± 9.8 dB in the control group. There were no statistically significant differences between the preoperative and the postoperative visual field results in either group    in the DM group and 59.6% (31 eyes) in the control group (5 eyes in) Figure  at the 5P = 0.041). There were 2 (2.3%) cases of endophthalmitis in the DM group. After vitrectomy, both patients lost light perception. There was no other patient who lost light perception in both groups . Anterior chamber reformation was performed in all cases with prolonged postoperative hypotony, which is always accompanied with prolonged shallow anterior chamber. All eyes with an encysted bleb underwent 1-time needling procedure without injecting any antifibrotic agent P = 0.01. There As far as we know, there is no report about the effect of trabeculectomy on diabetic PACG patients without retinopathy. There are few studies examining the effect of trabeculectomy on diabetic eyes with DM, but the results are conflicting –2, 12–14P = 0.046; log-rank test) at the 5-year follow-up. Mean postoperative IOP was 12.4 mmHg in the DM group and 15.0 mmHg in the control group (P = 0.024). The majority of trabeculectomy failures are characterized by a marked inflammatory response in the conjunctival dermis and Tenon's capsule [To our knowledge, this is the first study to investigate the outcome of trabeculectomy with MMC in diabetic PACG patients without retinopathy. The results showed that long-term IOP was less controlled in the eyes of diabetic PACG patients without retinopathy than in the eyes of those without DM. Mean postoperative IOP of the DM group was higher than that of the control group. The rates of qualified success were 42.9% in the DM group and 65.4% in the control group ( capsule , 17. It The results demonstrated that there were no statistically significant differences in the rate of complications such as hypotony, choroidal detachment, shallow anterior chamber, and conjunctival leakage between the two groups. The incidence of encysted bleb ranged from 2.5% to 29% in literature . In the There was a mild decrease of vision in the DM group and in the control group, and the decrease achieved statistical significance. Matlach et al. have reported a slight decrease in visual acuity after 12 months compared to baseline values . In a siAt the 5-year follow-up, there was a mild decrease of MD in both groups, but the decrease did not achieve statistical significance. The differences in MD between the two groups were not statistically significant at the last visit. Previous studies reported that lowering IOP slowed the advancement of visual field damage in glaucoma patients –25. HoweWhile the postoperative IOP, number of medications required, and surgical success were all worse in the DM group, the postoperative vision and visual field mean deviation were similar between the two groups. There are two reasons: first, the retrospective comparative nature of this study may have caused a bias. Second, a small number of included patients will affect the final statistical results.This study has some limitations. First, the retrospective comparative nature of the study may have caused a bias. Second, we were unable to assess the severity of the glaucoma by the visual field defect, although this has been suggested to play little part in the outcome of trabeculectomy surgery . A prospIn conclusion, diabetic PACG patients without retinopathy undergoing primary trabeculectomy with MMC may not achieve the same long-term IOP control that nondiabetic patients achieve, despite the use of MMC and the same surgical technique, and in the absence of diabetic retinopathy and with blood sugars under medical or diet control. However, further prospective studies are expected.


Introduction: Antiretroviral treatment (ART) reduces HIV transmission. Despite increased ART coverage, incidence remains high among men who have sex with men (MSM) in many places. Acute HIV infection (AHI) is characterized by high viral replication and increased infectiousness. We estimated the feasible reduction in transmission by targeting MSM with AHI for early ART.Methods: We recruited a cohort of 88 MSM with AHI in Bangkok, Thailand, who initiated ART immediately. A risk calculator based on viral load and reported behaviour, calibrated to Thai epidemiological data, was applied to estimate the number of onwards transmissions. This was compared with the expected number without early interventions.Results: Forty of the MSM were in 4th-generation AHI stages 1 and 2 +/4thG immunoassay (IA)-/3rdG IA–; 4thG stage 2, NAT+/4thG IA+/3rdG IA–) while 48 tested positive on third-generation IA but had negative or indeterminate western blot (4thG stage 3). Mean plasma HIV RNA was 5.62 log10 copies/ml. Any condomless sex in the four months preceding the study was reported by 83.7%, but decreased to 21.2% by 24 weeks on ART. After ART, 48/88 (54.6%) attained HIV RNA <50 copies/ml by week 8, increasing to 78/87 (89.7%), and 64/66 (97%) at weeks 24 and 48, respectively. The estimated number of onwards transmissions in the first year of infection would have been 27.3  with no intervention, 8.3 (6.4–11.2) with post-diagnosis behaviour change only, 5.9 (4.4–7.9) with viral load reduction only and 3.1 (2.4–4.3) with both. The latter was associated with an 88.7% (83.8–91.1%) reduction in transmission.Conclusions: Disproportionate HIV transmission occurs during AHI. Diagnosis of AHI with early ART initiation can substantially reduce onwards transmission. Antiretroviral treatment (ART) reduces HIV viral load and HIV transmission in HIV-discordant heterosexual couples –6. HowevAcute HIV infection (AHI) is characterized by high viral replication in blood and genital secretions and hence increased infectiousness to which characteristics of founder viruses may also contribute –26. HereStudy RV254/SEARCH 010 is a prospective cohort study conducted in Bangkok, Thailand, which enrols predominantly Thai participants in the earliest stages of HIV-1 infection. AHI staging was based on a combination of HIV RNA detection (nucleic acid testing (NAT) testing) 4th-generation (4thG) immunoassay (IA), third-generation (3rdG) IA and western blot results: 4thG AHI stages 1, 2 and 3  . Both 4tParticipants who presented for voluntary HIV counselling and testing (VCT) with 4thG IA at two sites in Bangkok, Thailand (the Thai Red Cross Anonymous Clinic and Silom Community Clinic) and gave informed consent had blood samples screened for AHI by pooled HIV NAT and sequential IA according to published methods ,32. AHI HIV RNA was measured in plasma at baseline and weeks 2, 4, 8, 12, 16, 20, 24 and every three months thereafter by either the COBAS® AMPLICOR HIV-1 Monitor Test v1.5  or the COBAS®AmpliPrep/COBAS® TaqMan® HIV-1 test v2.0  with analyses based on lower limit of detection of 50 copies/ml.The number and type of sexual partners , condom use and specific sexual behaviours were assessed by standardized written questionnaires asking quantifying information which were completed by participants in a private setting at baseline, week 24 and week 48. . The questionnaire asked data about number of partners; oral/anal intercourse practices, role  and condom use; and knowledge about HIV sero-status of each partner in the preceding four months.Questions regarding behavioural practices, such as unprotected anal intercourse, were asked as part of standard medical history from each participant by the study physicians at each visit. If crucial information was missing from either questionnaire or medical history, these were supplemented by each other in the final data set used for this analysis.Estimated date of infection was assessed from questionnaire and medical history.Study participants who reported sex with male partners and opted for immediate ART were included in the analysis. Statistical analyses were done using Stata/IC 12.1 for windows .Time-dependent individual-level plasma HIV viral load and reported transmission-related behaviour data were used to inform a risk-equation model. The model included the  transmission probability per-act and frequency of types of