Using ultraviolet photoelectron spectroscopy, we show that different iodide sources change the QD HOMO/LUMO levels, allowing their fine tuning. AFM measurements suggest that colloidally-passivated QDs result in formation of more uniform thin films in one-step deposition. The second part of this paper is devoted to the PbS QDs with colloidally-exchanged shells . We especially focus on QD optical properties and their stability during storage in ambient conditions. Colloidal lead iodide treatment is found to reduce the QD film resistivity and improve photoluminescence quantum yield (PLQY). At the same time stability of such QDs is reduced. MAI-treated QDs are found to be more stable in the ambient conditions but tend to agglomerate, which leads to undesirable changes in their optics.Iodide atomic surface passivation of lead chalcogenides has spawned a race in efficiency of quantum dot (QD)-based optoelectronic devices. Further development of QD applications requires a deeper understanding of the passivation mechanisms. In the first part of the current study, we compare optics and electrophysical properties of lead sulfide (PbS) QDs with iodine ligands, obtained from different iodine sources. Methylammonium iodide (MAI), lead iodide (PbI Type of the ligand and exchange treatment procedure govern quantum dot (QD) solid properties, such as energy structure, charge transport, and stability ,2. Long These days, LE can be performed with different methods and compounds . The mosOne strategy is post-deposition  LE, which strips the existing oleate ligands, simultaneously binding the desired new ligand , ethanedithiol) to the exposed QD surface ,15. Howe2 molecules [When QDs are used in optoelectronic devices, their environmental stability is of exceptional importance. Due to the high surface-to-volume ratio of the QDs, they become very susceptible to environment impact, which leads to undesired changes of QD properties. The lack of the degradation theory makes these changes unpredictable. Because of the lead-rich surface ,22,23, oolecules  and crea2), and TBAI have both advantages and disadvantages, indicating that a careful choice of both the LE procedure and the iodide source is required for different QD applications. Furthermore, we investigate how colloidal ligand exchange affects QD aging, both in colloidal and solid-state form.Despite the high demand of halide LE, there is a lack of direct comparison between different iodide treatments. In this paper, we investigate the impact of iodide source on the PbS QDs’ optical and electronic properties. We show that the use of methylammonium iodide (MAI), lead iodide  was performed as reported in [2 solution in the mixture of dimethylformamide (DMF) and n-butylamine (BA). To achieve LE, the resulting solution was shaken for two min. Precipitated QDs were redispersed in 1,2-dichlorobenzene (DCB) and BA mixture for deposition and storage. Colloidal MAI treatment was adopted from Lan et al. [Colloidal ligand exchange with lead iodide . All solutions were prepared with concentration of ∼10−6 M. Sartorius filter paper  was used as a porous matrix. QDs were transferred to the porous matrix via drop-casting the diluted solution (concentration of 10−6 M) onto the matrix. From this moment on, QDs in porous matrix will be referred to as ‘QD solids’. All of the samples were stored in ambient conditions without any light exposure.QD stability in the ambient atmosphere was studied for both QD solids and QD solutions. The reference sample of OA-capped QDs was dissolved in tetrachloromethane; the MAI-treated QDs were dissolved in a mixture of toluene and DMF (4:1 volume); and PbIFourier-transformed infrared (FTIR) spectra were acquired using Tensor 27 FTIR spectrometer . Atomic force microscopy (AFM) measurements were taken with Solver-Pro  atomic force microscope in the semicontact mode. UV-VIS-NIR absorption spectra were acquired using Shimadzu UV3600 spectrophotometer  equipped with the Shimadzu ISR-3100 integration sphere. The ultraviolet photoelectron spectra (UPS) were collected using PREVAC UPS system . QD HOMO level was determined directly from the UPS measurements.LUMO energy was calculated by adding QD bandgap energy to HOMO value. QD bandgap was determined from the position of the first excitonic absorption peak , adding −1, corresponding to the C–H stretching oscillations. FTIR absorption spectra for OA-, TBAI-, MAI-, and PbI2-passivated QD thin films are presented in 2 (87%) and TBAI (95%) treatments correspond well to the published protocols [LE efficiency was estimated by monitoring the residual OA ligands on the QD surface using the FTIR spectroscopy. OA had distinguishable absorption bands in the range of 3000–2800 cmrotocols ,27.2) demonstrated negligible changes in their absorption spectra (blue and red lines on Absorption spectra obtained for the QD films are presented in QDs with different ligand shells have different positions of the conduction band bottom (LUMO) and the valence band top (HOMO) . We show2 LE demonstrate almost no defects, as can be seen in 2, TBAI  (see reatment . In the 18 eV−1·cm−3). As we have already shown, MAI-treated QDs tend to agglomerate, which increases the carrier chance to be trapped in the nonradiative state. PbI2 treatment, in turn, reduces the number of deep traps by an order in comparison with TBAI and MAI treatments. We interpret this as a sign of a more efficient QD surface passivation.It has been shown ,34 that 2-treated QDs have slightly blue-shifted emission and absorption maxima, reduced Stokes shift, and increased PLQY  should be taken into account. Due to a certain size distribution, FRET can occur even in a quasi-monodisperse QD ensemble ,36. It l2-treated QDs are less stable and blue-shifting over the time of storage (2-treated QDs in pure n-butylamine and monitored their PL evolution (see Evolution of the PL parameters of the colloidal QDs is displayed in tion see . During tion see E. A negl2-treated QDs are also less stable in porous matrix. Such samples demonstrate a dramatic shift of PL peak position (The QD degradation is more pronounced in the solid-state form due to the direct contact with atmospheric oxygen see A. QDs lo2-treated sample demonstrates unusual behavior in PL decay evolution (see 2-treated QDs. These factors are the residual presence of n-butylamine, atmospheric oxygen, and the amount of iodide molecules on the QD surface.In contrast to their colloidal counterparts, QD solids suffer from a noticeable change of their PL decay during storage C. Averagtion see . Within 2 treatments result in QD film morphology and conductivity comparable to those achieved with common TBAI solid-state exchange. We found that PbI2-treated QDs demonstrate the highest PLQY and conductivity due to lower amount of the deep-trap states of as-exchanged QDs. As a drawback, PbI2-treated QDs possess poor environmental stability due to interactions with BA. In contrast, MAI-treated QDs show the highest stability of their PL responses, which is beneficial for creating more stable optoelectronic devices. These results indicate that a careful choice of LE procedure is required for each application. To achieve both high performance and stability, it is necessary to further develop LE techniques.To summarize, we performed a comparative study of iodide-passivated PbS QDs, prepared as both colloidal solutions and solids. We analyzed the influence of LE on the optical, electronic, and morphological properties of QD solids, as well as on the stability of QD properties. We show that more convenient and material-efficient colloidal MAI and PbI


