Whilst previous work has identified clustering of the active trachoma sign “trachomatous inflammation—follicular” (TF), there is limited understanding of the spatial structure of trachomatous trichiasis (TT), the rarer, end-stage, blinding form of disease. Here we use community-level TF prevalence, information on access to water and sanitation, and large-scale environmental and socio-economic indicators to model the spatial variation in community-level TT prevalence in Benin, Cote d’Ivoire, DRC, Guinea, Ethiopia, Malawi, Mozambique, Nigeria, Sudan and Uganda.We fit binomial mixed models, with community-level random effects, separately for each country. In countries where spatial correlation was detected through a semi-variogram diagnostic check we then fitted a geostatistical model to the TT prevalence data including TF prevalence as an explanatory variable.The estimated regression relationship between community-level TF and TT was significant in eight countries. We estimate that a 10% increase in community-level TF prevalence leads to an increase in the odds for TT ranging from 20 to 86% when accounting for additional covariates.We find evidence of an association between TF and TT in some parts of Africa. However, our results also suggest the presence of additional, country-specific, spatial risk factors which modulate the variation in TT risk. Chlamydia trachomatis infection, an organism that produces chronic inflammation of the tarsal conjunctiva. This is characterised by sub-epithelial follicles, which may meet the definition for the sign trachomatous inflammation—follicular (TF) [Trachoma is a blinding disease caused by recurrent ocular lar (TF) . TF is tlar (TF) . Throughlar (TF) , 4, conjlar (TF) . Correctlar (TF) , 6 or eplar (TF)  are usedOcular chlamydial transmission is declining in many countries –11 suggeC. trachomatis, and TT a proxy for historic transmission. The prevalence levels of these signs are therefore signals for C. trachomatis transmission intensity at different times . Even though TF prevalence and TT prevalence are markers of transmission at different time points or over different time scales, in areas where antibiotic mass drug administration (MDA) for trachoma [C. trachomatis transmission intensity has remained more or less constant over decades, and that TF prevalence and TT prevalence will therefore closely correlate. This assumption is reasonable if access to water, sanitation, hygiene and anti-chlamydial antibiotics at community level have been constant or have changed only gradually. However, such an assumption is not always valid [The pathogenesis of trachoma, implicitly conceptualized within WHO recommendations for district-level interventions, is of repeated episodes of active trachoma incrementally increasing the cumulative risk of TT. It should be noted that this may be a simplistic outlook on the complicated pathway to TT and additional elements may influence progression. However, active trachoma is a pre-requisite on TT’s causal pathway, with moderate to high prevalences of TF being a proxy for current transmission of ocular trachoma  has not ys valid , 15.Many national programmes  have sucThanks to the GTMP, there has been an increasing availability of high quality geolocated trachoma and water, sanitation and hygiene (WASH) data. The GTMP was launched in December 2012 with the aim of mapping the global prevalence of trachoma in all suspected-endemic districts, through completion of population based prevalence surveys. It systematically collected trachoma and WASH data across 1546 districts in 29 countries, nearly exclusively in areas where control activities, including antibiotic MDA, had not yet occurred . These dIn this study, we attempted to identify risk factors that, in addition to TF, might associate with variation in community-level TT prevalence. To this end we fit binomial mixed models, with random effects at community level, to GTMP baseline data from ten countries. We then test for residual spatial correlation and, in countries where this is detected, use geostatistical methods in order to model the variation in TT prevalence between countries.Ten GTMP collaborating countries provided data for this study: Benin, Cote d’ Ivoire, Democratic Republic of the Congo (DRC), Ethiopia, Guinea, Malawi, Mozambique, Nigeria, Sudan and Uganda. Data provided were from 15,051 clusters (or communities) within 624 trachoma elimination intervention-naïve evaluation units (EUs)  [Previous studies have shown that lower precipitation levels and higher temperatures can lead to an increase in the risk of TF . Therefodatabase . The ariIAR-CSI) . CGIAR-CIt has been suggested that frequent sandstorms that occur in some areas of Sudan cause eye trauma . IrritatWe speculate that access to healthcare and other services are associated with developed infrastructure, and therefore sought an infrastructure indicator. Light density at night has been shown to be correlated with local economic activity and gross production rate at different scales , 45. Nig2 resolution covariates, we estimated the mean value in a 5 km × 5 km window using the aggregate tool in the Spatial Analyst toolbox of ArcGIS 10.1 . When shrinkage of spatial resolution was needed for the 1 kmj2 is the fraction of explained variance in the j-th explanatory variables by the other explanatory variables.To identify collinearity among the selected variables, we used the variance inflation factor (VIF) , definedpi denote the probability of having TT, β0 is the intercept and Ui is community-level unstructured random effects (let i denote the i-th community). We fit the following nested binomial mixed models, where γTFi is the regression coefficient for the effect of TF prevalence on the log-odds of TT:dij in M3 are the explanatory variables described in the previous section. We use the log-likelihood ratio test to select among the three models defined above.Let In fitting M3, we also carried out variable selection using a backward stepwise approach, starting from the mixed effects model with all the variables included. The likelihood-ratio test was used to test for the significance of each variable, with terms removed one by one until all those remaining were significant at 5% level.Ui from the best model identified in the previous step, and then computed the empirical semi-variogram. A semi-variogram provides insights into the rate of decay of spatial autocorrelation in the data. It does this by computing the mean squared difference between pairs of residuals as a function of the distance between their associated geographical locations. A flat semi-variogram is interpreted as evidence against the presence of spatial correlation. To test for spatial correlation more formally, we also generated 95% confidence intervals under the assumption of spatial independence. These intervals were obtained by computing semi-variograms on 1000 randomly permuted point estimates of Ui, while holding the geographical locations fixed.To assess the presence of residual spatial correlation, we first obtained a point estimate of the community-level unstructured random effects Ui was modelled as a spatial Gaussian process with a stationary and isotropic correlation function. All the geostatistical models were fitted in the PrevMap [In cases where we found evidence of spatial correlation, we fitted geostatistical binomial logistic models, in which  PrevMap  package.The output for the cluster-level tests suggested that temperature, precipitation, aridity index, and PET interact with one another Table . Since aP < 0.05) between community-level TF prevalence and TT prevalence in all countries except Guinea and Uganda. In contrast, there was evidence of association with access to latrines in 4 of 10 countries ), with access to improved latrines in 2 countries (Nigeria and Uganda (p < 0.05)), and with water source variables in 3 countries ). Observed relationships with environmental factors were equally heterogeneous, with associations observed with aridity index in DRC, Ethiopia, Nigeria and Sudan; and with sand/soil fraction only in Benin and Ethiopia. Night light was associated in DRC, Ethiopia, Mozambique and Nigeria.The strength of association for variables in the full mixed effects model varied between countries Table . There wThe nested mixed effects models Table  show thaThe best models, selected using the likelihood ratio test, are shown in Table Semi-variograms generated with Pearson’s residuals of the best fitting non-spatial binomial models suggest presence of residual spatial correlation in Benin, DRC, Ethiopia, Mozambique and Sudan. The 95% confidence intervals generated under the assumption of spatial independence demonstrate spatial correlation in these countries Fig. .Fig. 2SeThe distance at which spatial correlation fell below 5% ranged from 3.0 km (in Ethiopia)  to 14.2 km (in Mozambique) , corresponding with a very rapid decline in spatial correlation with distance at larger scales, after accounting for covariates  a change in transmission dynamics over time, (2) population movement, (3) a pointer to the fact that we are using all-trichiasis as the dependent variable, rather than only trichiasis due to trachoma or (4) other factors are influencing TT aside from Streptococcus pneumoniae and Staphylococcus aureus [Importantly, in these models, the proportion of variance explained by TF ranges from 6% (in Nigeria) to 42% (in Benin). This range highlights the complex relationship of the distribution of TT and the distribution of TF. Environmental covariates, on average, explain an additional 9% (in Ethiopia) to 46% (in Nigeria) of variance. Our models suggest that while community-level TF prevalence is generally the strongest single predictor of TT, it does not fully explain the variation in community-level TT prevalence, and implying that occasionally, high-TT-prevalence populations will be found where TF is rare. It has been widely observed that dry conditions  is a risk factor for TF in children , 50–52. s aureus , could is aureus .M. sorbens breeding if the latrines are not appropriately maintained, thereby deterring some potential users whilst protecting householders from legal or peer pressure to build an adequate facility. The development of TT requires many previous C. trachomatis infections [High levels of self-reported latrine use by adults, aridity index and 1997 night light had strongly significant associations with TT prevalence in only three of ten countries. This suggests that hygiene practices, dry climate and historic infrastructure may link to increased community-level TT prevalence in some settings, but generally they do not. Previous studies have clearly shown the association between access to WASH and risk of TF , 39, 56 fections  and so pMany other studies have