transmission-related acts. Calculations were calibrated by adjustment of the parameter for transmission probability per-act associated with viral load set point to reflect HIV incidence estimates among MSM in Bangkok of 5.9 infections per 100 person years (95% CI 5.2–6.8) and an HIV prevalence estimate of 23.9% (95% CI 21.1–27.0) . This moTransmission risk behaviours of the cohort reported at the time of diagnosis were kept constant across the simulation in this counterfactual/no-intervention scenario. Two further hypothetical scenarios were simulated: (i) a scenario where only behavioural changes were considered as reported in the cohort data and (ii) a scenario in which behaviours were assumed to be as they were just prior to diagnosis but viral load reduction was considered as measured in the study. These scenarios were projected to distinguish between the influence of viral load reduction through treatment and diagnosis-related risk behaviour changes.We used an adaptation of Røttingen and Garnett’s previously reported equation  of time . Here,  where There is a strong relationship between HIV viral load and transmission ,36. Blaswhere Bangkok) ,33. The ercourse ,38.n = 48), the remainder in 4thG stages 1 (n = 19) and 2 (n = 21). Mean CD4+T cell count was 412 cells/mm3, mean plasma HIV RNA 5.62 log10 copies/ml and the majority of participants (73%) were infected with HIV-1 subtype CRF_01AE, the predominant circulating form in Thai MSM [Baseline characteristics of the 88 participants are summarized in Thai MSM . CompletAny condomless sexual intercourse in the preceding four months was reported by 72 participants (83.7%). Condomless intercourse with regular partners, casual partners, commercial sex workers, paying partners and partners who inject drugs was reported by 55 (64%), 49 (57%), 6 (7%), 6 (7%) and 9 (10.5%) participants, respectively. None reported abstinence from sexual intercourse in the four months preceding the study while 10 (11.6%) reported a single partner. Two to four partners were reported by 47 participants (54.7%) and ≥5 partners by 25 (29.1%) participants. Four participants (4.7%) declined to answer.p < 0.001) and 29/65  reporting a single partner or no partner at weeks 24 and 48, respectively. However, 25 (29.1%) of 86 participants reported ≥5 partners in the preceding four months at baseline while 9/65 (13.9%) reported ≥5 partners in the preceding four months at week 48 (p = 0.06); 4/9 participants were among the 25 who reported ≥5 partners at baseline. Any reported condomless sexual intercourse declined from 72/86 (83.7%) at baseline to 18/85 (21.2%) at week 24 (p < 0.0001) and 20/65 (30.8%) at week 48 (p < 0.0001)  reported multiple partners in the preceding four months, while 10/86 (11.6%) and 0/86 reported a single or no partners, respectively, and 4 (4.6%) declined to answer. This contrasts with 43/85  ; 16/18 aMean plasma HIV RNA decline is shown in Based on viral load and transmission-related risk behaviour of each individual within the cohort, we estimated the rate of infectiousness per infected individual per day . Figure In the baseline  scenario, the mean number of estimated new infections caused per infected individual over their first year of infection was 0.035, whilst in the hypothetical scenario of no early detection and treatment this value was estimated at 0.294. In the hypothetical scenario where only viral load reduction is considered (without changes to risk behaviour), the mean number of estimated new infections caused per infected individual was 0.066. The value for the scenario in which only post-diagnosis behaviour changes are considered was 0.093. We also calculated potential additional second-, third- and fourth-generation transmissions in the first year from the index cases in the cohort. In the baseline scenario, an estimated mean 3.00  direct infections and 0.08 (0.06–0.11) indirect infections are caused by the cohort one year after HIV exposure, giving a total of 3.08 (2.37–4.29) new transmissions. In the hypothetical scenario of no early detection and treatment intervention, the estimated total number of new infections caused by the cohort after one year was 27.26 (21.68–35.26): 25.34 (20.09–32.85) direct and 1.92 (1.59–2.41) indirect transmissions. These results translate into an estimated 88.7% (83.8–91.1%) reduction in onwards transmissions, one year after HIV exposure due to early detection and treatment .Figure Under the scenario of post-diagnosis behaviour change only, an estimated 8.26 (6.39–11.24) transmissions are caused by the cohort (approximately 70% reduction compared to no intervention), whereas the number of new infections in the viral load reduction-only scenario was 5.88 (4.42–7.91) (approximately 78% reduction compared to no intervention).Our study provides evidence of substantial impact in reducing HIV transmission among Thai MSM through diagnosing HIV during acute infection and early initiation of ART. Post-diagnosis behavioural changes can significantly reduce the number of new HIV infections, but viral load reductions through ART have the greatest impact and together are estimated to reduce transmission by 89% over the first year of infection.The broader population impact of such interventions may depend on the stage of the HIV epidemic. Current literature estimates the fraction of HIV transmissions in urban MSM populations resulting from AHI partners to be anywhere from 4% to 50% ,19,40–45Benefits of treatment as prevention for MSM are considered highly plausible ,46. HoweThe public health challenges of optimizing the engagement of care cascade to facilitate treatment as prevention strategies at the population level have been well described, recently with focus on engaging people with AHI ,51. WhilWhile we need longer follow-up data to assert whether the observed behavioural changes are sustained, it is very encouraging that they are at least sustained until the vast majority of participants have reached plasma HIV RNA below the level of detection. Screening for AHI and linkage to care and treatment have been routinely practised in other settings, including North America and Australia, showing both feasibility and cost efficacy –66.Our study is subject to limitations, which should be noted in interpreting results. The main finding of estimated transmissions averted is dependent on the relationship between viral load and HIV transmission probabilities. The relationship used was derived from heterosexual discordant couples in latent infection but adjusted to reflect higher transmission probability magnitudes for MSM in Bangkok and calibrated to local incidence and prevalence data. Calculations used numbers of self-reported transmission-related risk events, which are subject to potential bias. Social desirability and information bias cannot be excluded in gathering historical information on sexual behaviour. Given the sensitive nature of the information, we assume that self-reported behavioural data may underestimate number of partners and overestimate condom use. Standardized medical history taking at each follow-up visit readdressed these questions as a result of which such phenomena were minimized as much as possible. The duration of time from infection to diagnosis cannot be verified or estimated precisely, regardless of evidence of acute HIV detection among the individuals included in this study.There is strong evidence that ART not only has individual clinical benefits but also substantially reduces transmission risk. Given the large contribution of AHI to population incidence, we aimed to assess the potential effect of targeting detection of AHI in Bangkok-based MSM and initiation of early ART. We estimated that this approach could lead to 89% reduction in HIV transmissions during the first year of HIV infection when levels of transmission are highest. We believe that global elimination targets by 2030 require investment in prioritizing detection and treatment of early HIV infection. We have provided evidence that intensive efforts with such a strategy can lead to large changes in transmission and thus potential epidemic reversal among MSM.