The Belgian Superior Health Council (SHC) recently added a 13-valent pneumococcal conjugate vaccine (PCV13) to its recommendations for adult pneumococcal vaccination. This study addresses the policy question regarding whether a single dose of PCV13 should be reimbursed among Belgian adults aged 65–84 years with chronic comorbidities (“moderate-risk”) or immunosuppression (“high-risk”).A cohort model was developed to project lifetime risks, consequences, and costs of invasive pneumococcal disease (IPD) and pneumococcal community-acquired pneumonia (CAP). Parameter values were estimated using published literature and available databases, and were reviewed by Belgian experts. PCV13 effectiveness was assumed to be durable during the first 5 years following receipt, and to progressively decline thereafter with 15 years protection. The Belgian National Health Insurance perspective was employed.Use of PCV13 (vs. no vaccine) in moderate/high-risk persons aged 65–84 years  would be expected to prevent 527 cases of IPD, 1,744 cases of pneumococcal CAP and 176 pneumococcal-related deaths, and reduce medical care costs by €20.1 million. Vaccination costs, however, would increase by €36.9 million and thus total overall costs would increase by €16.8 million. Cost per QALY gained was €17,126. In probabilistic sensitivity analyses, use of PCV13 was cost-effective in 97% of 1,000 simulations.Reimbursement of PCV13 in moderate/high-risk Belgian adults aged 65–84 years would be cost-effective from the Belgian healthcare perspective. Streptococcus pneumoniae causes significant morbidity and mortality worldwide in both children and adults. Invasive pneumococcal disease (IPD)—including bacteremia and meningitis—is the most serious manifestation because of its high mortality rate, but is relatively uncommon. Nonbacteremic/noninvasive pneumococcal community-acquired pneumonia (CAP) has a much higher incidence rate and also is associated with significant morbidity, mortality, and healthcare costs. While widespread childhood use of pneumococcal conjugate vaccines has reduced the incidence of invasive pneumococcal disease, the burden of both invasive and noninvasive disease in older adults remains substantial )—which included adults with lung disease, heart disease, liver disease, diabetes, or asthma (self-reported)—compared with the overall study population, suggesting similar efficacy against vaccine-type CAP in at-risk adults as in the general population [The recommendation was based in large part on findings from the Community-Acquired Pneumonia Immunization Trial in Adults (CAPiTA), in which PCV13 demonstrated protection against community-acquired pneumococcal pneumonia  and IPD ,7. This pulation .While a single dose of PCV13 is now recommended for many age- and risk-specific adult subgroups in Belgium, the economic implications of vaccine reimbursement in these patient populations are unknown. Blommaert et al. have investigated the cost-effectiveness of PCV13 in Belgian adults aged >50 years, on an overall basis  as well as within the healthy subgroup, and concluded that PCV13 vaccination is unlikely to be cost-effective ,10. TheiThe model utilizes a deterministic cohort framework and a Markov-type process to depict expected lifetime risks, consequences, and costs of IPD and pneumococcal CAP, as well as the expected impact of PCV13 vaccination, in Belgian adults . Both IPThe model cohort is characterized in terms of age and risk profile . Low-risk includes persons who are immunocompetent without chronic comorbidities, moderate-risk includes persons who are immunocompetent with ≥1 chronic comorbidity, and high-risk includes persons who are immunocompromised . Low-risk persons may transition to the moderate-risk or high-risk groups during the modeling horizon based on the probability of newly developing a chronic comorbidity or immunocompromising condition, respectively; moderate-risk persons may similarly transition to the high-risk group. Persons in the model cohort may receive PCV13 or no vaccine at model entry; vaccination coverage may vary by age and risk profile. No vaccine was selected as the comparator because PPV23 is infrequently used among Belgian adults  and PCV13 will not replace PPV23 (cfr SHC recommendation). The current situation considering 10% vaccination with PPV23 and 90% no vaccination will be used as comparator in a scenario analysis .Expected clinical outcomes and economic costs are estimated for the model cohort on an annual basis, based on age, risk profile, vaccination status, and time since vaccination. IPD is stratified by condition (bacteremia vs. meningitis), and pneumococcal CAP is stratified by setting of care . Persons vaccinated at model entry may be at lower risk of future IPD and pneumococcal CAP; the magnitude of vaccine-associated risk reduction depends on clinical presentation , as well as time since vaccination, age, and risk profile. Risk of death from IPD, hospitalized CAP, and all other causes  depends upon age and risk profile.Expected costs of medical treatment for IPD and pneumococcal CAP are generated based on event risks and unit costs in relation to the setting of care , age, and risk profile. Costs of vaccination—including vaccine and vaccine administration—are tallied at model entry. Clinical outcomes and economic costs are projected over the modelling horizon for the model cohort under the vaccination strategies considered, and include: cases of IPD (bacteremia and meningitis) and pneumococcal CAP ; deaths due to IPD and CAP; life-years ; costs of medical treatment for IPD and CAP; and costs of vaccination.Rates of IPD and pneumococcal CAP , case-fatality rates, and disease-specific episodic costs—by age and risk profile —were estimated based on age-specific values from Belgium sources, age-specific relative rates/costs by risk profile from Belgium, UK and US sources, and age-specific population weights by risk profile from Belgium sources. The incidence and mortality values reflect a conservative approach . A detaiAge-specific rates of bacteraemia and meningitis were based on 2015 surveillance data from the Belgian National Reference Centre, and were extrapolated to the Belgian adult population . Age-speAge- and risk-specific rates of all-cause outpatient pneumonia were estimated using 2013 data from the INTEGO Database, which was assumed to be representative of the Belgian population ,14. EpisAge-specific rates of pneumococcal CAP requiring inpatient care  were estimated using data from the KCE report . Age-speEffectiveness of PCV13 against vaccine-type IPD for immunocompetent  persons with chronic comorbidities was based on the estimated efficacy of PCV13 against vaccine-type IPD among subjects in the per-protocol population of the CAPiTA study . The impEffectiveness of PCV13 against VT-CAP for immunocompetent persons with chronic comorbidities  was based on the estimated efficacy of PCV13 against vaccine-type CAP among subjects in the per-protocol population of a post-hoc analysis of the CAPiTA study . In the VE was considered stable during the first 5 years following vaccination , and theMedical-care costs were derived from the database of a large sickfund and interviews ,19,20, aThe reimbursed price of PCV13 was assumed to be €63.64 per dose, based on the public price (€75.44) . BecauseClinical outcomes and economic costs were projected over remaining years of life for moderate/high-risk Belgian adults aged 65–84 years at model entry, under two alternative vaccination strategies: (1) use of PCV13 at model entry (58% vaccination coverage level); and (2) no vaccination at model entry. Analyses were conducted from the perspective of the Belgian healthcare public payer; accordingly, only direct costs associated with the provision of medical care for IPD and pneumococcal CAP, and the costs of vaccination, were considered. Results were standardized to the Belgian population of interest based on age- and risk-specific weights. Medical-care costs were discounted at 3% annually, and life-years were discounted at 1.5% annually .The cost-effectiveness of PCV13 (vs. no vaccination) was calculated in terms of the cost per life-year gained and cost per QALY gained, respectively, which are derived as the ratio of the difference in total costs to the difference in total life-years/QALYs. In Belgium, a willingness-to-pay threshold—below which interventions are considered cost-effective—has not been formalized. However, the Belgian Health Care Knowledge Centre (KCE) has recently used three alternative thresholds in health technology assessments of pneumococcal vaccination in the elderly, namely 35,000€, 70,000€, and 105,000€, which correspond to 1x, 2x, and 3x the gross domestic product (GDP) per inhabitant [One-way deterministic sensitivity analyses were undertaken to evaluate the potential impact of parameter value uncertainty on study results for moderate/high-risk adults aged 65–84 years. In these analyses, key model parameter values were varied ±25% of their base case levels (unless otherwise noted