identified correlates of high TF prevalence, including potential socio-economic, demographic and environmental risk factors , 56–58, The variation between countries in directions of association of environment-related indicators and the variation in spatial structure indicate that fitting a single model to the whole set of data is inadvisable. These variations are presumably attributable to country context.There may be several explanations for the inconsistency of associations between large-scale indicators and community-level TT seen between countries. Studies have shown that post-operative recurrence of TT , 65, 66 We observed residual spatial correlation in only five countries , suggesting that in the remaining countries there are no outstanding large-scale environmental factors influencing progression to TT.In the geostatistical models, we identified a very rapid decline in spatial correlation with distance at larger scales, after accounting for covariates. This suggests that very closely adjacent communities have similar levels of TT.The lack of consistent risk factors beyond community-level TF raises concerns that the models identified artefacts that are not generalizable, such as non-trachomatous trichiasis, or that the clinical history of trachoma varies substantially between settings. This underlines the importance of understanding local context when designing interventions for at-risk populations. Whilst our findings are not generalizable across countries, they can provide general direction for where to initiate case finding activities. As has been found in the Guinea Worm eradication program, active surveillance and case finding will be essential as trachoma elimination endpoints draw closer ; these a


Cavia porcellus) and ram lambs (Ovis aries). Individuals of both species were administered r-met-hu/G-CSF daily for 4 days. During and after administration protocols, testicular function and development were assessed through hematological responses, hormonal profiles  testicular morphometry and germ cell kinetics. As expected, r-met-hu/G-CSF acutely mobilized white-lineage blood cells in both species. LH was increased by r-met-hu/G-CSF in guinea pigs (P<0.01) but T remained unchanged. In ram lambs gonadotropins and T increased in dose-response fashion (P<0.01) while cortisol values were stable and similar in treated and control animals (P>0.05). In guinea pigs there were no differences in testicular weights and volumes 2-mo after r-met-hu/G-CSF application (P>0.05). However, ram lambs showed a dose-response effect regarding testis weight (P<0.05). 66.66% of ram lambs had initial testes not yet in meiosis or starting the first spermatogenic wave. After 60-days only 25% of control animals were pubertal while all treated animals (1140-μg) had reached puberty. We propose an integrated hypothesis that G-CSF can stimulate spermatogenesis through two possible ways. 1) r-met-hu/G-CSF may go through the brain blood barrier and once there it can stimulate GnRH-neurons to release GnRH with the subsequent release of gonadotrophins. 2) a local testicular effect through stimulation of steroidogenesis that enhances spermiogenesis via testosterone production and a direct stimulation over spermatogonial stem cells self-renewal. In conclusion, this study shows that r-met-hu/G-CSF differentially affects prepubertal sexual development in hystricomorpha and ovine species, a relevant fact to consider when designing methods to hasten sexual developmental in mammalian species.The understanding of mammalian spermatogenesis niche factors active during sexual development may be leveraged to impact reproduction in farm animals. The aim of this study was to evaluate the effects of r-met-hu/G-CSF (filgrastim) on prepubertal sexual development of guinea pigs ( Spermatogenesis is a complex physiologic process in which spermatogonial cells differentiate into spermatozoa after mitosis, meiosis, and spermiogenesis . In mammStarting the prepubertal period the germinal epithelium initiates spermatogenesis for the first time under the influence of several hormone pulses  and when sperm cells appear in the seminiferous tubules, male animals reach puberty ,5,6. TheGranulocyte colony-stimulating factor (G-CSF) is a member of the hematopoietic growth factor family –11. G-CSCavia porcellus) and a farm animal ram lambs (Ovis aries).In order to gain insight into the possible roles of r-met-hu G-CSF on male sexual function and development during the early prepubertal period, the aim of the present study was to evaluate its effects by monitoring hematological responses, endocrine profiles, testicular tissue development and germ cell kinetics in a prepubertal laboratory animal, guinea pig , in strict accordance with the recommendations in the legal framework  currently in place in AGROCALIDAD Ethics Committee (MAGAP- AGROCALIDAD), for all Ecuadorian Public and Private Laboratories and Higher Education Institutions . "This research was performed under the Project License PROM-CEB-013-2014 from the National Secretariat of Higher Education, Science, Technology, and Innovation (SENESCYT), in strict accordance with the recommendations in the legal framework  currently in place and approved by AGROCALIDAD Ethics Committee (MAGAP- AGROCALIDAD), and under regulations for all Ecuadorian Public and Private Laboratories and Higher Education Institutions . Guinea pigs were castrated under general anesthesia (ketamine + xylacine). For castration, ram lambs were sedated with xylacine and local anesthesia was used (lidocaine). Analgesics were used for 24h after surgery "All reagents and media used in the present study were purchased from Sigma  unless otherwise stated. In all cases, reagents and media were used according to manufacturer recommendations.Cavia porcellus; n = 32) from Peruvian genetic-line (age 1-mo) were used for the different experiments. All animals were randomly sorted by experimental groups and kept in indoor individual boxes (0.6×0.75×0.5 m). Entire Corriedale breed ram lambs  were maintained in indoor individual pens (5×5 m). The buildings for the housing of the animals were disinfected with quaternary ammonium (day -7) every 7d. In all cases, vitamins and dextrose were given to attenuate transportation and initial stress. All animals were quarantined for 15d before the experiments started to ensure a disease-free status and adaptation to their new environment. During adaptation (8-days) all animals were dewormed, kept in an enriched environment and fed alfalfa forage, standard formulated concentrate-food and water ad libitum during the experimental period.Entire male guinea pig males . In guinea pigs, three r-met-hu G-CSF treatment groups  were administered 1 μg, 10 μg and 20 μg respectively per animal for 4 days . In ram lambs, three r-met-hu G-CSF treatment groups (n = 5 each) received 18 μg, 180 μg and 360 μg per animal respectively, daily for 4 days . The control group (n = 5) was administered with saline (NaCl 0.9% w/v), instead of r-met-hu G-CSF. In order to evaluate r-met-hu G-CSF treatment effect on testicular morphometrics all animals were castrated . After assessing the results, 12 or 17 animals  of the same ages, species, husbandry and sources were used for a second experiment to evaluate spermatogenesis through histological and stereological methods. On this second experiment, 4 or 6 initial control animals were castrated at day-0 , 4 guinea pigs were treated with the highest dose  and 4 were controls (ram lambs n = 6). r-met-hu G-CSF treated animals and controls were castrated at ages 3 mo (guinea pigs) and 4 mo (ram lambs). Irrespective of the species studied, individuals were randomly allocated to experimental groups. In a first experiment, 5 control and 15 treatment animals were used. In both species each treatment consisted in 4 daily SC applications of r-met-hu G-CSF  provided essential information to monitor bone marrow response to different r-met-hu G-CSF treatments. Changes in cell profiles were evaluated at days 1,2,3,4 and 5 post-treatment. Hemograms were carried out with a HumaCount 30TS® hematologic analysis system . The analysis included all available red blood cell (RBC) and differential white blood cell (WBC) counts and indices. RBC count and indices included mean corpuscular volume (MCV) and red cell distribution width (RDW) among others. Differential WBC counts included total WBC count, absolute granulocyte count (AGC), absolute lymphocyte count (ALC), absolute monocyte count (AMC). Wright stained smears were evaluated by using a Human Scope® microscope  to assess relative values.Blood samples (1 mL) from guinea pigs were aseptically taken for hormone analysis  by making a small incision at the interdigital space, once, 5 days after the first r-met-hu/G-CSF injection. In ram lambs, 5 mL-blood samples were obtained from the external jugular vein. For each individual, a blood sample was taken at days 1, 5, 15, 30 and 60 . A blood sample was taken to the group of animals castrated at the beginning of the experiments (before castration). Hormonal assays were performed at a private accredited bioanalysis laboratory. Hormonal assays were performed using the enzyme-linked immunosorbent assay (ELISA) kits . For the direct quantitative determination of FSH, LH, T and Cortisol, specific monoclonal antibodies  were used. A two-step assay was carried out for the quantification of each hormone. The first monoclonal specific antibodies were used as first immobilized factors onto the microwell plate and the second specific antibodies, conjugated with radish peroxidase (RP), were designed for a different specific region of each hormone.oC before histological fixation. The tunica vaginalis and epididymis were excised and testis weight recorded. Volume was estimated by the water displacement method using a graduated cylinder. Testes did not show any apparent macroscopical abnormalities. Testes were transversally excised and tissue samples were fixed in neutral buffered formalin  or Bouin’s fixative and stored in 70%v/v ethanol (4 oC). Testicular tissue pieces (~0.5 cm3) were transferred into biopsy cassettes for further histological processing. Samples were dehydrated in alcohol series and xylene and infiltrated with paraffin in a histoprocessor . Samples were embedded in paraffin blocks and cut 5-μm thick with a microtome . Slides were deparaffinised, stained with Harris hematoxylin and mounted with Secure Mounttm, .Sixty days after the last r-met-hu G-CSF application all animals were castrated (control and treatment groups). Guinea pigs were castrated in a surgical area, under general anesthesia . For castration, ram lambs were sedated with xylacine (0.1mg/kg/IV) and local anesthesia was used (lidocaine) in a clean environment (barn). Guinea pigs were administered aspirin . Ram lambs were given flunixin  immediately after the surgery and a second dose after 24h. Animals were monitored and showed no signs of pain or abnormal behavior during the first 72h after surgery . Testes were obtained early in the morning and kept at ~4 http://imagej.nih.gov/ij/) in an average of 80 and 200 cross-sections of tubule profiles/animal (guinea pigs and ram lambs respectively).Testicular tissues were analyzed for general morphology. Stereologic and morphometric parameters were estimated. Images were captured with a digital video camera . Tubule diameters were measured on the digital images. Tubular diameters were calculated as the average of the major and minor axes of randomly chosen near-round cross-section profiles of seminiferous tubules. Diameters were measured with image-J software . During the meiotic phase animals show primary spermatocytes as the most advanced GC types. Animals were considered in spermiogenesis when spermatids (in any step) were the predominant GC type. Pubertal animals were those with at least 10% of seminiferous tubule cross-sections with sperm in the lumen.Cavia porcellus: 12500 μm3 each, total of 200000 μm3 per image; Ovis aries: 200000 μm3 each, total of 3200000 μm3 per image) and by multiplying this density by the testicular volume. Clearly, each spermatid produces one sperm while the time divisor (which is considered a constant for a species) is the sum of the duration of the stages (in days) were round spermatids appear. To this end, the duration of the cycle and frequency of the stages is required. For guinea pigs, the time divisor is 2.57  in reference to the total duration of the cycle (8.45 days) [Daily sperm production (DSP) and efficiency of spermatogenesis (DSP/g of testicular parenchyma) were estimated and compared among treatments in animals with full spermatogenesis. To this end, digital images of 20 random microscopic fields at 400x were captured and used for round spermatid counting through the physical dissector . Each im45 days) . For rampost-hoc test.As the effect of r-met-hu G-CSF on the reproductive status of farm and lab animals has not been widely studied and there are not enough data available , power calculation for animal number estimation was not performed previous to the start of our experiments. Numbers of animals used for the treatments used are specified in the figure legends. Every animal participating on this study was considered an experimental unit. All statistical analyses were performed using SPSS for Windows® software, v17. General statistical analysis of haematological values, hormonal levels, stereologic and morphometric variables was performed using one-way ANOVA. The level of significance was set at P<0.05 and when the analyses were statistically significant, multiple comparisons were performed by means of Bonferroni In r-met-hu G-CSF treated guinea pigs white blood cells (WBCs) increased in numbers between days 1 and 2, irrespective of the doses used compared to the control (P<0.05) to later on decrease to control levels. WBCs response was mostly based in an increase in granulocytes which had a sustained response compared to the controls until day 3 (P<0.01), when levels dropped back to control values . Red bloThe patterns of bone marrow response after administering r-met-hu G-CSF differed in ovine species. WBCs in treated ram lambs had consistent higher values after day 3, significant with highest doses of r-met-hu G-CSF (720 μg and 1140 μg), (P<0.01), . This pans. Testosterone values were unchanged in treated guinea pigs with respect to controls (P>0.01), , an effect more clear with the lowest dose (4 μg), while FSH values showed a similar pattern, though statistically P>0.01), .In r-met-hu/G-CSF treated ram lambs, gonadotropins and testosterone values constantly and clearly increased during the first 5 days compared to the controls, in a doses–response fashion (P<0.01), . At day r-met-hu G-CSF had different sets of effects depending on the species studied. In guinea pigs there were no differences in treated animals compared the control group regarding testicular weight and volume 2-mo after the r-met-hu/G-CSF administration (P>0.05), , while iSeminiferous tubule diameters were significantly lower in guinea pigs treated with the lowest doses of r-met-hu/G-CSF compared to control animals . However, the diameters of animals treated with higher doses were similar to those of the control . Ram lam6 and 11.9 ±2.8 x 106, respectively.In guinea pigs at day 0, 100% of the animals were already in spermiogenesis. After 60d, both control and r-met-hu/G-CSF treated guinea pigs (80 μg) were pubertal, with near 20% of them with incipient sperm production (less than 10% of seminiferous tubule cross-sections with sperm presence) and 80% with clear adult spermatogenesis, regardless of the treatment group . Puberta6 and 1.1 ±0.2 x 106, respectively.No ram lamb testis before experiments was beyond meiosis: 50% of the animals were in neonatal stage (gonocytes as the most advanced cell type), 25% in spermatogenic stage and only 25% had entered meiosis. Thus, 75% of animals were on a pre-meiotic phase. After 60 days of experiments , 33.33% of non treated animals had entered meiosis, 33.33% were in spermiogenesis and 33.33% were pubertal while r-met-hu/G-CSF treated animals were all pubertal . Ram lamCavia porcelus) and farm (Ovis aries) animals to better understand the role of this growth factor in a non-hematopoietic tissue. Monitoring reproductive function and sexual development is crucial for the improvement of animal production programs [In this work we report for the first time the effects of r-met-hu G-CSF on sexual development by following haematological responses, testicular function, endocrine profiles and germ cell kinetics in prepubertal laboratory . Accordingly, peripheral WBCs increase in numbers within days of administration as reported in humans, pigs, horses, rats and mice –43. The Unchanged cortisol levels throughout experiments with ram lambs showed that the animals were under normal, steady stress conditions and also that the stress hormonal  axis was kept intact under the effect of r-met-hu/G-CSF in this species . However, there is evidence in the literature supporting both involvement  or not  of the aEven though blood cell dynamics was similar in guinea pigs and ram lambs treated with r-met-hu/G-CSF, reproductive hormone patterns differed significantly. Guinea pigs had one blood sample taken 5 days after r-met-hu G-CSF administration. In that specific time window, only LH was elevated in low r-met-hu/G-CSF -dosed animals while FSH and testosterone were unaltered, regardless of the dose. On the other hand, ram lambs had gonadotropin and testosterone values above those of control animals 5 days on after r-met-hu G-CSF administration. Overall effects of r-met-hu/G-CSF on guinea pig sexual development were not conclusive, perhaps due to species-specific sensitivity to the drug. Accordingly, with normal testosterone values found in r-met-hu/G-CSF treated guinea pigs, testis size and seminiferous tubule compartment were unchanged during the experiments. r-met-hu G-CSF treated guinea pigs did not show variations in the duration of the first spermatogenic wave leading to puberty since all animals were initially in spermiogenesis and the end of the experiments (after 2 mo) both groups (r-met-hu/G-CSF treated and control) were pubertal. Furthermore, in order to see the effect of r-met-hu G-CSF at the cell dynamics level, daily sperm production (DSP) and the efficiency of spermatogenesis (DSP/ g of testicular parenchyma) were estimated. Evaluation of spermatogenesis through these stereologic methods is based on key literature research through. Castration is often used and important/practical information derived in animal science . For ins-/- mice show a general disregulation of the Hypothalamus-Pituitary-Testis axis that results in 90% lower testosterone concentrations compared to the wild type [scc, in the conversion of cholesterol to pregnelonone [r-met-hu/G-CSF effects on the sexual development of ram lambs showed a different pattern. Reproductive hormone progression in r-met-hu G-CSF treated animals is consistent with an activation of the Hypothalamus-Pituitary-Testis axis. Accordingly, higher testis weights were observed in treated animals. Moreover, r-met-hu/G-CSF treated animals were all pubertal at the end of the 2 mo experimental period while only about 25% of control animals were producing sperm. Interestingly, control animals were in the expected testis developmental age-range reported in the literature for several ovine breeds  but r-meild type . Furtherild type  and probild type . CSF-1 anelonone . CSF-1 mr-met-hu/G-CSF treated ram lambs, which had increased reproductive hormone values also had had heavier testes. Within the testis, CSF-Rs are located in SSCs ,63,64. CIn the present work, r-met-hu G-CSF effect in extra-bone marrow tissues such as the male gonad seems to be long-term contrary to short-term hematopoietic effects. Species-specific effects were clearly observed. Guinea pigs long term reproductive unresponsiveness was probably due to non-equivalent developmental periods during the experiments compared to ram lambs, lower drug-sensibility, higher doses requirements or different cytokine (of human derived) -receptor affinities. Rams had their hypothalamus—pituitary–gonad axis activated and enhanced spermatogenesis with higher doses of r-met-hu G-CSF. More studies are needed in different species and under a wider range of r-met-hu G-CSF concentrations.In conclusion, the present work suggests r-met-hu G-CSF administration contributes to the unset of male mammalian puberty associated with highly species-specific responses during testicular development. Drug concentrations will require adjustments in order to modulate and improve the developmental kinetics of GCs with the aim of accelerating puberty arrival. Moreover, this study shows that the developmental effects derived from administering r-met-hu G-CSF in prepubertal male individuals leads to specific and differential endocrine responses and testicular developmental rates among in hystricomophs and ovine species. This supports the fact that species-specific protocols in the administration of r-met-hu G-CSF should be considered. r-met-hu G-CSF effects on sexual development were strongly evident in ovine species. With the due optimization, the use of r-met-hu G-CSF represents an efficient and functional method seeking to accelerate sexual developmental in mammalian species.S1 ARRIVEThis file contains information on experimental animal welfare including methods to alleviate pain or suffering as well as animal monitoring schedules as presented in this manuscript.(PDF)Click here for additional data file.