Gabon is an endemic area for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) and the risk of co-infection is high.Between November 2015 and April 2016, we conducted retrospective study on HCV infection among people living with HIV/AIDS (PLHA). A total of 491 PLHA were included in this study and tested for the presence of HCV infection. HIV viral loads were obtained using the Generic HIV viral Load® assay and the CD4+ T cells count was performed using BD FACSCount™ CD4 reagents. HCV screening was performed using the MP Diagnostics HCV ELISA 4.0 kit. HCV genotypes were determined by sequence analysis of NS5B and Core regions. The Mann-Whitney test was used to compare the groups. Chi-2 test and Fisher's Exact Test were used to compare prevalence.HCV seroprevalence was 2.9% (14/491), :1.4–4.3%). The percentage of HCV viremic patients, defined by the detection of HCV RNA in plasma, was 57% (8/14), representing 1.6% of the total population. HCV seroprevalence and replicative infection were not statistically differ with gender. The percentage of co-infection increased with age. No correlation with CD4+ T cells count and HIV viral load level was registered in this study. Identified HCV strains were predominantly of genotype 4 (87.5%) including 4k, 4e, 4g, 4p, 4f and 4c subtypes. Only one strain belonged to genotype 2 (subtype 2q). Analysis of the NS5B region did not reveal the presence of resistance-associated substitutions for sofosbuvir.A systematic screening of hepatitis C is therefore strongly recommended as well as genotyping of HCV strains in order to adapt treatments for the specific case of people living with HIV/AIDS in Central Africa. People living with human immunodeficiency virus (HIV) often become infected by hepatitis viruses, whether hepatitis C (HCV), and/or hepatitis B (HBV), occasionally associated with hepatitis Delta (HDV). In addition, reactivation of hepatitis virus in patients infected with HIV is now considered significant ,2. TheseHCV infection alone is a leading cause of chronic liver disease in the world. The World Health Organization (WHO) estimated that in 2015 about 170 million people were infected by HCV. In sub-Saharan Africa, the overall HCV prevalence is 5.7% with 32 million people affected, and therefore this region is considered as highly endemic . There wModes of transmission of HCV are related to blood contact: reuse of needles , blood transfusions, tattoos, piercing, and transplantations. Sexual transmission is less often implicated, excepted in cases of men having sex with men. Finally, the vertical transmission rate is low, around 5%, but it increases by 2 to 5 times in cases of HIV-HCV co-infection .The HCV viruses are classified into 7 genotypes, each devised in subtypes . HCV genotype 1a is most commonly detected in Europe and the USA, while genotype 1b is distributed worldwide with high prevalence in Europe and the USA as well as in Japan. Genotype 2 is common in Europe, Japan, North America and in West Africa. Genotype 3 is most common in India, Indonesia and South-East Asia. Genotype 4 appears to be prevalent in Central Africa and the Middle East, while genotype 5 is most frequently reported in South Africa. Genotype 6 is found in Hong Kong and South-East Asia . RecentlIn Central Africa, several studies report that HCV prevalence varies widely between countries, ranging from 0.6% to 50.6%, depending on the populations studied, the age and the transmission routes . On averIn Gabon, HCV prevalence varies from 2.1% among pregnant women in urban areas, to 11.2% in rural areas, and can reach 20.4% in very remote villages –19. PrevA recent study performed in Gabon, to provide comprehensive guidance for HCV testing, indicated that the HCV prevalence was 7.3% among patients living with HIV in semi-rural areas .Gabonese guidelines on HIV/AIDS recommend hepatitis C screening. Recently, oral direct-acting antiviral (DAA) combination regimen of sofosbivir (SOF) and daclastavir (DCV) is affordable.In addition, the development of new technical platforms in Gabon allows the virological follow-up of the treated patients.In this context, it is important to know the HCV prevalence among people living with HIV/AIDS (PLHA) in order to establish future needs. For this, we propose to determine the prevalence of HCV in PLHA and to describe the diversity of HCV strains circulating in Libreville, capital of Gabon.This retrospective study was carried out from November 2015 to April 2016 in Libreville, Gabon. HCV antibody detection was performed on 500 blood samples taken from PLHA who came to the laboratory as part of their virological and immunological follow-up . For 9 of them, the data were insufficient and were excluded from the study. For 491 patients, the data on gender, age and antiretroviral treatment were provided by clinicians. The study was approved by the Ethics Committee of Gabon (N°0021/2013/SG/CNE). Written informed consent was obtained from each patient.Whole blood specimens were collected in EDTA-containing tubes. After centrifugation at 800g, 10 min, resulting plasma was frozen at -80°C. All plasma samples were tested for HCV IgG antibodies using the third generation ELISA test  based on double antigen “sandwich” principle and according to the manufacturer’s instructions. Specimens with a signal-to-cutoff S/CO ratio <0.7 were considered non-reactive, and those with an S/CO ratio ≥1.0 were considered reactive. All specimens with an S/CO ratio ≥1.0 including one specimen with S/CO ratio between 0.7 and 1.0 were further tested with an immunochromatographic assay  for confirmation. The specimen with S/CO ratio between 0.7 and 1.0 was non-reactive and was considered as negative.Viral RNA extractions were performed from clarified plasma, (centrifugation at 3000g/10 min) using Arrow Viral NA™ in NorDiag Arrow automatic extraction system . HIV-VLs were obtained using the Generic HIV viral Load® assay with a detection limit of 100 copies/mL .The CD4+ T cells count was performed using a BD FACSCount™ cytometer with a total of 50 μl of whole blood using BD FACSCount™ CD4 reagents  according the recommendations of the manufacturer.g for 10 min) with the QIAamp RNA viral Mini Kit , as recommended by the manufacturer. 