below), each in turn, including: rates of ambulant and hospitalized pneumococcal CAP; age-specific utility values; case-fatality rates; vaccine effectiveness ; vaccine effectiveness  for high-risk adults relative to immunocompetent adults; medical-care costs; and herd effect for vaccine-type IPD and pneumococcal CAP (varied from 0–100%). Probabilistic sensitivity analysis  was employed to account for uncertainty surrounding disease rates and costs, case-fatality rates, and vaccine effectiveness and other key model parameters in estimation of clinical outcomes, economic costs, and incremental cost-effectiveness ratios.In addition, scenario analyses were conducted to evaluate the impact of methodological uncertainty on study results for moderate/high-risk adults aged 65–84 years. In these analyses, alternative data sources and/or assumptions were employed in estimating key variables, including: current situation as comparator ; higher case-fatality rates (based on Blommaert 2016a), PCV13 serotype coverage against pneumococcal CAP , higher rate of waning for PCV13 , vaccine coverage , discount rate , and population comprising persons aged 65 years only. Scenario analyses focusing on moderate-risk Belgian adults aged 65–84 years and high-risk Belgian adults aged 65–84 years, respectively, were also conducted.Without vaccination, the expected lifetime numbers of total cases of disease among moderate-risk and high-risk Belgian adults aged 65–84 years  would be: IPD, 7,352; hospitalized pneumococcal CAP, 33,662; and outpatient pneumococcal CAP, 17,617 . ExpecteOn balance, therefore, use of PCV13—as described above—would reduce lifetime cases of IPD by 527, hospitalized pneumococcal CAP by 1,074, and outpatient pneumococcal CAP by 669; disease-related deaths by 176; and medical-care costs (excluding vaccination costs) by €20.1 million. Medical-care and vaccination costs (aggregated) would increase by €16.8 million. Cost per QALY gained  was €17,126.In all of the aforementioned one-way deterministic sensitivity analyses, use of PCV13 among moderate-risk and high-risk Belgian adults aged 65–84 years was found to be cost-effective versus no routine vaccination . Cost-efIn the scenario analysis comparing use of PCV13 (58% coverage) versus use of PPV23  among moderate/high-risk Belgian adults aged 65–84 years, use of PCV13 was found to be cost-effective. Similarly, in the scenario analysis comparing use of PCV13 versus no vaccine in a cohort of moderate/high-risk Belgian adults aged 65 years, and those analyses focusing (separately) on moderate-risk Belgian adults aged 65–84 years and high-risk Belgian adults aged 65–84 years, use of PCV13 was found to be cost-effective. Results from all sensitivity analyses and all scenario analyses are set forth in the In the probabilistic sensitivity analysis, all of the 1,000 replications generated estimates of incremental cost per LY/QALY gained that were located in the northeast quadrant of the scatterplot, and thus projected higher costs and higher LY/QALY with use of PCV13 versus no routine vaccination . In thisSince April 2015, the SHC in Belgium has included PCV13 in their recommendations for pneumococcal vaccination among adults of various ages and risk profiles. While Blommaert et al. ,10 have Our findings suggest that, under conservative assumptions concerning disease burden and reasonable assumptions concerning vaccine effectiveness and costs, implementation of a strategy targeting Belgian moderate-risk and high-risk adults aged 65–84 years for vaccination with PCV13 would reduce the numbers of cases of pneumococcal disease and pneumococcal-related deaths, would—on an overall basis—partly offset the cost of the vaccination from the healthcare public system perspective, and would be a cost-effective use of scarce healthcare resources. Notwithstanding differences in model structure, model population, model estimation, and vaccination strategies, our conclusions are largely consistent with those from several recent evaluations in which adult PCV13 use (mainly persons aged ≥65 years) was found to have a reasonable cost-effectiveness profile based on current disease epidemiology ,24–26. IThe results of analyses described herein are based on data and assumptions specific to the vaccination environment  in Belgium, and thus reflect the recommendations and use of PCV13, PPV23, and PCV10 in that setting. While findings were projected assuming conservative values for several key model parameters, and were robust when varying key model parameters in sensitivity analyses (including higher waning of PCV13 effectiveness and herd effect from PCV10 use), caution should be exercised in generalizing the results of this study to other countries in which vaccine recommendations and use may be different among children and adults. The results of this assessment are based on certain assumptions regarding the level and durability of PCV13 effectiveness among adults, and the level and persistence of indirect vaccination effects from the Belgian pediatric program, and thus are limited to the scenarios considered herein until proven otherwise. To the extent new data become available, study results will require updating.A modelling approach was chosen because, for several reasons, a trial-based  pharmacoeconomic evaluation focusing on the population of interest was not possible. CAPiTA enrolled only subjects in the Netherlands, and the epidemiology of pneumococcal disease and the associated use of healthcare resources may be different in important aspects in Belgium. CAPiTA enrolled only subjects who were immunocompetent, and thus high-risk persons were not included in the trial population . Follow-up in CAPiTA extended for only 4–5 years (on average), and thus the long-term/life-time benefits of vaccination were not captured. Moreover, we note that the modelling approach described herein—in terms of the structure of the model and the methods of estimation—is similar to that employed in other recently published economic evaluations of alternative strategies for pneumococcal vaccination in other countries ,20,28–33We believe that the major limitation of this assessment is the uncertainty concerning some of the parameter estimates. Perhaps the area of greatest uncertainty concerns the assumed effectiveness of vaccination with PCV13, which was based primarily on data from the CAPiTA trial ,34. EffeS. pneumoniae among Belgian adults is largely unknown; the conservative estimates from Blommaert et al. were applied [S. pneumoniae, the veracity of which is unknown. Because the purpose of this study was to compare the risks and costs of IPD and pneumococcal CAP over remaining years of life for a given cohort of Belgian adults assumed to receive PCV13 versus no vaccine, persons were precluded from aging into the model population.Another area of parameter uncertainty concerns rates of IPD and pneumococcal CAP, not only in the first year of the modelling horizon, but also future levels of disease that may be prevented with vaccination. The percentage of CAP that is attributable to  applied . Rates o applied  with PCVWe also note that PCV13 coverage levels were based on the assumption that the vaccine will be reimbursed, and that its use will be comparable to other vaccines that are currently reimbursed in Belgium . Finally, our model, like all cohort models, simplifies reality to some extent. Our model does not, for example, take into account serotype replacement over time; to date, replacement by non-PCV serotypes appears to be limited in the Belgian adult population, however, and thus its consideration in the model would be expected to have a limited impact on study findings based upon the data and time period that were considered . Long teThe results of the pharmacoeconomic assessment described herein suggest that, under conservative assumptions concerning disease burden , and reasonable assumptions on vaccine effectiveness, and cost of pneumococcal infection, implementation of a strategy targeting Belgian moderate-risk and high-risk adults aged 65–84 years for vaccination with PCV13 would reduce the numbers of cases of pneumococcal disease and pneumococcal-related deaths and would be—on an overall basis—cost effective from the healthcare public system perspective.S1 File(DOC)Click here for additional data file.