Hepatitis E virus (HEV) genotype 4 (HEV-4) is an emerging cause of acute hepatitis in China. Less is known about the clinical characteristics and natural history of HEV-4 than HEV genotype 3 infections in immunocompromised patients. We report transmission of HEV-4 from a deceased organ donor to 5 transplant recipients. The donor had been viremic but HEV IgM and IgG seronegative, and liver function test results were within reference ranges. After a mean of 52 days after transplantation, hepatitis developed in all 5 recipients; in the liver graft recipient, disease was severe and with progressive portal hypertension. Despite reduced immunosuppression, all HEV-4 infections progressed to persistent hepatitis. Four patients received ribavirin and showed evidence of response after 2 months. This study highlights the role of organ donation in HEV transmission, provides additional data on the natural history of HEV-4 infection, and points out differences between genotype 3 and 4 infections in immunocompromised patients. Orthohepevirus) is a major cause of hepatitis globally  identify other infected recipients from the same donor, 2) confirm that the infection was indeed donor derived, 3) investigate the natural history of HEV-4 infection, and 4) study outcomes of infected patients who received ribavirin. In Hong Kong, Queen Mary Hospital is the liver and heart–lung transplantation center, Princess Margaret Hospital is a kidney transplant center, and Tuen Mun Hospital offers specialist follow-up services for kidney transplant recipients. HEV diagnostic testing for this study was performed at the University of Hong Kong Department of Microbiology, based at Queen Mary Hospital. We retrieved patient identifiers for the organ donor and recipients from the organ donor registry and retrieved clinical details for donor and recipients from the electronic patient record. Archived serum samples from the donor and all recipients were retrieved and subjected to HEV quantitative real-time reverse transcription PCR (qRT-PCR) and serologic testing. Ethics approval for this study was obtained from the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.http://www.ystwt.cn/HEV.html). In addition, we tested donor and recipient serum samples by using IgG and IgM Western blots as previously described  on tissue sections, as previously described  level was 18 U/L (reference range 8–58 U/L), aspartate aminotransferase (AST) 22 U/L (reference range 15–38 U/L), and bilirubin 16 μmol/L (reference range 4–23 μmol/L). On hospitalization day 4 , her heart, liver, lungs, and kidneys were harvested and transplanted to 5 recipients. After hepatitis E in the organ recipients was recognized, HEV qRT-PCR was performed on archived donor serum collected on the day of organ harvest; viral load was 2.87 × 10ern blot  Figure 17 copies/reaction. Because the patient’s liver function test results were spontaneously improving . Prothrombin time was elevated to 14.2 seconds. Liver histology on posttransplantation day 122 showed moderate inflammation at portal tracts , panel Amproving , panel Amproving , panel AA 59-year-old man with a history of bronchiectasis and chronic obstructive pulmonary disease underwent sequential bilateral lung transplantation. The procedure was complicated by bleeding requiring exploratory thoracotomy for hemostasis. For postoperative immunosuppression, the patient was administered tacrolimus, mycophenolate, and prednisolone. The patient required prolonged hospitalization for ventilator-associated pneumonia. Starting at posttransplantation day 65, he had low-grade hepatitis. ALT was elevated to 65 U/L, AST to 45 U/L, while bilirubin remained within normal limits. Over the next 3 months, ALT elevations persisted , panel BA 6-year-old boy with focal segmental glomerulosclerosis underwent kidney transplantation at Princess Margaret Hospital. Immunosuppression was induced with methylprednisolone and basiliximab and maintained with oral tacrolimus, mycophenolate, and prednisolone. Mycophenolate was switched to azathioprine because of BK virus reactivation. Starting at posttransplantation day 67, persistent ALT level abnormalities were noted. Because hepatitis E had been diagnosed in 2 other recipients of organs from the same donor, HEV qRT-PCR testing of plasma collected on posttransplantation day 177 was performed, and results confirmed the diagnosis of persistent HEV infection. Administration of tacrolimus and azathioprine was stopped, and immunosuppression was continued with cyclosporin A, everolimus, and prednisolone. On posttransplantation day 180, oral ribavirin (100 mg 2×/d) was started. Liver function test results improved; HEV viral load underwent a 1-log reduction after the patient had received ribavirin for 1 month , panel CA 54-year-old woman with chronic glomerulonephritis underwent kidney transplantation at Princess Margaret Hospital. Preoperatively, she received antithymocyte globulin and methylprednisolone. Postoperative immunosuppression included tacrolimus, mycophenolate, and prednisolone. During follow-up at Tuen Mun Hospital on posttransplantation day 54, abnormal parenchymal enzymes were first noted, coinciding with an increasing trend in tacrolimus levels detected in May , panel D8 copies/mL. Because the hepatitis had persisted for >3 months, on posttransplantation day 181, oral ribavirin  was started, and all immunosuppressant dosages were reduced. At the end of the second month of ribavirin treatment, ALT and HEV RNA loads were improving.A 49-year-old woman received a heart transplant for severe myocarditis at Queen Mary Hospital. Intraoperatively, she received 500 mg intravenous methylprednisolone; postoperatively, as an outpatient, she received immunosuppression with tacrolimus, mycophenolate, and prednisolone. On posttransplantation day 34, abnormal liver function test results were first noted; ALT was 123 U/L, AST 94 U/L, and ALP 123 U/L, but bilirubin levels were within reference range. Subsequent blood testing showed ongoing low-grade hepatitis , panel EThis hepatitis E outbreak affected 5 transplant recipients who had received organs from a donor with HEV viremia. Two previous studies have described HEV transmission from organ donors to recipients  between organ donation and recognition of the outbreak. Because archived blood samples obtained from recipients shortly after transplantation were not available, we were unable to comprehensively examine the viral load dynamics of HEV infection. The variable performance of HEV serologic assays and lack of a standard serologic test is a limitation of our study, which we tried to minimize by using 2 independent assay formats. Although some research findings suggest that the analytical sensitivity and specificity of the Wantai HEV IgG and IgM ELISAs are good, the work of Cattoir et al. shows that discordant results with other commercial assays are not uncommon (We recognize that definitive conclusions on HEV-4 infections cannot be based on limited case series. To delineate intergenotypic differences in clinical characteristics, natural history, and ribavirin responsiveness, prospective studies of HEV-4 infection in cohorts of immunocompromised patients are needed. Additional methods and results for study of donor-derived genotype 4 hepatitis E virus infection, Hong Kong, China, 2018.


PGRN) is predominantly expressed by microglia in the brain, and genetic and experimental evidence suggests a critical role in Alzheimer's disease (AD). We asked whether PGRN expression is changed in a disease severity‐specific manner in AD. We measured PGRN in cerebrospinal fluid (CSF) in two of the best‐characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). In carriers of AD causing dominant mutations, cross‐sectionally assessed CSF PGRN increased over the course of the disease and significantly differed from non‐carriers 10 years before the expected symptom onset. In late‐onset AD, higher CSF PGRN was associated with more advanced disease stages and cognitive impairment. Higher CSF PGRN was associated with higher CSF soluble TREM2 (triggering receptor expressed on myeloid cells 2) only when there was underlying pathology, but not in controls. In conclusion, we demonstrate that, although CSF PGRN is not a diagnostic biomarker for AD, it may together with sTREM2 reflect microglial activation during the disease.Progranulin ( CSF PGRN did not differ between the three ADAD‐associated genes  among the MC participants  as well as their interaction with mutation status. The first‐order (i.e. linear) model best fitted the data (P = 0.041), indicating that the changes in CSF PGRN as a function of EYO differ between MC and NC. We compared the estimated CSF PGRN at 5‐year EYO intervals by t‐tests and observed that CSF PGRN started to be significantly increased in MC compared to NC at EYO = −10 (Table We determined how CSF PGRN changes as a function of the estimated years from expected symptom onset (EYO) in MC compared to NC. Mean estimated levels of CSF PGRN at consecutive 5‐year interval EYO were computed by a linear regression model including mutation status, EYO and gender as predictor variables see  section.et al , we testthe data . The int10 Table .et al, et al, et al, Following the approach of previous DIAN studies . A least significant difference (LSD) pair‐wise post hoc comparisons revealed that all MC groups  had significantly higher CSF PGRN than the NC . No differences were found between the MC groups at different clinical stages. Likewise, CSF PGRN levels were not associated with Mini‐Mental State Examination  or CDR sum of boxes  among MC.Next, we compared CSF PGRN in MC at different clinical stages defined by the clinical dementia rating (CDR) score with that of the NC. We hence compared four groups: NC, cognitively normal (CDR = 0) MC, very mild dementia (CDR = 0.5) MC and mild‐to‐severe dementia CDR > 1) MC. Participants with a CDR ≥ 1 were grouped together because of the low number of subjects in these groups , but was not associated with age .To further validate our findings in subjects with late‐onset AD, we studied a total of 1,017 participants of the ADNI study , P‐tau181P (T) and T‐tau (N). The aggregated tau (T) and neurodegeneration (N) groups were merged together to simplify the number of groups to compare . Bonferroni corrected pair‐wise post hoc comparisons indicated that CSF PGRN was significantly higher in the “AD CDR = 1” group compared to the “healthy controls” (P = 0.006) and “Preclinical AD A+/TN−” (P < 0.0001) groups , where significant differences were observed when “AD CDR = 1” was compared to “healthy controls” (P = 0.030) and “Preclinical AD A+/TN−” (P = 0.0001) groups in Bonferroni corrected pair‐wise post hoc comparisons. Similarly, CSF PGRN remained lower in “Preclinical AD A+/TN−” compared to “AD CDR = 0.5” (P = 0.002) and “AD CDR = 1” (P = 0.0001) groups and a tendency existed when compared to “Preclinical AD A+/TN+” (P = 0.082). Consistent with previous reports , such that the CSF PGRN mean level of the GRN rs5848 TT carriers  was significantly lower than that of the rs5848 CT carriers  and rs5848 CC carriers  groups (Bonferroni corrected pair‐wise post hoc comparisons). Together, the CSF PGRN changes across biomarker‐defined clinical stages are consistent with the increase we found in ADAD throughout EYO.Given that continuum category ; and additionally adjusted for CSF Aβ1–42 and CSF T‐tau  and executive function as assessed by ADNI‐EF  in an unadjusted model  for ADNI‐EF, ADAS‐Cog 11 and ADAS‐Cog 13, and a tendency existed for ADNI‐Mem and MMSE   Fig A, but noet al, continuum, this is an unbiased comparison that does not assume any particular sequence of biomarkers in the course of the disease. et al, et al, et al, continuum category as a whole. CSF PGRN significantly differ between categories  and Alzheimer's continuum categories (P < 0.0001). Finally, we also tested the association between CSF PGRN and cognitive function and neuroimaging biomarkers within the SNAP category, with the same linear regression models we applied in the analysis focused on the Alzheimer's continuum category. Strikingly, and in contrast with the results in the Alzheimer's continuum, CSF PGRN was not associated with cognitive decline in the SNAP group in any of the models tested (P = 0.203). These results suggest that, despite the fact that CSF PGRN is increased in SNAP, it selectively associates with disease severity in AD.In the second approach, we examined CSF PGRN between biomarker profile  × 100a2 (%) in plate x = [(concentration of IS2 in plate x)/] × 100a3 (%) in plate x = [(concentration of IS3 in plate x)/] × 100a4 (%) in plate x = [(concentration of IS4 in plate x)/] × 100The mean of the percentages (Ax) for all the IS  in plate x was calculated, and the following correction factor was computed for each individual plate: P < 0.0001).The raw values were multiplied by the correction factor of the corresponding plate; the corrected values are provided as the variable “MSD_PGRNCORRECTED” in the ADNI database. Due to the low interplate CV, the corrected and raw PGRN values in the ADNI sample are highly correlated  was similar to that published with other assays . The mean intraplate CV was 3.1%, and all duplicate measures had a CV < 15%. Alike the CSF PGRN ELISA, four CSF IS samples were loaded in all plates. The interplate CV for each of the IS was 11.4, 12.2, 10.5 and 7.1%. We corrected the raw measurements based on values of the four IS that were loaded on all plates in a similar manner as in CSF PGRN measurements. Consequently, the corrected values were used and are available in the ADNI database as variables “MSD_sTREM2CORRECTED”.1–42, T‐tau, P‐tau181P). Within each of the samples, we determined the CSF PGRN outliers, as defined as values differing 3 standard deviations from the mean (3 outliers in the DIAN sample and 11 in the ADNI sample). In order to rule out that our results are not driven by extreme values, all the analysis described in the main text are performed without these outliers. Nevertheless, including them did not affect the main results (as shown in the main text and in P = 0.062). In contrast, CSF PGRN in ADNI did not follow a normal distribution . After a log10 transformation, it followed a normal distribution . All the statistical analyses in the ADNI sample were hence performed with the log10‐transformed values.In both DIAN and ADNI samples, we only included in our study participants that had the following data available: age, gender and the three AD CSF core biomarkers .The data from the DIAN sample (Data Freeze 9) were studied in a similar approach to that we previously applied  and cubic (EYO3) terms and their interactions with mutation status. We determined the model that best fitted the data by forward selection of the predictors, and the final model was chosen based on the Akaike information criterion  and we therefore used simple linear regression in all the following analysis.In order to test how CSF PGRN levels change as a function of EYO  for pair‐wise comparisons. Individuals falling into CDR = 1 to CDR = 3 were grouped together due to the low number of subjects in these stages.We compared CSF PGRN levels between NC and MC in different clinical stages  in an analysis of covariance (ANCOVA) controlling for age, gender, continuum, we applied an ANCOVA including the biomarker‐defined stages of AD, gender and APOE ε4 status as fixed effects and age as covariate, followed by Bonferroni corrected pair‐wise post hoc comparisons. An additional analysis was performed adding the GRN rs5848 genotype as a covariate. A similar approach was used for comparisons between biomarker categories shown in Fig In ADNI, demographic, clinical and biochemical data group comparisons were performed by one‐way ANOVA or Pearson's chi‐square tests, as appropriate. To test whether CSF PGRN changed across the Alzheimer's APOE ε4 and education as covariates, and Model 3 included the former covariates and also CSF Aβ1–42 and CSF T‐tau.To study the associations between CSF PGRN and cognitive and functional scores, we applied three linear regression models. Model 1 was unadjusted, Model 2 included age, gender, APOE ε4 and education. Analyses including hippocampal volume were adjusted for intracranial volume.The association between CSF PGRN and temporo‐parietal FDG‐PET and total hippocampal volume was tested with a linear regression adjusted for age, gender, The diagnostic value of CSF PGRN to discriminate AD (ADAD or late‐onset AD) from controls was tested with a receiver operating characteristic (ROC) analysis. We computed areas under the curve (AUC), and we tested whether they were significantly different from the null hypothesis that the AUC equals 0.50, which corresponds to a random test.181P, Aβ1–42) was studied with a linear model adjusted for age, gender and APOE ε4 status. The analysis was performed stratifying for the mutation status (DIAN) or classifying the subjects in the healthy controls, Alzheimer's continuum or SNAP categories (ADNI). The standardized regression coefficients (β) are reported. In order to rule out that the associations were driven by extreme values, we performed the analysis both including and excluding biomarker outliers (defined as AD CSF core biomarkers 3 standard deviations below or above the group mean) and the analysis yielded similar results . Figures were built using GraphPad Prism or R. All tests were two‐tailed, with a significance level of α = 0.05.Statistical analysis was performed in SPSS IBM, version 20.0, statistical software and the free statistical software R  is the most frequent neurodegenerative disorder. Besides β‐amyloid and tau deposits, all AD brains invariantly show neuroinflammatory symptoms associated with microgliosis. Progranulin (PGRN), a secreted protein whose loss of function is associated with frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis, is also genetically involved in AD. In the brain, PGRN is predominantly expressed in microglia. In this study, we investigated PGRN concentration in the cerebrospinal fluid (CSF) of two of the largest and best characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN), which includes families with autosomal dominant AD (ADAD), and the Alzheimer's Disease Neuroimaging Initiative (ADNI), which studies the evolution of late‐onset AD.In carriers of ADAD causing dominant mutations (DIAN), we found that CSF PGRN increased 10 years before the expected symptom onset. In late‐onset AD (ADNI), higher CSF PGRN was associated with more advanced disease stages as well as cognitive impairment. CSF PGRN was associated with levels of proteolytically generated soluble TREM2 (sTREM2), a protein described to be a central regulator of microglial function and known to increase 5 years before the expected symptom onset. Importantly, the association between CSF sTREM2 and CSF PGRN was only observed when there was underlying pathology, but not in controls.CSF PGRN together with CSF sTREM2 may serve as a microglia activity marker in AD and could be used to prove target engagement in clinical trials aiming to modulate microglial activity.http://adni.loni.usc.edu/.All data of the ADNI study (including the CSF PGRN and sTREM2 reported in this study) are publicly available in AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen associated with immunocompromised and chronically ill patients, as well as nosocomial outbreaks. In this study, we characterized 23 MDR C. striatum isolated of bloodstream and catheter-related infections from a hospital of Rio de Janeiro.C. striatum isolates were identified by 16S rRNA and rpoB genes sequencing. The dissemination of these isolates was accomplished by pulsed-field gel electrophoresis (PFGE). All isolates were submitted to antimicrobial susceptibility testing by disk diffusion and by minimum inhibitory concentration using E-test strips methods. Antimicrobial resistance genes were detected by polymerase chain reaction. Quantitative tests were performed on four different abiotic surfaces and the ability to produce biofilm on the surface of polyurethane and silicone catheter was also demonstrated by scanning electron microscopy.gyrA gene and was negative in the search of genes encoding antibiotic resistance. The other 22 isolates were positive to resistance genes to aminoglycoside, macrolides/lincosamides and chloramphenicol and showed mutations in the QRDR of the gyrA gene. Scanning electron microscopy illustrated the ability of MDR blood isolate partaker of the epidemic clone (PFGE profile I) to produce mature biofilm on the surface of