10μl of RNA were used to perform both RT-PCRs.Specimens positive for HCV antibodies were submitted for HCV genotyping. RNA was extracted from 140 μL of clarified plasma . Pairwise alignments were performed using MEGA 6.06 software : 37–52) with no significant difference between females (42 years [IQR: 36–50]) and males (48 years [IQR: 43–55]). Among them, 72.9% (n –230500) .In our study, antibodies against HCV were found in 2.9% of PLHA . Out of these, the HCV RNA was detected in 57.1% (8/14), 1.6% of the total population (95% CI: 0.5–2.7%) .st line ART treatment. No patient was taking a 3rd line ART treatment (The medians of CD4+ T cells and HIV-VL were respectively 350 cells/μL (IQR = 93–561) and 1520 copies/mL (IQR = 99–355000). HCV prevalence was 2.1%  in female and 4.3%  in male. HCV viral RNA was detected in 1.2% of female and 2.4% of male, respectively. However, HCV seroprevalence and replicative infection were not statistically differ with gender (p = 0.25 and p = 0.45). Serological and molecular prevalence were higher in older patients but without statistical significance (p = 0.21 and p = 0.06). No correlation with CD4+ T cells count and HIV viral load level was registered . All datreatment .Out of the fourteen HCV seropositive patients, we successfully amplified HCV RNA in 8 patients for the three regions studied . Firstly, to determine the genotype we performed alignment of our sequences with reference sequences of HCV genotypes in core and NS5B regions. As shown in Figs et al. 2012, including the representative strains from all the provincial capitals of Gabon.In addition, we compared our NS5B sequences with 211 sequences obtained by Njouom Two our HCV strains were closely related to subtype 4k, one respectively to subtype 4g, 4p, 4f, 4c, and the last one to 4e . Our onlThis study was conducted to assess the impact of HCV co-infection in PLHA. Through this study, we recorded a low HCV prevalence (2.9%). This prevalence increased significantly with age but without statistical significance with regard to gender (p = 0.25). Our result in PLHA is similar to those described previously in pregnant women, from Libreville, with an average prevalence of 2.1% (1.3% among 14–20 years old and 6% among those over 35 years old) . ConversRecently, a study conducted in PLHA from Franceville (Haut-Ogooué province) showed an average prevalence of 7.3% . This prA similar trend was found in Cameroun, where PLHA living in cities were less likely to be infected with HCV compared to those in rural villages although the difference was not statistically significant .HCV spreAverage prevalence of HCV for PLHA in Central Africa was estimated at 5.87% (95% CI: 3.71‒8.04) ,31. Our Out of 14 HCV seropositive patients, 8 were viraemic (57.1%), corresponding to the overall prevalence of 1.6%. This rate of replicative infection corroborates those from previous studies ,18. HowePhylogenetic analyses indicate the circulation of two HCV genotypes (4 and 2) in PLHA in Libreville. Genotype 4 was predominantly found in 7 of 8 (87.5%) HCV viraemic patients, followed by genotype 2 in one patient. These HCV genotypes were previously found in all studies in Gabon ,20. The Genotype 2 has already been described, with a prevalence of 2.2% in Gabon, but not in the area of the Estuary . We did For the purpose of eradicating HCV, the oral HCV direct-acting antiviral combination with sofosbuvir/daclastavir is available in Gabon. The absence of naturally occurring sofosbuvir resistance-associated variants for both genotypes 4 and 2 is comforting for the choice of this strategy and its long-term effectiveness.However, there is some limitation to this study. Information on some known risk factors for HCV was missing and should be incorporated into future studies In addition, wider sampling will enable more statistical power in the analysis of the impact of HCV in PLHA. Finally, future studies should incorporate biochemical values of liver function as well as HCV viral load values.Presently, one of the challenges is the implementation of an epidemiological and molecular surveillance of Gabonese population. Diagnosis of HCV genotypes is clinically relevant as genotypes differ in their response rates to the current standard treatment . AdditioContrary to expectations, the prevalence of HCV in PLHA is low in Libreville. The risk factors for the acquisition of this infection in urban areas should be sought as they appear to be clearly different to those in rural areas. However, as the circulating genotypes are identical to those previously found in Gabon we assume that the origin of these HCV strains remains local. Finally, systematic screening, quantification of HCV RNA and genotype characterization in PLHA must be integrated to the health system in order to obtain quality care.S1 Table(XLS)Click here for additional data file.S1 FigNeighbor-joining phylogenetic tree constructed using the Kimura two-parameter method of estimating genetic distance. Numbers next to the nodes of the tree represent bootstrap values (1000 replicates). Gabonese strains are in red. Our sequences in bold are preceded by a red spot.(TIFF)Click here for additional data file.S2 FigNeighbor-joining phylogenetic tree constructed using the Kimura two-parameter method of estimating genetic distance. Numbers next to the nodes of the tree represent bootstrap values (1000 replicates). Gabonese strains are in red. Our sequence in bold is preceded by a green spot.(TIFF)Click here for additional data file.