The gender and age effect was also examined. MMP-1, MMP-9, and IL-6 were expressed in higher levels compared to OPN and TNFa as well as in symptomatic (with type II and III carotid plaque classification) than asymptomatic (type IV) patients with differences considered statistically significant . A significant positive correlation between MMP-1 and IL-6 (with Pearson correlation coefficient 0.748) is also notable. The data give further insight into the possible role of specific biomarker and enhance the need for further studies in order to clarify the proper one(s) for detection of the vulnerable plaque and help identify patients at risk for cardiovascular events.Atherosclerosis is an inflammatory process resulting in local plaque deposition in the vessel wall of arteries with symptoms to various areas of vascular tree. Identification of patients with progressive advanced atherosclerotic disease is mainly based on the known characteristics of the vulnerable or recently ruptured plaque. Molecular and cellular features associated with the vulnerable plaque are considered potential diagnostic markers for plaque rupture and thrombosis. Here, protein expression levels of the metalloproteases MMP-1, MMP-9, osteopontin (OPN), and cytokines TNF Atherosclerosis is a chronic, inflammatory disease of the medium and large arteries, such as the aorta and carotid arteries. It is a major contributor to the development of cardiovascular diseases and the leading cause of death worldwide. Although atherosclerotic plaque development is a local process in the vessel wall that can give rise to symptoms in one specific area, it is also a systemic disease with simultaneous plaque formation in different areas of the vasculature –3. MoreoAtherosclerotic plaques are characterized by intimal thickening from the progressive accumulation of lipids , 2 togetIn other reports, for example, serum markers such as high-sensitive C-reactive protein, interleukin-6 –9, and IOther markers that have been previously associated with cardiovascular events and are now associated with carotid atherosclerotic plaques are matrix metalloproteinases (MMPs) , tissue It has been also reported that carotid plaque composition contains predictive information for future cardiovascular events elsewhere in the vascular system, independent from established risk factors and medication. Moreover, patients undergoing carotid endarterectomy, with a local plaque containing intra plaque hemorrhage or marked intra plaque micro vessel formation, demonstrated an increased risk of secondary cardiovascular events with high hazard ratios , 22. ThuThis study looks at evaluating carotid plaques in symptomatic and asymptomatic patients after endarterectomy in order to potentially detect vulnerable plaques, based on the differences in protein expression levels of five biomarkers.α were purchased from Acris Antibodies , while anti-osteopontin was purchased from Millipore. The rabbit polyclonal antibody raised against actin and the secondary anti-rabbit antibody conjugated with alkaline phosphatase were purchased from Sigma-Aldrich Chem Co . All other chemicals used in the study were also purchased from Sigma-Aldrich.Rabbit polyclonal antibodies that recognise MMP-1, MMP-9, IL-6, and TNF24 patients  age (55-85) were enrolled from the Konstantopoulio General Hospital, Department of Vascular Surgery, Athens, Greece, who had undergone standard carotid ultrasound examination and subsequently carotid endarterectomy. All patients were entered into a prospective database and were on best medical treatment. Colour Doppler ultrasonography and Power Doppler were used in addition to serial ultrasonographic measurements, in order to monitor and evaluate the carotid plaque type and vulnerability preoperatively. Following the carotid endarterectomy, the carotid plaque specimen from each patient was immediately washed and stored at -80°C under liquid nitrogen, until tissue homogenization and subsequent analysis.  Nonidet P-40 according to Sigala et al. [Frozen carotid plaque samples were homogenized in 50 mM Hepes pH 7.5, 150 mM NaCl, 15 mM a et al. . The homa et al. , using bα (Acris), and OPN (Millipore). The anti-actin antibody (Sigma-Aldrich) was used for assessing equal loading of total protein per sample. Gel-Pro Analyzer was used to evaluate and record the intensity of each band.Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS as described by Laemmli et al. . ProteinStatistical analysis was performed by SPSS (version 23) software and Microsoft Excel 2011 for Mac and the data were expressed as mean values ± standard deviation (SD). Independent samples T-test was used to determine differences in the variables. P values less than 0.05 were considered significant at confidence level 95%. Pearson correlation analysis was used to assess correlation between the biomarkers.Carotid duplex ultrasound provides quantitative measurements of carotid artery stenosis and intima-media thickness. The diagnosis of carotid artery stenosis is important in clinical practice because it determines the need for surgical intervention while carotid intima-media thickness measurements provide a quantitative measure of disease . This stStatistical significant differences in MMP-1 levels between symptomatic and asymptomatic patients, P<0.05 (P=0), were recorded .Higher protein levels of MMP-9 were also detected in the samples of symptomatic than in asymptomatic patients , with thThe expression level of OPN was low and the variation within samples from symptomatic and asymptomatic patients was statistically insignificant at a confidence level 95%, with P=0.2. Previous reports show that carotid plaque osteopontin level is a predictor of cardiovascular events after carotid endarterectomy  and high (data not shown).Cytokines are key regulatory glycoproteins that modulate all aspects of vascular inflammation. Many cytokines have been implicated in atheroma formation and complication. In this study, the cytokines IL-6 and TNFa were measured. IL-6 is a phosphoglycoprotein with MW ranging from 21 to 29 kDa, depending on the degree of glycosylation and phosphorylation and IL-6 dimer forms a complex with the dimer of IL-6 receptor and the dimer of gp-130 to initiate signal transduction , 37. IL-Expression levels of these five biomarkers, both in symptomatic and asymptomatic patients, were also evaluated regarding the gender of patients. There is no statistical significant difference between males (n=11) and females (n=5) in the values of protein expression levels in all cases of symptomatic patients. The boxplot of the mean MMP-1 values in male and female symptomatic patients, indicatively, is shown in Pearson correlation values were used to examine linear correlation between the biomarkers. Pearson correlation coefficient value of 0.748 for MMP-1 and IL-6 and 0.606 for MMP-9 and IL-6  indicateThe results demonstrate that expression levels of MMP-1, MMP-9, and IL-6 were significantly more pronounced than OPN and TNFa, respectively, and statistical significant differences in the values of the first three between symptomatic and asymptomatic patients were also observed. The findings support that elevated levels of the three biomarkers identify symptomatic patients and may be used for vulnerable plaque classification and the prediction of patients' risk for cardiovascular events.It is worth mentioning that despite the prominent differences in the protein expression level of the biomarkers studied, no control samples from healthy individuals could be used in order to have a threshold value to compare with, indicating that a standard control when evaluating a biomarker is still of major concern. This is due to obvious ethical issues and carotid plaque analysis is therefore limited to patients who undergo vascular surgery.Thus, these data analyses prompt further studies, evaluating larger groups of patients and compare the protein expression levels of the biomarkers in carotid plaques versus the circulating ones which may give rise to tissue specific biomarkers. Therefore, it may provide evidence towards patient selection and the treatment planning in order to decrease the perioperative risk.