polyurethane and silicone catheter.Eleven PFGE profiles were found. The PFGE profile I was the most frequently observed among isolates. Five different MDR profiles were found and all PFGE profile I isolates presented susceptibility only to tetracycline, vancomycin, linezolid and daptomycin. Only the multidrug-susceptible isolate did not show mutations in the quinolone-resistance determinant region (QRDR) of the C. striatum is a case of concern.Genotyping analysis by PFGE revealed the permanence of the MDR PFGE profile I in the nosocomial environment. Other new PFGE profiles emerged as etiologic agents of invasive infections. However, the MDR PFGE profile I was also found predominant among patients with hematogenic infections. The high level of multidrug resistance associated with biofilm formation capacity observed in MDR The online version of this article (10.1186/s12879-019-4294-7) contains supplementary material, which is available to authorized users. Corynebacterium genus consists of Gram-positive aerobic or anaerobic facultatively pleomorphic rods with a high G + C content DNA. Some species are part of human skin or mucosa [Corynebacterium striatum has been increasingly associated with severe infections in both immunocompetent and immunocompromised hosts [C. striatum isolates have been included among the etiologic agents of bacteremia with or without central venous catheter (CVC) in place [C. striatum as an emerging multidrug-resistant (MDR) pathogen related to nosocomial outbreaks in several countries [r mucosa , 2. Coryed hosts , 4. Howein place , 6, endoin place , breast in place , septic ountries –16.C. striatum infections highlighted by Verroken et al. [C. striatum and its involvement in nosocomial infections require appropriate interpretive criteria to the selection of the adequate antibiotic therapy [The main risk factors for acquisition MDR n et al.  were: prn et al. . The eme therapy .C. striatum mainly encompassed the respiratory tract [C. striatum [C. striatum mostly isolated from tracheal aspirates samples was initially verified in 2009 [C. striatum isolates were noticed in the same hospital. In the present study, we aimed to investigate the clonal relationship, antimicrobial susceptibility profiles, ability of biofilm formation, molecular detection of resistance genes to amynoglycosides, quinolones, compounds of the MLSB group (macrolides and lincosamides) and chloramphenicol of these C. striatum invasive isolates.Most reports of nosocomial infections and outbreaks caused by ry tract , 13, 18.striatum , 19–21.  in 2009 . SubsequC. striatum isolates recovered from blood and catheter samples of 21 hospitalised patients with signs and symptoms of bacteremia (n = 13) and catheter-related infections (n = 10) were analysed for microbiological features (Table 1). Two of these isolates  were previously studied by Baio and co-workers [n = 22) and Hospital Municipal Jesus (HMJ) (n = 1), both located at the metropolitan area of Rio de Janeiro, RJ, Brazil. The 2296 isolate came from a patient with CVC-related infection from HMJ and it was sent to the Laboratório de Difteria e Corinebactérias de Importância Clínica/UERJ for identification of the pathogen. C. striatum was isolated from blood samples and catheters segments of patients with signs and symptoms of bacterial infections as part of the medical care procedures of both hospitals. The consent to participate was not required because all the investigated isolates were taken as a part of standard care (diagnostic purposes). This study focused on bacteria and no identifiable human data were used. All the isolates were deposited in the CBAS/Fiocruz  culture collection.Twenty three -workers . The patClinical specimens were inoculated in Bactec Plus anaerobic/aerobic vials and analysed in a Bactec 9240 continuous-monitoring system . Corynebacterium-like colonies were selected for identification when they were grown in significant numbers (> 15 colonies) or in pure culture from blood or catheter samples, as recommended by Maki’s semi quantitative method to distinguish infection (> 15 colonies) from contamination of catheter-tips . Bacterincbi.nlm.nih.gov) using the BLAST algorithm and/or the Eztaxon server [Molecular identification was performed according with protocols previously described . The 16Sn server .Pulsed-field gel electrophoresis was performed as described by Baio and co-workers . PFGE baStaphylococcus spp. These antibiotics are not considered in Clinical and Laboratory Standards Institute (CLSI) and BrCAST/EUCAST guidelines for Corynebacterium spp. Linezolid 30 μg was interpreted in accordance to criteria defined by CLSI [Staphylococcus spp. The BrCAST document recommends the use of linezolid 10 μg for antimicrobial susceptibility testing, but this is not found commercially available in Brazil.Antimicrobial susceptibility profiles were determined by the disk diffusion and by minimum inhibitory concentration (MIC) using E-test strips methods in cation-adjusted Mueller-Hinton agar supplemented with 5% sheep blood using inoculum equivalent to a 0.5 McFarland standard. Seven antibiotic disks  were used: clindamycin (2 μg), moxifloxacin (5 μg), gentamicin (10 μg), rifampicin (5 μg) and vancomycin (5 μg), according Brazilian Committee on Antimicrobial Susceptibility Testing – BrCAST . Another by CLSI  for StapAntimicrobial susceptibility to daptomycin, penicillin, ciprofloxacin and tetracycline were determined by MIC using E-test strips . Interpretation of values to penicillin, ciprofloxacin and tetracycline were performed also according BrCAST . Due to gyrA gene was performed following protocols described by Sierra and co-workers [gyrA gene were identified by aligning of amino acids sequences and compared to the sequence of C. striatum ATCC 6940 (GenBank accession number AY559038) using Clustal X program [C. striatum isolates also were screened for the presence of genes codifying antibiotic resistance, ermX (macrolides and lincosamides), aphA (amynoglycoside) and cmx (chloramphenicol), by PCR using primers specific for each gene [The amplification and sequencing of the QRDR region of the -workers . Mutatio program . The 23 ach gene , 34. Forach gene .6 CFUml− 1 of C. striatum and incubated at 37 °C for 48 h then Maki’s semi-quantitative roll plate technique were performed. Basically, after washing (three times) with phosphate buffered saline (PBS) 0.1 M pH 7.2, contaminated abiotic substrates were rolled up on Columbia agar plates supplemented with 5% sheep blood  for 48 h at 37 °C were analyzed presence of bacterial carpet [Sterile 4 cm segments of 16-gauge percutaneous nephrostomy polyurethane and silicone catheters were immersed in TSB containing 10l carpet , 35.p < 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism version 6 .Quantitative analysis of viable sessile cells of representative isolates of PFGE profile I (2023) and II (2038) was evaluated by quantitative tests based on previously described methods  and usinC. striatum isolates from blood, 2023 (PFGE profile I) and 2038 (PFGE profile II), were demonstrated by Scanning Electron Microscopy (SEM). Specimen preparation and staining protocol for SEM were performed as described by Souza and co-workers [C. striatum 1987 (PFGE profile I), 2369 (PFGE profile II), 1961 (PFGE profile III) and 1954 (PFGE profile IV) isolates were used as positive controls [The biofilm produced (48 h incubation) on the surface of in vitro prepared on fragments of polyurethane and silicone catheters by -workers . Cathetecontrols .rpoB genes sequences from 23 isolates from blood and catheter samples exhibited the highest similarity values with C. striatum ATCC 6940 type strain, ranging from 99.77–99.12% and 99.28–98.65%, respectively. GenBank accessions for C. striatum 16S rRNA and rpoB genes were deposited: KJ934779 to KJ934789, KM001910 to KM001914, KJ855309 to KJ855313, KR010627 to KR010645, KR020513 and KR020514.The analysis of 16S rRNA and C. striatum isolates were designated I, Ia, Ib, II, V, VI, VIa, VII, VIII, IX and X (Table n = 9) observed among isolates (Table C. striatum: PFGE profiles VI (2230 and 2237 isolates) and IX (2425 and 2432 isolates), respectively. PFGE profile X was related to a case of catheter-related infection in a 3 months-old patient from Neonatal ICU. C. striatum PFGE profile VII was isolated only from patient with CVC infection from HMJ-RJ (Table C. striatum PFGE profile I isolates were detected in patients with bloodstream and/or catheter-related infections during the years of 2009 (n = 1), 2010 (n = 2), 2011 (n = 5), 2013 (n = 1) but not 2012.Eleven PFGE profiles among  X Table  and 2. Tes Table . The PFGRJ Table . C. striC. striatum isolates are displayed in Table C. striatum isolates, independent of their PFGE profiles and hospital settings, showed non-susceptibility to at least one agent in three or more antimicrobial categories and were consequently identified as MDR pathogens. Twenty-two C. striatum isolates exhibited five different multiresistance levels corresponding to five different MDR profiles: A, with resistance to 9 antimicrobial agents; B and B1, resistance to 8 antimicrobial agents; C, resistance to 7 antimicrobial agents; and D, with resistance to 6 antimicrobial agents. All C. striatum isolates related to bloodstream and catheter-related infections, representative of different PFGE profiles, showed susceptibility to tetracycline, vancomycin, linezolid and daptomycin. C. striatum isolates, independent of PFGE profiles showed resistance to penicillin (100%), ciprofloxacin and moxifloxacin (95.6%), clindamycin (95.6%), erythromycin (95.6%), gentamicin (91.3%), rifampin (52.2%), imipenem (76.2%) and chloramphenicol (95.6%). MDR profile A was demonstrated for 8 C. striatum PFGE profile I isolates. Moreover, MDR profile A was also shown for C. striatum isolates PFGE profiles Ib, II and VIII.Antimicrobial susceptibility profiles of 23 gyrA gene of 22 isolates characterized as resistant to fluoroquinolones were compared to that of the quinolone-susceptible C. striatum ATCC 6940 (MIC = 0.094 mg/L) and with our multidrug-susceptible (MDS) 2376 isolate (MIC = 0.125 mg/L). The relationships between the mutations in the QRDR region gyrA gene and the MICs of ciprofloxacin of the 23 isolates and C. striatum ATCC 6940 are