Chlorambucil (CHB), a standard front-line therapy for elderly patients at trial initiation, was used as a comparator.7Lenalidomide ((LEN) Revlimid; Celgene Corporation, Summit, NJ, USA), an oral immunomodulatory agent, demonstrated activity in phase 2 trials in patients with chronic lymphocytic leukemia (CLL) in front-line therapyThe study protocol is summarized in the The data monitoring committee observed an imbalance in deaths between the treatment arms favoring CHB in February 2013. As a result, patients aged ⩾81 years discontinued study treatment in April 2013, and all patients receiving LEN discontinued treatment in July 2013. By March 2014, all patients in the CHB arm had also stopped treatment. Patient flow and the rationale for the data cutoff dates are provided in Median age was 72 years (range 65–90) and 73 years (range 65–92) in the LEN and CHB arms, respectively. Baseline characteristics were well balanced between treatment arms . More paP=0.323). There were 35 deaths (16.5%) in the LEN arm and 21 (9.8%) in the CHB arm. The HR for OS was 1.69  in favor of CHB. Similar results were seen at the April 2013 cutoff date, when all patients aged ⩾81 years discontinued treatment  was 30.8 months in the LEN arm versus 23.0 months in the CHB arm. The hazard ratio (HR) for PD or death favored the CHB arm but was not statistically significant  0.88–1.66; reatment . As of MP=0.883; .P=0.026). Complete response (CR) was achieved in 6 (2.7%) LEN-treated patients and 22 (9.8%) CHB-treated patients. Median time to first response was 8.9 weeks for LEN-treated patients versus 8.1 weeks for CHB-treated patients. Median duration of response had not been reached for LEN-treated patients versus 87.1 weeks for CHB-treated patients. The HR for duration of response favoring LEN was 0.74 .Overall response rate at the April 2013 cutoff date was significantly lower in the LEN arm than the CHB arm , with greater frequency of treatment discontinuations due to treatment-emergent adverse events (TEAEs) in the LEN arm. Median number of treatment cycles was 7 in the LEN arm and 10 in the CHB arm. Median dose intensity was 4.9 mg per day in the LEN arm. More patients in the LEN arm than in the CHB arm had study drug interrupted and/or reduced due to adverse events . Neutropenia was the most frequently reported TEAE, leading to dose reduction/interruption in either treatment arm. More patients in the LEN arm had dose reductions/interruptions due to neutropenia than patients in the CHB arm .TEAEs were reported by more LEN-treated patients than CHB-treated patients . The most frequently reported (⩾2%) TEAEs leading to discontinuation were thrombocytopenia, pneumonia and tumor flare for LEN and neutropenia, anemia and thrombocytopenia for CHB.At the April 2013 cutoff date, 61 deaths had occurred—36 (16.0%) in the LEN arm and 25 (11.1%) in the CHB arm. The primary causes of death are shown in More deaths were observed among older LEN-treated patients: in the overall LEN-treated population, 11.6% of patients were aged >80 years; however, 27.8% of those patients who died were >80 years. Of the 26 LEN-treated patients aged >80 years, 10 patients (38.5%) died compared with 26 (13.1%) of 199 LEN-treated patients aged ⩽80 years. Over-representation of deaths in older patients was not observed in the CHB arm where the proportions of patients >80 years (11.1%) and patients >80 years who died (12.0%) were similar. Fewer deaths occurred in responders than in non-responders: 10.5 vs 22.8%, respectively, in the LEN arm, and 6.1 vs 20.8%, respectively, in the CHB arm.LEN-treated patients who died were more likely to have had baseline comorbidities requiring treatment and/or a more complex medical history than CHB-treated patients . A higheA greater proportion of patients who died had moderate renal impairment (CrCl ⩾30 to <60 ml/min). In the LEN arm, 39.1% of patients overall had moderate renal impairment, whereas of patients who died, 58.3% had moderate renal impairment. In the CHB arm, the incidence of moderate renal impairment was 43.6% overall, and 56.0% in the patients that died.As of March 2014, a total of 20 (8.9%) patients in the LEN arm versus 32 (14.3%) in the CHB arm experienced ⩾1 second primary malignancy (SPM). A lower proportion of patients in the LEN arm experienced an invasive SPM (hematologic and solid tumor) versus the CHB arm . Hematologic SPMs occurred in 3 (1.3%) vs 4 (1.8%) patients in the LEN versus CHB arm, respectively; solid tumor SPMs occurred in 5 (2.2%) versus 10 (4.5%) patients, respectively.At the March 2014 cutoff date, approximately one-third of patients in the LEN (36.6%) and CHB arms (33.6%) had received subsequent anticancer therapies. As there was no apparent imbalance between the treatment arms, the use of subsequent anticancer therapies was not believed to have confounded the OS results.On the basis of these interim results of a trial that was terminated early, it appears that LEN did not prolong PFS and was associated with a lower response rate, a higher incidence of grade ⩾3 AEs and a higher number of deaths compared with CHB. Although LEN demonstrated clinical activity in a subset of patients in this trial, LEN monotherapy is not recommended as first-line therapy for patients with CLL, particularly those who are elderly and/or frail. Further research will be required to determine whether future immunomodulatory agents could have a role in the treatment of CLL.


To improve endometrial quality and implantation rates after theadministration of platelet-rich plasma in patients with refractoryendometrium.in vitro fertilization, aged between33 and 45 years with a history of refractory endometrium, to whom plateletrich plasma was given by infusion with a catheter into the uterine cavity onthe tenth day of the hormone replacement therapy, and then 72 hours afterthe first administration.19 patients undergoing Endometrial thicknesses >7mm was reported with the first use; and in allcases, endometrial thicknesses >9mm were evident after the secondadministration. The entire study group qualified for Embryo Transfer at theblastocyst stage. We had 73.7% of positive pregnancy tests, of which 26.3%yielded live births; 26.3% ongoing pregnancies; 10.5% biochemicalpregnancies; 5.3% anembryonic pregnancies and 5.3% had fetal death (16weeks).Platelet-rich plasma and its biostimulation effects on the endometrialmicrovasculature seems to be beneficial to patients with refractoryendometrium, providing an increase in endometrial receptivity and aconsequent increase in implantation rates. As an autologous resource, theyare easy to obtain and inexpensive. Thus, we recommend it to be included inthe different protocols for endometrial preparation, including those inwhich a natural cycle is preferred. The refractory endometrium, characterized by atrophy with endometrial interfacemeasurements below 6 mm by ultrasound; and/or the presence of intrauterine adhesionsand synechia through hysteroscopy, which is the so-called Asherman syndrome,continues to represent a serious problem for the practitioner who does theendometrial preparation for patients undergoing Assisted Reproductive Techniques(ART). This type of endometrial pattern is still responsible for many embryotransfer (ET) cancellations or implantation failures . These dOriginally, PRP was defined as "the volume of autologous plasma having a plateletconcentration above the basal line". It was later defined as a group of soluble anddiffusible polypeptide substances able to regulate the growth, differentiation andphenotype of numerous