Ageing, one of the largest risk factors for many complex diseases, is highly interconnected to metabolic processes. Investigating the changes in metabolite concentration during ageing among healthy individuals offers us unique insights to healthy ageing. We aim to identify ageing-associated metabolites that are independent from chronological age to deepen our understanding of the long-term changes in metabolites upon ageing. Sex-stratified longitudinal analyses were performed using fasting serum samples of 590 healthy KORA individuals (317 women and 273 men) who participated in both baseline (KORA S4) and seven-year follow-up (KORA F4) studies. Replication was conducted using serum samples of 386 healthy CARLA participants (195 women and 191 men) in both baseline (CARLA-0) and four-year follow-up (CARLA-1) studies. Generalized estimation equation models were performed on each metabolite to identify ageing-associated metabolites after adjusting for baseline chronological age, body mass index, physical activity, smoking status, alcohol intake and systolic blood pressure. Literature researches were conducted to understand their biochemical relevance. Out of 122 metabolites analysed, we identified and replicated five  and four  significant metabolites in women and men respectively. Arginine decreased, while ornithine increased in both sexes. These metabolites are involved in several ageing processes: apoptosis, mitochondrial dysfunction, inflammation, lipid metabolism, autophagy and oxidative stress resistance. The study reveals several significant ageing-associated metabolite changes with two-time-point measurements on healthy individuals. Larger studies are required to confirm our findings. Ageing, the deterioration of functional capacity and stress resistance over a lifetime ,2, is a However, due to the high metabolic heterogeneity of individuals, the development of ageing biomarker remains a great challenge at technical level ,10,11. MTargeted metabolomics, which is a data-driven approach to analyse biochemically known and annotated metabolites in a living organism ,16,17, cConsequently, previous studies have used metabolomics to explain metabolic changes associated with age in humans ,20,21,22The primary objective of the study is to identify metabolites that commonly change among healthy individuals over time. The secondary objective is to understand the potential biochemical roles of the identified metabolites in ageing.In KORA, a total of 317 healthy women and 273 healthy men were included for analysis A. ReplicThe characteristics of study participants at two time-points for KORA and CARLA studies are shown in p < 4.1 × 10−4) associated with ageing in women and men, respectively  was ornithine, with a yearly average increase by 0.14 standard deviations in KORA in women and men, respectively and by 0.24 and 0.22 in CARLA in women and men, respectively. In addition to the replicated metabolites mentioned above, additional five metabolites (namely octadecenoylcarnitine (C18:1), phenylalanine, valine, lysoPC a C16:0 and lysoPC a C18:0) were significant in the CARLA study but in opposite trends between discovery and replication studies: phenylalanine  .Based on literature search and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database, we found that three amino acids, ornithine, serine and arginine, are involved in different pathways related to the ageing processes. Their metabolic pathways and/or cell-signalling functions are shown in Ageing is difficult to be captured due to high individual variances along time. Limited by the study design, previous large-scale, cross-sectional metabolomics studies of ageing ,23 were Hence, with the availability of two-time-point measurements, our study has made the capture of the “ageing” phenotype with consideration of individual heterogeneity in ageing possible. By pooling the individual data of metabolite changes over time after adjusting for potential confounders that may affect ageing rate and metabolite concentration, namely chronological age at baseline, BMI and lifestyle factors, what we observe is, consequently, the average of changes in metabolite concentration within a healthy population during ageing, irrespective of individual differences. Therefore, the metabolites we identified are associated with ageing process in general.In both KORA and CARLA studies, we observed similar degree of change in replicated metabolites. However, it was noteworthy that a number of metabolites  demonstrated significant but opposite changes between KORA and CARLA participants. There are some potential factors that may account for the differences, for instance, the concentrations of these metabolites are systematically different between fasting (in KORA) and non-fasting samples (in CARLA) because they could be influenced by the time since the last food intake and activities ,26. FurtConsistent with previous studies that suggested metabolic profiles are highly sex-dependent ,15,28,29Furthermore, it is also noteworthy that serine, which was discovered and replicated in women only, also commonly increased in both sexes over time and was significant in men under FDR-adjusted P value in discovery and replication studies. This indicates that despite its insignificant increase in women over time, this metabolite could also be associated with ageing in general. However, our study power to detect the changes was potentially limited by the sample size.Arginine and ornithine are involved in the metabolism of polyamines  whose supplementation in several model organisms led to increased lifespans . DecreasWhether ageing-associated increase of the C18 in women and decrease of PC aa C36:3 and PC ae 40:5 in men of the KORA and CARLA studies could be related to low polyamines or developing pre-diabetes  remain tAgeing KORA and CARLA individuals also demonstrated increased levels of serine, a non-essential amino acid and a predominant source of one carbon groups for the de novo synthesis of purine nucleotides that is essential for cell proliferation. Serine has been recently linked with decreased lifespan in yeast models through sensitization to oxidative stress and activation of the TOR-S6 signalling pathway .Meanwhile, ageing-related increase of tyrosine in KORA and CARLA women could be due to alterations in the activity of tyrosine hydroxylase (TH) and synthesis of catecholamines such as dopamine and other neurotransmitters involved in stress response. In mouse models, a haplo-insufficiency in TH was beneficial for regulation of blood pressure and increased lifespan .This study adds to the relatively less investigated field of human ageing by metabolomics using a longitudinal study design. It has the privilege of making use of the longitudinal targeted metabolomics data of healthy individuals from two independent human cohort studies. It also fills the current knowledge gap by capturing the change in metabolites during ageing by making the observation of the individual changes in metabolite concentration over time possible and accounting for the intra- and inter-personal variances, such as BMI and lifestyle changes over time. Furthermore, the similarity of participant characteristics in both studies has made our results generally comparable. Moreover, both KORA and CARLA studies made use of serum samples, which are generally more subject to homeostatic control and hence generally less sensitive to intervention-induced metabolic phenotype changes compared to urine samples. Despite urine samples have an advantage of non–invasive sampling method, appropriate normalization methods for the metabolite concentration profiles should be considered when adopted ,42. FutuAnother major strength of this study is the replication from another independent CARLA study. After taken into account the potential influences of inter-kit differences on metabolite concentration in KORA study, CARLA study serves as an excellent replication study, since the metabolites in CARLA were measured with the same assay at both time-points. Hence, the fact that several amino acids were successfully replicated with similar degree of changes over time further adds robustness to the evidence in the study. Nevertheless, our study also exhibits several limitations. Firstly, the difference between fasting and non-fasting samples may influence the concentration level of certain metabolites, especially those from the small lipid class. Secondly, since only two time-points were available, it provided limited information on whether individuals experience consistent changes in metabolite concentration along time. Thirdly, the generalizability of the results was limited to the German population where individuals may share similar lifestyle factors and genetic backgrounds. Fourthly, given the complexity of ageing process, future studies should consider performing systems analyses on multi-OMICs data to examine multiple biomarkers apart from metabolomics. Finally, it is noteworthy that despite some sex-specific metabolite changes, many of them also displayed changes in the same direction in both sexes, even though they were borderline significant. Our literature review of published studies pointed to a very low reproducibility of ageing-related metabolites, especially those in lipid class. Thus, future studies with larger sample size and multi-time-point data from other fasting populations are highly recommended to confirm our results.All participants in KORA and CARLA studies gave written informed consent. The KORA study was approved by the local ethics committee of the Bavarian Medical Association, Germany. The project identification codes for KORA S4 and KORA F4 are 99186 and 06068, respectively. The CARLA study was approved by the local ethics commission of the Medical Faculty of the Martin Luther University of Halle-Wittenberg and was carried out in accordance with the declaration of Helsinki. The ethic codes for CARLA-0 and CARLA-1 are 117/24.10.01/11 and 164/12.10.05/1, respectively. 