summarized in Table gyrA gene were deposited in GenBank/NCBI under numbers MG010347-MG010369.The sequences of the QRDR region of the ermX (macrolides and lincosamides), aphA (amynoglycoside) and cmx (chloramphenicol) were found except in MDS 2376 isolate. Twenty-two isolates resistant to erythromycin, clindamycin and chloramphenicol presented ermX and cmx genes. These isolates were also aphA-positive and resistant to gentamicin, except for one isolate. The 2130 isolate was aphA-positive and susceptible to gentamicin, confirmed by MIC using E-test strip gentamicin. Analysis of genes sequences exhibited high similarity values (above 99%) with those deposited in NCBI. The sequences were deposited in GenBank/NCBI under numbers MF872250 to MF872252.The genes codifying for antibiotic resistance, C. striatum isolates 2023 and 2038  are shown in Fig. 2. Viable sessile bacterial cells were detected 48 h post-infection on surfaces of all types of abiotic substrates tested, but at different levels. C. striatum 2023 isolate (PFGE profile I) showed a higher ability to adhere to glass surface, a hydrophilic and positively charged abiotic surface (p < 0.001). In addition to glass, C. striatum 2023 isolate also exhibited a significantly (p < 0.001) greater level of adherence to an abiotic hydrophilic (polyurethane) positively charged surface compared with C. striatum 2038 isolate (PFGE profile II). Conversely, C. striatum 2038 isolate exhibited a significantly (p < 0.001) greater level of adherence to negatively charged and hydrophobic polystyrene (Thermanox) surface compared with 2023/PFGE profile I isolate. Interestingly, both C. striatum isolates (PFGE profiles I and II) were able to strongly adhere and survive on the metal surface with similar intensities (p = 0.374)  on fragments of polyurethane and silicone catheters, as illustrated in Fig. Results of the semi-quantitative roll plate method (> 15 CFU) showed extensively adherent viable sessile forms for both lates 202 and 2038C. striatum 2023/PFGE profile I  or in significant numbers.Antimicrobial resistance has a major impact on human health . At pres-workers , our finC. striatum from hospitalized patients with signs and symptoms of infection was observed in some studies [C. striatum in Rio de Janeiro, Brazil. MDR clones related to the profiles of PFGE I and II were predominant. In that opportunity only two isolates of PFGE I and II profiles were isolated from blood samples [C. striatum isolates in HUPE, current investigation of the clonal relationship of these C. striatum isolates revealed the permanence of the MDR PFGE profiles I and II in the nosocomial environment as invasive clones. However, the PFGE I profile was found predominant among patients with hematogenic infections. In addition, other new MDR PFGE profiles (V to IX) emerged as etiologic agents of bloodstream and catheter-related infections. Interestingly, one non-multiresistant clone (PFGE profile X) was also related to a case of catheter-related infection in the newborn.In Brazil the isolation of  studies –40. In a samples . Due to C. striatum isolates [C. striatum isolates genotyping. Thus, the method is only applicable to compare isolates for regional epidemiology surveillance. Gomila and co-workers described the development of a multilocus sequence typing (MLST) scheme for C. striatum. [gyrA and rpoB) that comprise it. PFGE has limitations but it is the tool available so far for the discrimination of this bacterial species. Future researches are required to evaluate genotyping methods that provide useful data for global surveillance of infections caused by C. striatum.PFGE is a valuable tool to investigate the clonal relatedness of microbial strains during nosocomial outbreaks. Several nosocomial infections and outbreaks studies have employed this methodology for typing of isolates , 16, 18.triatum. . HoweverCorynebacterium spp. in many laboratories [C. striatum isolates. However, studies suggest that according to the severity of the infection, empiric treatment should be carried out with vancomycin and linezolid because of low levels of susceptibility to other antimicrobials [Antimicrobial susceptibility testing remains rarely performed on ratories . The metratories , 42–44. ratories , 29, excratories , 16, 45.crobials , 46. In crobials , 48.C. striatum isolates has been related in some countries, such as Japan [C. striatum isolates showed resistance to imipenem . Combination therapy that includes imipenem for the treatment of MDR C. striatum infections in Brazil should be more prudent.Resistance to imipenem by as Japan , Spain [as Japan , 10 and as Japan . Most ofC. striatum isolates showed susceptibility to daptomycin. However, resistance to daptomycin in C. striatum has been documented and may occur during therapy in patients with invasive infections. Consequently, some authors recommend caution in daptomycin monotherapy for treatment of these infections [Effective treatment of corynebacterial infections with daptomycin has been reported in the literature , 50. In fections , 51.Corynebacterium species. Studies of the sequences encoding the A subunit of the gyrase enzyme in strains of C. striatum, Corynebacterium amycolatum and Corynebacterium macginley have shown that resistance to fluoroquinolones is associated with mutations of a spontaneous nature in this gene and depends on the number of mutations and the type of amino acid that was exchanged [gyrA gene, respectively, found in three of our isolates have not been described in the literature for Corynebacterium species until the present moment, conferring resistance to ciprofloxacin and moxifloxacin.Some mechanisms of antimicrobial resistance have been reported in xchanged , 52, 53.ermX gene (erythromycin ribosome methylation) encoding the rRNA methylase enzyme leads to simultaneous resistance to macrolides, lincosamides and streptogramins B (MLSB) [ermX gene can be involved in the clindamycin and erythromycin resistance phenotype of our isolates.The B (MLSB) , 54. ThiC. striatum isolates resistant to aminoglycosides. Consequently, the use of aminoglycosides as second-line complementary antimicrobial for treatment of C. striatum infections should be cautious [aac (acyl-coenzyme A-dependent acetyltransferase), ant (nucleoside triphosphate-dependent nucleotidyl transferases) and aph (nucleoside triphosphate-dependent phosphotransferases). These enzymes are often disseminated by various mobile genetic elements and many aminoglycosides can be inactivated by more than one enzyme [aph gene was found in 22 isolates, except in the MDS 2376 isolate, but one aph-positive isolate showed susceptibility to gentamicin, confirmed by MIC E-test strip . Future analyzes of the region where the gene is located and other mechanisms of resistance to aminoglycosides in all aph-positive isolates should be made.Antimicrobial susceptibility studies have shown cautious , 56. Thee enzyme . In thiscmx gene is responsible for coding the efflux protein to chloramphenicol and has already been found in transposons, plasmids and genomes of Corynebacterium species [cmx-positive were resistant to chloramphenicol. The cmx gene sequence showed similarity above 99% with the sequences of cmx gene found in chromosomes, plasmid and genomic island of Pseudomonas aeruginosa and other Corynebacterium species deposited in GenBank/NCBI.The  species –60. All C. striatum PFGE profiles I to IV formed biofilm on hydrophilic and hydrophobic surfaces. C. striatum PFGE profile I, predominant isolated from nosocomial outbreak, showed the greatest ability to adhere to all surfaces produced much more biofilm than the other profiles [C. striatum isolates identified as predominant PFGE profile had high ability to produce biofilm in glass cover-slips after 72 h post-incubation [C. striatum PFGE profiles I and II isolated from patients with bloodstream infections. Similar to C. striatum PFGE profile I isolated from patients undergoing endotracheal intubation procedures, PFGE profile I isolated from bloodstream and catheter-related infections also showed a higher ability to adhere to and to survive on abiotic surfaces of medical devices including those used in invasive procedures. MDR C. striatum viable cells were able to multiply and to produce mature biofilms on both types of catheter surfaces.Biofilm is a structure that facilitates several bacterial processes influencing virulence and resistance to antimicrobials such as adhesion capacity, metabolite exchange, cellular communication, protection to antimicrobials, protection against host immune attacks. Consequently, the formation of bacterial biofilms leads to an increase in healthcare costs and extend hospitalization , 62. Preprofiles . In Japacubation . In the C. striatum clones to produce biofilm on different types of abiotic surfaces may contribute to the pathogenicity favoring bacterial invasive potential and establishment of bloodstream and catheter-related infections, in addition to permanence in hospital environment and dissemination of antimicrobial resistance. The potential of C. striatum to cause infection should not be underestimated. Therefore, antimicrobial susceptibility testing should be performed on clinically significant C. striatum isolates. Medical surveillance programs should include control strategies in order to decrease potential risk factors of nosocomial infections and outbreaks due to C. striatum.The ability of some Additional file 1:Figure S1. Pulsed-field gel electrophoresis (PFGE) profiles of Brazilian Corynebacterium striatum isolates from blood and catheter segments. Lane 1: λ DNA ladder PFGE marker; lane 2: profile VIII (isolate 2103); lanes 3, 5 and 6: profile I ; lane 4: profile Ia (isolate 2324); lane 7: profile X (isolate 2376); lane 8: profile VIa (isolate 2390); lane 9: profile IX (isolate 2425) and lane 10: profile Ib (isolate 2454). (TIF 523 kb)Additional file 2:Figure S2. Pulsed-field gel electrophoresis (PFGE) profiles of Brazilian Corynebacterium striatum isolates from blood and catheter segments. Lane 1: λ DNA ladder PFGE marker; lanes 2, 3 and 7: profile I ; lane 4: profile V (isolate 2130); lanes 5, 6 and 9: profile VI ; lane 8: profile VII (isolate 2296) and lane 10: profile IV (isolate 1954 – control). (TIF 469 kb)