cell types vis-à-vis those that do stimulate all phases of regeneration (platelets),explains its ability to reinforce tissue regeneration , phosphatidicacid and lysophosphatidate. These substances have an anti-apoptotic effect onendothelial cells, they are involved in the chemotaxis of endothelial cells, andtheir proliferation and promotion of adhesion bonds form capillary-like structures.Sphingosine appears to be critical to the onset of angiogenesis, since it is theligand of endothelial differentiation gene (EDG) receptors. Finally, theoverexpression of VEGFR2 renders the endothelium sensitive to the production of VEGF to initiate angiogenesis from endothelial cells and (IVF Caracas) in the period from February 2016 toFebruary 2017.To improve endometrial quality and implantation rates after the administration ofplatelet-rich plasma in patients with refractory endometrium undergoing Prospective Design.This study was carried out in the Maternal Reproductive Assistance Unit (inSpanish UNAMATER) in Merida, Venezuela and at the Venezuelan Institute ofFertility Caracas (in Spanish IVF Caracas) in Caracas D.C, Venezuela.In Vitro Fertilization), aged between 33 and 45years with a previous history of refractory endometrium and at least one failedIVF attempt. To these patients, an autologous PRP serum was applied in aquantity of 1 cc by infusion with a catheter into the uterine cavity, on thetenth day of HRT with Estradiol Valerate, and again at 72 hours of the firstadministration (day 12 of HRT). Endometrial thickness was determined bytransvaginal ultrasound.The study population consisted of 19 infertile patients undergoing HighComplexity ART (This study was carried out for one (1) year, from February 2016 to February2017.All patients with failed IVF, either performed in our Units of Human AssistedReproduction or in External Units, who expressed a desire to undergo a newIVF+ET treatment, in whom preliminarily refractory endometrium was detected.During the period from February 2016 to February 2017.Patients without unsuccessful IVF attempts.Patients with previous IVF but without refractory endometrium.Clinicians, Embryologists, Anaesthesiologists, Nurses, Cardiologist,Psychologist, among others.Assisted Human Reproduction Units: Maternal Reproductive Assistance Unit(UNAMATER) in Merida, Venezuela and Venezuelan Institute of Fertility Caracas(IVF Caracas) in Caracas D.C, Venezuela.The data was collected in a form especially designed for this study.The patients included in this study consented with their signature and that of awitness; an additional written Informed Consent was used for the application ofautologous PRP serum.After completing collection, the data was transcribed to a database andstatistically analysed using the SPSS software version 20.0. The descriptivestatistical results are presented in the form of tables of frequencydistribution, figures and measurements.In the population studied, endometrial thicknesses >7 mm was obtained with thefirst application of PRP, and in all cases endometrial thicknesses greater than 9 mmbecame evident after the second application of the autologous preparation. Theentire study group qualified for ET in the blastocyst stage.Of a total of 19 patients with refractory endometrium who underwent IVF+TE during theperiod between February 2016 and February 2017 and who received two PRP applicationsas adjuvant in the endometrial preparation, 14 positive pregnancy tests, whichcorresponded to 73.7%; and 5 presented negative pregnancy tests, which wasequivalent to 26.3%. The average age of the patients studied was 39.21±3.22years (range 33-45 years). When categorizing age values into two age groups, 57.9%(n=11) were grouped around 33-39 years and 42.1% (n=8) aged 40-45 years .With regards to the number of previous IVF attempts, 57.9% (n=11) had had one (1)previous IVF; followed by 31.6% with 2 previous IVF and 10.5% with 3 previous IVF. In relaAs for the type of pregnancy, 26.3% (n=5) presented healthy live births; 26.3% (n=5)had ongoing clinical pregnancies with satisfactory evolution; 10.5% (n=2) hadbiochemical pregnancies; 5.3 % (n=1) corresponded to anembryonic gestation and 5.3%(n=1) had fetal death at 16 weeks, for which the results of the embryogenic studywere expected .vs. 10.5% (n=2) of oocyte receptors had had one previous IVF.15.8% (n=3) with their own oocytes vs. the same percentage (n=3) of oocyte receptorshad had 2 previous IVF and an equal percentage of 5.3% (n=1) for each grouppresented had had 3 previous IVF  of the patients with their own oocytesious IVF .When coious IVF .Regarding oocyte origin and its relation to the type of pregnancy, 21.4% (n=3)correspond to ongoing clinical pregnancies in patients using their own oocytes,14.3% (n=2) for ongoing clinical gestation in oocyte receptors, 14.3% (n=2) of livebirths with their own oocytes vs. 21.4% (n=3) in oocyte receptors. Same percentageof 7.1% (n=1) for each biochemical gestation group. For anembryonic gestation we had7.1% (n=1) and 7.1% of fetal death (n=1) was present only in the group of patientsusing their own oocytes .vs. 10.5% (n=2) with negative pregnancy test. Finally, 10.5%(n=2) of the patients with positive pregnancy tests presented a history of (3) IVFprevious vs. no case in patients with negative pregnancy tests (In relation to the number of previous IVF and the result of the pregnancy test a42.1% (n=8) with a positive test had a history of (1) previous IVF, 15% (n=3) with anegative pregnancy test. A 21.1% (n=2) with positive test presented (2) previous IVFcy tests .Regarding the type of pregnancy related to the number of previous IVF, the highestpercentages of 21.4% (n=3) and 14.3% (n=2) of ongoing clinical pregnancies and livebirths corresponded to patients with one (1) previous IVF, followed by 14.3% (n=2)of live births and ongoing clinical pregnancies in patients with two (2) previousIVF. Biochemical pregnancies with 14.3% (n=2) and anembryonic pregnancy of 7.1%(n=1) in patients with (1) previous IVF. Fetal death at 16 weeks added up to 7.1%(n=1) in patients with three (03) previous IVF. .When referring to patient age in relation to the number of oocytes, the highestpercentage was 52.6% (n=10) of own oocytes in the age group between 33-39 years oldand 15.8% (n=3) of own oocytes in the group between 40 - 45 years. A 5.3% (n=1)oocyte donation in the age group of 33-40 years vs. 26.3% (n=5) oocyte donation inthe age group of 40 - 45 years .When contrasting the age of the patients and the number of previous IVF, 42.1% (n=8)of the patients aged between 33-39 years had previous IVF, 10.5% (n=2) of the sameage range group presented 2 previous IVF and 5.3% (n=1) of patients presented 3previous IVF. Similarly, a 15.8% (n=3) of the patients between 40-45 years had ahistory of 1 previous IVF, 21.1% (n=4) of this age group presented 2 previous IVFand a 5.3% (n=1) had 3 previous IVF procedures .Regarding the age of the patient in relation to the result of the Pregnancy Test,42.1% (n=8) of the patients between 33-39 years of age had a positive test comparedto 15.8% (n=3) in this age group that had negative pregnancy tests. The 31.6% (n=6)of patients between 40 - 45 years of age had a positive test versus 10.5% (n=2) ofnegative pregnancy tests in the same age group .Finally, when correlating the age of the patients and type of pregnancy, we foundthat the highest percentages in both age groups corresponded to ongoing clinicalpregnancies and live births, with a 14.3% (n=2) and a 21.4% (n=3) in the age groupbetween 33-39 years. 