4.2. KORA Studies The “Kooperative Gesundheitsforschung in der Region Augsburg” (KORA) Study is a large population-based cohort conducted in Augsburg region, Germany . It consThe CARLA-Study  is a population-based cohort study in an elderly population of the city Halle/Saale in eastern Germany . Study dIn KORA, blood was drawn into S-Monovette® serum gel tubes  in the morning between 8:00 a.m. and 10:30 a.m. after a fasting period of at least eight hours . Blood sIn CARLA, metabolic profiles in the blood serum of the study participants were measured. Blood samples were taken after a supine rest of 30 min. Followed by a 10 min centrifugation , the samples was collected and deep frozen to −80 °C on the same day until analysis of the metabolites .IDQTM p180 Kit and AbsoluteIDQTM p150 Kit , respectively [IDQTM p150 Kit [Targeted metabolomics measurements have been performed in the Metabolomics Platform of the Helmholtz Zentrum München. In KORA, serum samples from participants at the baseline (KORA S4) and follow-up (KORA F4) studies were measured with the Absoluteectively ,28. In Cp150 Kit . Serum sp150 Kit ,48.IDQTM p180 Kit (KORA S4) and AbsoluteIDQTM p150 Kit (KORA F4). A detailed QC for AbsoluteIDQTM p180 Kit was reported previously in Quality control (QC) procedures were performed for both studies and details were reported elsewhere ,17. In KIDQTM p180 Kit and AbsoluteIDQTM p150 Kit) were used for quantitative measurements of metabolites in KORA S4 and KORA F4, respectively, inter-kit normalization was performed. For both assays, the same reference samples  were measured five times on each kit plate. QC procedures were performed on these reference samples. In total, 114 and 351 reference samples were initially analysed for AbsoluteIDQTM p180 Kit and AbsoluteIDQTM p150 Kit respectively. Sample values that exceed the range of median ±1.5 inter-quartile range were identified as outlier values. Samples which contained more than or equal to 10 outlier values were defined as outlier samples and were excluded . After exclusion of outlier samples, coefficient of variation (CV) of each metabolite in each kit were calculated separately using the remaining 111 and 328 samples respectively. Metabolites which had a CV larger than 25% in either KORA S4 or KORA F4 were excluded from the analysis. Accordingly, one metabolite (C9), which had a CV value of 36.96% (>25%) in KORA F4 (refer to Since different assays (Absoluterefer to  was exclIDQTM p180 Kit (used in KORA S4) by the median of reference sample values in AbsoluteIDQTM p150 Kit (used in KORA F4). Afterwards, the metabolite concentration of each sample in KORA F4 was adjusted by multiplying the concentration values to the NF of their corresponding metabolites  for each metabolite were calculated by dividing the median of reference sample values in Absoluteabolites . Median abolites .Since some metabolites had skewed distribution, all metabolite concentrations were natural-log transformed and scaled (z-transformed) for a more symmetric distribution before statistical analyses. In KORA, a total of 1125 individuals whose serum samples were collected at both baseline and follow-up studies were included for analysis. Since diets have notable influences on metabolite concentration and may lead to confounding results, we excluded 126 individuals with non-fasting samples at either time point A. In ordincluded A.In CARLA, the same criteria were applied except non-fasting samples were used as majority of CARLA samples were non-fasted. Among the 1409 individuals whose blood samples were available in both baseline and follow-up studies, 395 individuals with cardiovascular disease or diabetes, 241 individuals with hypertension and 50 individuals with obesity in either or both baseline and follow-up were excluded B. In addOwing to the significant metabolic ,28 and bGeneralized estimation equation (GEE) model was performed on each metabolite  to identify any significant changes in metabolite concentration during normal ageing process in terms of years, after adjusted for the intra- and inter-personal confounders within two time-points. Baseline chronological age , BMI 1111 and poMetabolite | id = ß0 + ß1*(year) + ß2* + ß3*(BMI) + ß4* + ß5*(smoking status) + ß6* + ß7*(SB pressure) + ε.The outcome variable of the model was the concentration of each metabolite, while the major predictor variable of interest was the beta estimate of year, which represents the overall changes in metabolite concentration each year. To identify significantly changed metabolites in both women and men, in the discovery study (KORA), we adopted stringent Bonferroni correction for determining the cut-off of significance level. After identifying the significantly changed metabolites based on stringent Bonferroni correction, to further determine whether a metabolite is replicated in the replication study (CARLA), less stringent false discovery rate-adjusted P value (pFDR) will be adopted, given the smaller sample size in CARLA study and the discovery nature in this paper. Nevertheless, both significant P values after Bonferroni correction and FDR adjustment were displayed and indicated for reference.http://www.r-project.org/) . Packages used were broom , tableone , joineR , gee , gdata , scales  and graphics .All analyses were performed and the graphs were generated using the R programming language version 3.1.3 (http://www.genome.jp/kegg/pathway.html). Potential metabolic pathways related to the identified metabolites were investigated using literature search and a web database for pathway analysis: KEGG Pathways (The study reveals several significant ageing-associated metabolite changes with two-time-point measurements on the same individuals in two German populations. For instance, the decrease in arginine and the increase in ornithine and serine over time may be involved in ageing processes: apoptosis, mitochondrial dysfunction, inflammation, lipid metabolism, autophagy and oxidative stress resistance. Future studies with larger sample size and multi-time-point data should be conducted to confirm our findings.


We re-examined the participants of a clinical trial four years after enrolment to identify which of the two most commonly used eyelid surgery procedures to treat the blinding stage of trachoma , the posterior Lamellar Tarsal Rotation (PLTR) and Billamelar Tarsal Rotation (BLTR), gives better results in the long-term.A randomised, controlled, single masked clinical trial was done in Ethiopia. At baseline, adults (aged >18 years with upper lid unoperated TT were recruited from a community-based screening. Participants were randomly assigned (1:1), to either BLTR or PLTR surgery, stratified by surgeon. At 4 years an independent assessor masked to allocation examined the trial participants’ eyes using the same procedures as for the baseline and earlier follow-ups. The primary outcome was the proportion of individuals who had recurrence  at the 4-year examination, or a history of repeat surgery in the 4-year period. The intervention effect was estimated by logistic regression, controlled for surgeon as a fixed effect in the model. The trial is registered with the Pan African Clinical Trials Registry (number PACTR201401000743135).p = 0•0006, with 8•6% (95%CI 3•8–13•5) risk difference.1000 participants with TT were enrolled, randomly assigned, and treated (501 in the BLTR group and 499 in the PLTR group) between Feb 13, 2014, and May 31, 2014. At year 4, 943 (94.3%) participants were re-examined  and included in the primary outcome analysis. PTT had developed in 169/943 (17•9%) study eyes, among which 129 (76•3%) had minor trichiasis (≤5 lashes touching the eye). PTT was significantly more frequent at 4-year in the BLTR arm (105/472 [22•2%]) than the PLTR arm (64/471 [13•6%]), adjusted OR 1•82 ; The PLTR surgical procedure had superior long-term outcomes to the BLTR with significantly lower risk of PTT supporting the current WHO guideline that the PLTR should be the procedure of choice for training new surgeons in the programmatic management of TT. Our study group conducted and published a systematic review of the management of trachomatous trichiasis searching CENTRAL, Ovid MEDLINE, EMBASE, ISRCTN registry, ClinicalTrials.gov, WHO ICTRP. The only randomised trial which compared variants of the Bilamellar Tarsal Rotation (BLTR) and Posterior Lamellar Tarsal Rotation (PLTR) surgical procedures performed by ophthalmologists in a teaching hospital in Ethiopia was conducted on 153 patients