This could allow for detection of diffuse amyloid deposits in the early stages of AD dementia, particularly in the striatum where diffuse amyloid is most commonly observed.[ Both “Original” Flutemetamol PET was performed at a mean of 7.5 months before death. Seventy-eight subjects (73.6%) had a history of dementia and 53 (50%) had a clinical diagnosis of dementia due to AD flutemetamol performance relative to either CERAD method revealed a sensitivity and specificity of approximately 90%, indicating that neuritic Aβ plaques can explain the majority of the PET signal. This is supported by the correspondence between neocortical [18F]flutemetamol PET signal and neuritic Aβ plaques at autopsy , seven false negatives (FNs)) [18F]flutemetamol PET signal from diffuse Aβ plaques resulted in positive PET reads. The third FP case ) , 7. AmylThe seven FN reads reducing sensitivity to approximately 90% were due primarily to the presence of brain tissue atrophy. In this study, use of structural images in support of PET image interpretation was at the discretion of image readers . Thus, wIn the context of AD pathology evaluation in living subjects, the use of amyloid PET to detect both neuritic and diffuse Aβ plaques enhances our ability to monitor the continuum of pathology progression. Diffuse plaques also contain Aβ fibrils  and may 18F]flutemetamol [11C]PIB [18F]flutemetamol PET signal in the striatum had a high specificity (100%) when compared to striatal pathology as SoT. The lower sensitivity (83–87%), however, is consistent with the idea that the PET ligand’s binding to diffuse Aβ plaques is lower than to denser amyloid fibrils in neuritic Aβ plaques. The threshold for striatal amyloid positivity is implicated in Thal phase 3, which has been reported to correspond to clinical transition from cognitively normal to AD dementia PIB , 16. In [11C]PIB , [18F]flCPIB , 16dementia , 16.18F]Flutemetamol PET performance metrics. This reinforces the high accuracy of this PET tracer and addresses the importance of pathology-based SoT methodologies and the nuances of PET image reading.The research described here summarises the understanding of [


The optimized tert-butylhydroquinone concentration in the extraction solvent was 20 µM. The addition of 20 µM ascorbic acid or co-addition of tert-butylhydroquinone and ascorbic acid (20 µM each) were also effective in preventing the oxidation of ubiquinol-10, but ascorbic acid alone gave poor reproducibility. Good within day reproducibility was observed, and day-to-day analytical variance was excellent.The redox balance of coenzyme Q10 in human plasma is a good marker of oxidative stress because the reduced form of coenzyme Q10 (ubiquinol-10) is very sensitive to oxidation and is quantitatively converted to its oxidized form (ubiquinone-10). Here we describe an HPLC method for simultaneous detection of ubiquinol-10 and ubiquinone-10 in human cerebral spinal fluid to meet a recent demand for measuring local oxidative stress. Since the levels of coenzyme Q10 in human cerebral spinal fluid are less than 1/500 of those in human plasma, cerebral spinal fluid extracted with 2-propanol requires concentration for electrochemical detection. Using human plasma diluted 500-fold with physiological saline as a pseudo-cerebral spinal fluid, we found that addition of  Oxidative stress is defined as a disturbance in the pro-oxidant-antioxidant balance in favor of pro-oxidants. The redox balance of coenzyme Q10 (CoQ10) is a good marker of oxidative stress because its reduced form (CoQ10H2) is highly reactive with oxygen radicals and is converted to the oxidized form (CoQ10). In fact, the incubation of human plasma at 37°C under aerobic conditions in the presence of 5 µM cupric ion initially produces a decrease in vitamin C (VC), followed by a decrease in CoQ10H2 and the concomitant production of an equal amount of CoQ10. This observation indicates that the percentage of CoQ10 in total coenzyme Q10 (%CoQ10) represents a good marker of early stage oxidative stress. We therefore developed a simple and reliable method for the simultaneous electrochemical detection of plasma CoQ10H2 and CoQ10 and applied the method to patients with various diseases. Significant increases in %CoQ10 were observed in patients with hepatitis, cirrhosis, hepatoma, juvenile fibromyalgia, amyotrophic lateral sclerosis (ALS), Parkinson disease, post cardiac arrest syndrome, and sepsis as compared to age-matched healthy controls. It is interesting that newborn babies have significantly higher plasma %CoQ10 than adults and %CoQ10 increases with age.,13Increases in oxidative stress have been suggested to contribute to aging and degenerative diseases such as heart attack, stroke, neurodegenerative diseases, diabetes, and cancer.Recently, the measurement of local oxidative stress is sought in order to gain a more precise understanding of the role of reactive oxygen species. For this purpose human cerebral spinal fluid (CSF) represents a good target compartment. However, the levels of total coenzyme Q10 (TQ10) in human CSF are less than 1/500 of those in human plasma. Therefore, CSF extracted with 2-propanol will require concentration prior to electrochemical detection. We prepared pseudo-CSF by preparing a 500 fold dilution of human plasma, which was used to optimize the analytical procedure.tert-butylhydroquinone (TBHQ) was effective in preventing the oxidation of the reduced coenzyme Q10 during the extraction and concentration steps. We found the optimal TBHQ concentration to be 20 µM. A column switching system was useful in avoiding interference by TBHQ in the detection of vitamin E (VE), CoQ10H2, and CoQ10. Good within-day reproducibility was observed and day-to-day variance of the analytical method was excellent. Finally, we demonstrated that our method is applicable to human CSF.Plasma VC was useful to prevent the oxidation of the reduced form of coenzyme Q10 in 2-propanol extracts (unpublished data). Although, CSF contains ~300 µM ascorbic acid, we found that the addition of 2 and CoQ10 were generous gifts from Kaneka . TBHQ, 2-propanol (IPA), sodium perchlorate (NaClO4) and other chemicals were of the highest grade commercially available. We prepared pseudo-CSF by diluting human plasma with 499 volumes of 0.9% physiological saline. Human CSF was obtained from the Department of Neurology, Graduate School of Medicine, The University of Tokyo. Human CSF samples were obtained after written informed consent was obtained from participants and the research project was approved by the institutional review board of the University of Tokyo, School of Medicine.CoQ10HThree hundred µl of Pseudo-CSF (or CSF) and 1.2 ml of 20 µM TBHQ in IPA were mixed in a 2.0 ml-polypropylene tube. After centrifugation at 3,000 rpm for 10 min at 4°C, 1.2 ml of supernatant was transferred into a 1.5 ml-LoBind tube . The supernatant was dried under a stream of nitrogen gas, and the residue was admixed with 120 µl of IPA. Aliquots (60 µl) were injected into the HPLC system described below.2 and CoQ10 using an octylsilyl column, a column switching method was employed  and was delivered at a flow rate of 0.4 ml/min. The mobile phase for the separation columns was 50 mM sodium NaClO4 in methanol/IPA  and was delivered at a flow rate of 0.8 ml/min. The columns were maintained at 20°C.The mobile phase for the cleanup column was 50 mM NaClOt test.Data presented are mean values and SD. Statistical analysis was performed with a Student’s 2 by the in line reduction column, thereby making the resulting CoQ10H2 detectable with the ECD. Almost identical slopes for CoQ10 and CoQ10H2 were observed  was delivered. In this condition, analytes eluted in the following order: VE, CoQ10H2, and CoQ10 as shown in the typical HPLC chromatograms of extract from pseudo-CSF . Therefore, %CoQ10 was lowest at 20 µM TBHQ in the extraction solvent. Under this condition, the molar ratio of TBHQ to CoQ10H2 is calculated as 20 µM × 1,200 µl/(6 nM × 300 µl) = 13,000, indicating that sufficient amounts of TBHQ were present to inhibit the oxidation of CoQ10H2. However, more than 50 µM TBHQ in the extraction solvent appeared to induce autoxidation of TBHQ and consequently resulted in an increase of %CoQ10. Therefore, the optimal TBHQ concentration in the extraction solvent was deemed to be 20 µM.Figure 2, CoQ10 and %CoQ10 values in the presence of either the individual reductants or both reductants. However, poor reproducibility in CoQ10 detection was observed when VC was used as a reductant in the extraction solvent. When TBHQ was employed as the reductant, the reproducibility in CoQ10 detection was excellent. More importantly, the results in the presence of both VC and TBHQ were identical to those with TBHQ alone, indicating no interference with the detection of CoQ10H2 and CoQ10. Levels of VC in human CSF are reported to be about 200 µM, and we confirmed this observation. 200 µM VC in CSF corresponds to 50 µM VC in the extraction solvent. This may be sufficient to prevent CoQ10H2 oxidation during the extraction and concentration procedure. However, we chose to add 20 µM TBHQ to the extraction solvent to prepare for the case of minute VC levels being present in patients’ CSF. We would like to also emphasize that the addition of 20 µM TBHQ gave excellent reproducibility.Next we compared TBHQ, VC, and TBHQ + VC as reductants (20 µM each) in the extraction solvent, and the results are shown in Fig. 2 and 1.24 nM CoQ10 added to pseudo-CSF. The recovery of spiked CoQ10H2 and CoQ10 to pseudo-CSF were excellent (n = 3), as was TQ10 by extension.Table 2 and CoQ10 in pseudo-CSF (n = 3). The CV for VE, CoQ10H2, CoQ10, TQ10 and %CoQ10 were less than 10%, showing excellent within-day reproducibility. Table 2, CoQ10, TQ10 and %CoQ10 were less than 15%, indicating that the method is reliable.Table -CSF n = . The CV Figure 2 and CoQ10 was described. Since the levels of TQ10 in human CSF is ~2 nM, the IPA extract required concentrations prior to analysis. To prevent the oxidation of CoQ10H2 during extraction and concentration, 20 µM TBHQ was added to the extraction solvent (IPA). Good within-day and day-to-day reproducibility was observed and the analytical variance was excellent. We demonstrated our method is applicable to human CSF.A method for the simultaneous electrochemical detection of CoQ10H