21.4% (n=3) a 14.3% (n=2) respectively, for the age group 40-45years. Biochemical gestation in the same percentage 7.1% (n=1) for both age groups.A 7.1% (n=1) anembryonic pregnancy and a fetal death of 7.1% (n=1) occurred only inthe 33-39 age group .in vitro fertilization + embryo transfertreatment. In this sense, the detection performed for one year, showed improvementsregarding endometrial thickness in the population studied, after the use of theautologous PRP. Regarding the implantation rates expressed in positive pregnancytests, 73.7% (n=14) presented very satisfactory results, matching the findingsobtained in the few previous serial clinical studies on the matter.The main objective of this study was to improve endometrial quality and implantationrates after the administration of platelet-rich plasma in patients with refractoryendometrium undergoing The main advantage of this detection would be the availability of an autologousresource of low cost, which offers obvious benefits regarding the development of areceptive endometrium that favours embryo implantation. PRP administration as an adjuvant in the endometrial preparation of patients withrefractory endometrium, demonstrated a good sensitivity for increasing endometrialthickness, as well as, to obtain positive pregnancy tests, clinical pregnancies andlive births. The use of the PRP preparation and its biostimulation effects on theendometrium microvascular endothelium seems to offer benefits to refractoryendometrium, providing an increase in endometrial receptivity and a consequentincrease in implantation rates. Being an autologous resource, therefore harmless tothe patient, easy to obtain and of very low cost, it is recommended to be includedin the different protocols for endometrial preparation in assisted reproductiontechniques, even in those where the natural cycle is preferred, that is, without therequirement of hormone replacement therapy.Further studies are recommended in terms of the number of population under study andthe time frame evaluated, as well as the approach of comparative studies betweendrugs and autologous preparations, to envisage effective therapeutic alternativesfor refractory endometrium. Despite having the latest resources, uterinetransplantation, something unimaginable a while ago, arises today in response tofertility preservation as far as uterine factor is concerned. It is necessary toestablish preliminary protocols with the use of a wide range of therapeuticalternatives, within which the autologous preparations are becoming increasinglyimportant today based on the positive results obtained.


To evaluate the single incision laparoscopic appendectomy (SILA) using existing instruments, the 10-mm laparoscope, and glove port technique.SILA was performed on 16 patients  between June 2012 and September 2015. A 20-mm incision was made in the umbilicus and a wound retractor was placed. A 10-mm trocar for the laparoscope and two 5-mm trocars were fixed to the three fingers of the latex gloves and it was attached to the wound retractor. Another thin forceps were inserted from right low abdomen.3, and average CRP was 1.81 ± 3.70 mg/dL. Preoperative body temperature was 36.8 ± 0.5°C. The mean appendix size was 9.6 ± 2.3 mm and none of the patients had an abscess on preoperative CT. The CT also revealed a fecal pellet in 5/16 (31%) of patients. Mean operation time was 66.4 ± 25.4 minutes, and minimal intraoperative bleeding was observed in all patients. Average hospital stay was 5.3 ± 1.9 days and none of the patients had complications.Average age of patients was 32.6 ± 17.7 years. Preoperative average white blood cell was 13,325 ± 4,584 /mmSILA using the 10-mm laparoscope and glove port technique may be a safe and feasible operation for mild to moderate appendicitis. First reported by Semm in 1983In this study, we aimed to confirm the feasibility of performing SILA using existing instruments, a 10 mm laparoscope system, and the glove port technique.A retrospective chart review was carried out on patients who underwent SILA for acute appendicitis from June 2012 to August 2015 at the Ehime Rosai Hospital in Niihama, Japan and Saiseikai Shimonoseki General Hospital in Shimonoseki, Japan. The study comprised 16  patients with mild to moderate symptoms and catarrhal or phlegmonous appendicitis. Patients who underwent multi-port laparoscopic appendectomy or open abdominal appendectomy for necrotic or perforated appendicitis were excluded from the study. Patient characteristics , as well as preoperative- , surgical- , and postoperative-related factors  were retrospectively analyzed.® XS) was placed at the umbilical wound. A 10-mm trocar for the laparoscope and two 5-mm trocars were fixed to the three fingers of the size 5.5 latex gloves  patients. The appendicular root was laparoscopically ligated and cut in 9 cases, and was removed and inverted at the umbilical wound under direct visualization in seven cases. Mean operation time was 66.4 ± 25.4 minutes, and there were minimal intraoperative bleeding in all patients. Only one patient (6%) required a drainage tube in the Douglas pouch. Average hospital stay was 5.3 ± 1.9 days. None of the patients developed complications.Between June 2012 and September 2015, 8 male and 8 female patients were enrolled in the study. The average age of patients was 32.6 ± 17.7 years and the mean body mass index was 21.1± 4.5 kg/mAcute appendicitis is one of the most common diseases encountered by surgeons. Although conservative therapy using antibiotics has been widely accepted for acute appendicitis, about 30 % patients eventually received surgical operations within a year.It currently supersedes open appendectomy for acute appendicitis because of obvious benefits, such as relief of postoperative pain, better cosmetic results, and shortened hospital stay.® was guided to the appendicular root or if the intestine was displaced by the forceps (Acute appendicitis is most common among those between 10-19 years forceps .2. However, all patients who underwent simple ligation without inversion did not present with postoperative residual abscess. We believe that serious postoperative complications in laparoscopic appendectomy did not develop even among those without stump inversions.® simple ligation and stump closure with invaginating suture and those who had open appendectomy, except for the significantly longer operation time in the invaginating suture group.12Many Japanese surgeons invert the appendix stump using the purse-string suture during open appendectomy in order to avoid postoperative residual abscess. Similarly, we also performed this from the umbilical wound and with direct visualization. This technique was utilized among thin patients with an average BMI of 19.8 kg/mIn conclusion, although we did not include patients with severe appendicitis that was complicated by an abscess or advanced adhesion, SILA using the 10-mm laparoscope and glove port technique may be a safe and feasible operation for mild to moderate appendicitis. We believe that this technique should be aggressively pursued, particularly for younger patients who were not managed in affluent hospital and do not have the capability to buy a new 5-mm laparoscope system.Takaaki Tsushimi and Hirohito Mori performed surgical procedure.Takaaki Tsushimi analyzed and interpreted the patient data and drafted the manuscript.Manabu Sudo, Minami, Koichi Ueki and Makoto Tamai participated substantially in conception, design, and execution of the study and in the analysis and interpretation data.All authors read and approved the final manuscript.