in 2002, which found no evidence of a difference in outcome after three months. We conducted a randomised, controlled, single masked clinical trial between February and May 2015 to determine the relative effectiveness of the PLTR and BLTR Ethiopia in larger sample (1000 patients) in a programmatic setting in Ethiopia. The 12-month results showed that the PLTR was superior to BLTR giving a substantially lower trichiasis recurrence rate by one year and fewer intra and immediate post-operative complications. There is no data on the long-term outcome of these two surgical procedures from a head to head comparison trial.The one-year results of our trial led to a shift in international guidance from BLTR being the treatment of choice to treat trachomatous trichiasis to a preference for new trainees to be taught PLTR. However, there was much international interest to see the long-term outcome of the PLTR and BLTR. Our trial participants were examined four years after randomised intervention to assess the long-term outcome of these two procedures and to ascertain whether the superiority of PLTR would be sustained beyond one year. The results showed that the PLTR surgical procedure had still superior long-term outcomes to the BLTR with significantly lower risk of recurrent trichiasis four years after surgery.The available evidences support the current WHO guideline that the PLTR should be the procedure of choice for training new surgeons in the programmatic management of TT.1Trachomatous Trichiasis (TT), the blinding stage of trachoma, is mainly treated with corrective eyelid surgery ,2. Many Four years ago, we conducted a randomised, controlled, single masked clinical trial to compare the relative effectiveness of the PLTR and BLTR procedures. One year after surgery we found that the cumulative rate of recurrent trichiasis  was more frequent in the BLTR group than in the PLTR group with a 9.5% risk difference There are about 3 million un-operated cases of TT globally In this long-term follow-up of a randomised controlled surgical trial, we followed and examined trial participants four years after enrolment to investigate the long-term outcomes of BLTR and PLTR surgery, and to ascertain whether the superiority of the PLTR outcome was sustained beyond one year.22.1The trial methods have been previously described in detail ,20. In s2.2Participants were randomly assigned (1:1) to either PLTR or BLTR surgery. Randomisation was stratified by surgeon and sequences were computer-generated by an independent statistician with random block sizes of 4 or 6. Allocations were concealed in sequentially numbered, sealed, opaque envelopes. Examiners who were responsible for clinical observations at baseline and follow-ups were masked to allocation. The surgery was performed by six experienced nurse/health officer trichiasis surgeons. These were already trained, certified, and regularly performing PLTR surgery. They were trained rigorously on the BLTR procedure using the WHO trichiasis surgery training manual 2.3Participant eyes were examined (EH) at baseline prior to randomisation using 2•5 × binocular loupes and torch, and graded using the detailed World Health Organisation (WHO) Follicles Papillae Cicatricae (FPC) Grading System Participants were re-examined at 10-days, 6-months, and 12-months postoperatively, following the same assessment procedures as per baseline. The only additional elements were assessment for granuloma, level of eyelid correction, and post-operative eyelid contour abnormalities (ECA). ECA were graded according to the PRET trial methodology The four-year follow-up was approved by the Ethiopian National Health Research Ethics Review Committee, the London School of Hygiene & Tropical Medicine Ethics Committee, Emory University Institutional Review Board, and the Ethiopian Food, Medicine and Healthcare Administration and Controls Authority. The study was conducted in compliance with the Declaration of Helsinki and International Conference on Harmonisation–Good Clinical Practice.Trial participants were re-contacted and invited to attend a four-year follow-up assessment at a local health facility. Those who were not able to come to the health facilities were examined in their homes. Reasons for loss to follow-up were identified and documented. Written informed consent in Amharic had been obtained at baseline, before the initial enrolment from participants. The participants were re-consented at the four-year follow-up. If a participant was unable to read and write, the information sheet and consent form were read to them and their consent recorded by thumbprint.k = 0.98).Participants were asked about any repeat surgery, epilation in the last 6-month, and satisfaction with their surgical outcome. They were examined following the same procedure as outlined above. Outcome assessment was conducted by an independent examiner (BA) who was masked to the intervention allocation and who had no prior involvement in randomisation allocation, outcome assessment, and data analysis. The four-year examiner received rigorous training and was standardised with the baseline and 12-month outcome assessor (EH). They had very strong agreement for the primary outcome , or a history of repeat surgery in the 4-year period.Prior to the start of the 4-year follow-up, a single primary end point was prespecified in the approved protocol. The primary end point was postoperative TT Secondary outcome measures included: PTT difference by surgeon and baseline disease severity; under correction; eyelid contour abnormality prevalence and regression at 4-year, corneal opacity and vision changes; effect of PTT on corneal opacity and vision changes, factors influencing long term outcomes; and patient-reported outcomes.3.1Sample size determination was described in the 12-month results paper p<0.2 into a multivariable model. Likelihood ratio test was used to decide on the covariates that should be included in the final multivariable model to determine the best fitting predictive model of risk factors for PTT at 4 years.A modified intention-to-treat analysis was performed (modified meaning that participants who died during follow-up or were not seen at follow up for another reason were excluded). Otherwise all trial participants were analysed in the groups they were originally randomised and included in the analysis if they were seen at the 4-year follow-up  and at least at one follow-up time point . The effect of the intervention on primary outcome and binary secondary outcomes  was analysed using logistic regression to estimate the odds ratio (OR) and 95% CI. All comparisons between the two surgical procedures were controlled for surgeon as a fixed effect in the model, to account for the stratified randomisation. Vision and corneal opacity changes between baseline and 4-years were categorised as worse, same, better. Effect of the intervention on ordered categorical secondary outcomes  were analysed using ordinal logistic regression. Intervention effects on ECA  prevalence and regression at 4-year were analysed using multinomial logistic regression to estimate relative risk ratio (RRR) and 95% CI. A non-prespecified sign test was used to analyse if ECA regression between 6- and 12-month, and 12-month and 4-year is statistically significant in all study participants. In order to identify potential predictors of PTT at 4 years, first a univariable logistic regression was performed using PTT at 4 years as an outcome and factors with possible association with PTT (covariates) as exposures and was done separately for each intervention arm, before including all covariates that were associated with the outcome with The trial was registered on the Pan African Clinical Trials Registry (PACTR201401000743135) and overseen by independent data and safety monitoring committee.4The funder of this long-term follow-up had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.5Between Feb 13, 2014, and May 31, 2014, 5168 people were examined for eligibility, of whom 4166 were ineligible and 1002 were eligible among which two (<1%) declined surgery. Thus, 1000 trichiasis cases consented, were enrolled, and randomly assigned: 501 in the BLTR group and 499 in the PLTR group), Baseline demographic and clinical characteristics of participants seen at 4-year were balanced between the two groups, p = 0•0006). The risk difference for recurrent trichiasis between BLTR and PLTR procedures was 8•6% (95%CI 3•8–13•5).At the 4-year follow-up, PTT was observed in 169/943 (17•9%) study eyes, among which 129 (76•3%) had minor trichiasis (1 - 5 lashes), and 23 (13•6%) had previously received repeat surgery in the study eye between baseline and 4-year follow-up. PTT was more frequent in the BLTR arm (105/472 [22•2%]) than the PLTR arm (64/471 [13•6%]), after adjusting for surgeon the OR was 1•82 , with PTT risk difference of 11•6% (95%CI 6•3–16•7). The cumulative PTT was greater in the BLTR arm than the PLTR arm at all follow-up time points , By four years, cumulative PTT had developed in 238/996 (23•9%) study eyes. PTT was more frequent in the BLTR arm (148/499 [29•7%]) than the PLTR arm (90/497 [18•1%]), after adjusting for surgeon the OR was 1•91 , and corneal opacity grade changes (p = 0•92) from baseline