Doses were at physicians' judgments. Sleep duration and latency were evaluated based on patients' sleep diaries over 14 days; sleep quality was evaluated at 28 ± 1 days. A total of 409 subjects were enrolled. The groups were balanced at baseline for age, sex, weight, and sleep disturbances. At day 14, both groups reported improved sleep latency and duration; latency was reduced from baseline by 37.3 ± 36.3 min with Neurexan and by 38.2 ± 38.5 min with valerian. The duration of sleep increased by 2.2 (±1.6) h in the Neurexan group and by 2.0 (±1.5) h in the valerian group. Differences between the groups in improvement on sleep duration were significantly in favor of Neurexan therapy at days 8, 12, and 14. At day 28, quality of sleep was improved in both groups with no significant differences between the treatments. Significantly more patients reported lack of daytime fatigue with Neurexan than with valerian therapies . For patients favorable towards a CAM-based therapy, Neurexan might be an effective and well-tolerated alternative to conventional valerian-based therapies for the treatment of mild to moderate insomnia.Insomnia is prevalent and complementary therapies are common, but data are lacking on the effectiveness and tolerability of preparations beyond valerian. Here we report on an open-label, prospective cohort study in 89 German centers offering both conventional and complementary therapies. Subjects received the homeopathic preparation Neurexan


S)-β-phenylalanine, easily accessible by lipase-catalyzed kinetic resolution, a chiral triamine was assembled by a reductive amination and finally cyclized to form the title compound 10. In the crystals of the guanidinium benzoate salt the six membered rings of 10 adopt conformations close to an envelope with the phenyl substituents in pseudo-axial positions. The unprotonated guanidine 10 catalyzes Diels–Alder reactions of anthrones and maleimides (25–30% ee). It also promotes as a strong Brønsted base the retro-aldol reaction of some cycloadducts with kinetic resolution of the enantiomers. In three cases, the retro-aldol products (48–83% ee) could be recrystallized to high enantiopurity (≥95% ee). The absolute configuration of several compounds is supported by anomalous X-ray diffraction and by chemical correlation.Starting from ( The besosolvent . By simphed data –24. Inte10  and C–H···O contacts . For the remaining combinations  only the Michael products 29–31 could be observed .The chiral guanidine es 22–24  [19,25–2products . Our resproducts . The subs 99% ee –35. In t10 we started with the structurally simplest case, the reaction of anthrone 20 and N-phenylmaleimide (22) which finally turned out to be the most complex one (25 were obtained in CHCl3 (37% ee) and CH2Cl2 (34% ee). Much better enantioselectivities of the Michael product 28 were found for reactions in solvents of increased polarity. Assuming that 28 is formed from 25 in a retro-aldol step, one might expect similar ee values for both compounds. This is clearly not the case. In THF the 83% ee for compound 28 strongly deviates from the low value for 25 (6% ee). Obviously, in the reaction of 20 and 22, a Diels–Alder step with low to moderate stereoselectivity is superimposed with a kinetic resolution of enantiomers in the subsequent retro-aldol reaction. Both are catalyzed by the chiral guanidine 10. As a result, the numbers shown in To determine the enantioselectivity of guanidine plex one . The besrac-25 reacted with 0.1 equiv of 10 in THF at −15 °C, at 22% conversion ee values of 18% and 70% were found for compounds 25 and 28. The dominant isomer of Michael product 28 is shown below to be R configurated which corresponds to the slower running peak in R isomer of 28 is formed from the S,S isomer of 25 with retention of configuration  and 27 (28% ee) in CH2Cl2 when no secondary conversion occurs. In contrast, when compound 21 reacts with 24 ring opening is so fast that no Diels–Alder product accumulates. Here the ee value of the Michael product 31 (21%) must correspond to the stereoselectivity of the Diels–Alder step. Due to low stereoselectivities and fast retro-aldol reactions guanidine 10 is not optimal for the preparation of Diels–Alder adducts.When guration . The cha THF see , at 45% 20 and maleimide 22 reacted with 0.1 equiv of 10 in THF at −15 °C for 64 h. Chromatographic separation then yielded 51% of 25 and 42% of Michael product 28 (83% ee). Recrystallization afforded 32% of 28 with 99% ee. Thus, 12.5 mg of catalyst 10 produced 51 mg of almost pure R-28. Analogous treatment of 20 and 23 yielded 21% of 29 (61% ee). After recrystallization 15% of 29 with 98% ee were obtained. Finally, Michael product 30, obtained from 20 and 24  yielded 26% of material with 95% ee.We therefore focussed our efforts on the Michael products: Anthrone Assignment of absolute configurations . Both compounds must result from the S,S configurated Diels–Alder adducts. In the case of 25 this is the faster running isomer on Chiralpak IA. In an earlier study [33, we have assigned the S,S configuration to the major isomer of product 32. By hydrogenolysis, we could now convert such a sample (40% ee) into 25. The product S,S-25 was formed as the dominant product (35% ee) thus confirming our previous assignment. Under mild basic conditions, S,S-32 finally could be opened into R configurated Michael product 30 [urations : Well grspersion . This isoduct 30  (31% ee)S)-β-Phenylalanine (13), readily accessible by enantioselective hydrolysis of ester rac-12, is the starting material for an efficient synthesis of the chiral TBD analog 10. The cyclization steps in particular, which convert the triamine 19 into the final guanidine 10, give considerably higher yields compared to guanidines with five-membered rings such as compounds 6–9. Apart from the better synthetic accessibility the formal ring expansion to the six-membered structure of 10 does not increase the stereoselectivity when it is used as a catalyst. We could not observe the near-perfect selectivities of the Tan catalyst 8 in the base-induced reaction of anthrones and maleimides. Nevertheless ee values of up to 83% for the formal Michael products allowed us to isolate 3 compounds in almost enantiopure form after a single recrystallization. The Tan guanidine 8 [33 [S,S isomer of Diels–Alder adduct 25. Although guanidine 10 has the opposite configuration, it also forms S,S-25 preferentially. Accordingly, transition-state structures in the reactions of guanidines 8 and 10 must differ from each other..X-ray data of guanidine File 329 (CCDC-1482612).X-ray data of compound File 430 (CCDC-1482613).X-ray data of compound