to 4-year between the two intervention arms.The secondary outcomes, all adjusted for surgeon effect, are shown in p = 0•033). But there was no statistically significant difference in the risk of clinically significant ECA between the two groups (4•0% vs 4•9%), p<0•0001). Similarly, among the clinically significant ECA diagnosed at 12-month, 48•3% in the PLTR group, and 51•5% in the BLTR group regressed to normal or mild ECA (sign test p<0•0001). At 4-year, 51•7% and 48•5% of cases with clinically significant ECA at 12-month remained unchanged in the PLTR and BLTR arm respectively.The BLTR surgery had a lower risk of ECA than the PLTR surgery  that may predict ECA regression at 4-year were analysed by intervention group and for all participants (data not provided) and there was not strong evidence found of an association of any of these with ECA regression.6It is possible that outcomes may change a few years after TT surgery. However, the data from this long-term follow-up indicate PLTR remains superior to the BLTR with a significantly lower risk of postoperative trichiasis both cross-sectionally at and cumulatively by 4-years after trichiasis surgery and is consistent with the one-year results we have previously reported The major factors predicting long-term outcomes were also similar to those reported at 1-year which included preoperative disease severity and surgical factors such as peripheral dissection and under-correction ECA has been a major issue for surgical programmes. It is cosmetically disfiguring, posing probably a greater concern than PTT for patients and surgeons. The good news is ECA, regressed between 6- and 12-months, and 1- and 4-years after surgery in about 73% and 66% of the cases in PLTR and BLTR surgeries respectively. However, about 50% of the clinically significant ECA cases at 12-month in both procedures remained un-changed. The regression seen in clinically significant ECAs between 6 and 12-month in the PLTR arm (15•0%) was much less than we have found in one of our recent trials which used the PLTR (40•6%) The strengths and limitations of this trial with regard to its design have been discussed in detail elsewhere ,20. We mOverall, there is strong evidence that PLTR remains superior to BLTR with reduced long-term risk of postoperative trichiasis supporting the current WHO guideline that the PLTR should be the procedure of choice for training new surgeons in the programmatic management of TT. Surgical programmes need to provide attention in improving outcomes and establish a system to comprehensively manage cases with poor surgical outcomes. The majority of PTT cases had five or less metaplastic lashes indicating that most of these cases can be treated with less invasive non-surgical methods. A relatively simple surgical procedure that can be used in trachoma endemic settings is needed to address un-resolving clinically significant ECA cases.The Amhara Regional Health Bureau Ethics Committee requires that all data sharing requests are reviewed and approved by them before data can be shared. Data is available to any researcher under reasonable request. To facilitate the data access process please contact ethics@lshtm.ac.uk.This four year follow-up was funded by the Coalition for Operational Research on Neglected Tropical Diseases (COR-NTD), at the Task Force for Global Health (NTD-SC 129D). The initial trial was funded by The Wellcome Trust .Esmael Habtamu: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing - original draft, Writing - review & editing. Tariku Wondie: Data curation, Investigation, Project administration, Supervision, Validation, Writing - review & editing. Zerihun Tadesse: Data curation, Project administration, Supervision, Writing - review & editing. Bezawit Atinafu: Data curation, Investigation, Project administration, Writing - review & editing. Bizuayehu Gashaw: Data curation, Supervision, Writing - review & editing. Abebaw Gebeyehu: Writing - review & editing. E. Kelly Callahan: Funding acquisition, Supervision, Writing - review & editing. David Macleod: Formal analysis, Writing - review & editing. Matthew J. Burton: Conceptualization, Funding acquisition, Methodology, Supervision, Writing - review & editing.We declare no competing interests.


The aim of the study was to compare short and long-term mortality and readmissions in patients with non-ruptured abdominal aortic aneurysm (AAA) treated with endovascular aortic repair (EVAR) or open aneurysm repair (OAR).Retrospective survival analysis based on prospectively collected medical records of the national Polish public health insurer.st 2011 and March 22nd 2016. The data on mortality, selected concomitant diseases and readmissions were collected. A total of 7805 patients  underwent OAR (n = 2336) or EVAR (n = 5469). A median follow up was 27.5 months (IQR range 10.0–38.4 months).In the National Health Fund database we identified all patients who underwent elective open or endovascular treatment of AAA between January 1The primary outcome variable was all-cause mortality, secondary outcomes included 30-day mortality and readmissions. Kaplan–Meier (K-M), Cox proportional-hazards and propensity score analyses were performed for primary and secondary outcomes adjusting for repair type of AAA (OAR vs. EVAR), age, sex and concomitant diseases.EVAR patients had higher all-cause mortality  compared with OAR. The mortality risks for OAR patients decreased below those for EVAR patients after 9.9 months. Of all the tested confounding factors only age independently and significantly influenced long-term mortality. Readmissions occurred more often in EVAR than in OAR  independently from other covariants. Survival and readmissions Kaplan-Meier curves remained statistically different between OAR and EVAR patients after propensity score matching.Survival benefit of EVAR over OAR disappeared early during the first year after procedure, particularly in patients below 70 years of age, accompanied by an increased frequency of readmissions of EVAR patients. Our data suggest re-evaluation of the strategy for AAA management in vascular units in the country. Abdominal aortic aneurysm (AAA) is a common disease in the Western population, with a prevalence of 2% to 5% in men ≥ 65 years of age, with very high mortality rates related with AAA rupture . Recent Although RCTs are considered the "golden standard" for comparing medical procedures they may not reflect every-day practice of AAA at population level so the applicability of their results in clinical practice may be limited . PatientIt is of note that patients with AAA in RCTs must be good candidates for both procedures, so the method of treatment might be suboptimal for the specific patient. RCTs are usually carried out in high-volume centers employing vascular surgeons experienced in endovascular technique and equipped with the best hardware. Of 4799 patients assessed for eligibility into the EVAR-1 study as many as 1795 were judged unsuitable for EVAR device, 457 were unfit for open repair and finally only 1082 (22.5%) were randomized .The results obtained from national registries may be easier to generalize than the results from RCTs as they reflect every-day practice and not just the practice of centers of excellence . HoweverMoreover, management and results of treatment of AAA varies between countries ,9,10. ThThe long-term survival and outcomes of EVAR and OAR have rarely been studied on a population level, and they either included selected age groups  or were In Poland EVAR is reimbursed on a regular basis by the NHF since 2009 and the data regarding medical procedures is available in digital form .The aim of the study was to compare short and long-term mortality and readmissions in patients with non-ruptured abdominal aortic aneurysm treated with EVAR vs. OAR using population data from the entire country.We used data collected by the National Health Fund (NHF), the only public and obligatory health insurer in Poland. In case of medical procedures related to the treatment of AAA, the NHF is practically the single payer that signs contracts with public and private healthcare providers.st 2011 to March 22nd 2016. Inclusion criteria were patients with International Classification of Diseases Tenth Revision (ICD-10) diagnosis code of I71.4 , who underwent either open or endovascular treatment of AAA with International Classification of Diseases Ninth Revision procedure (ICD-9) codes of 38.424 for OAR or 39.711 for EVAR .Survival analysis was performed for primary and secondary outcomes adjusting for repair type of AAA (OAR vs. EVAR), age, sex and concomitant diseases.nd 2016, 30-day all-cause mortality at 30 days. Readmissions were censored at death or at the end of the study.All-cause mortality outcomes were censored at the end of the study on March 22https://www.r-project.org), graphs with “survminer” and propensity score analysis with "MatchIt" R packages.Variables were compared using Fisher’s exact test and Mann–Whitney U test. Estimates of cumulative event rates were calculated by means of the Kaplan–Meier method with the log-rank comparison of survival curves. Cox proportional-hazards analyses were performed for primary and secondary outcomes controlling for repair type (OAR vs. EVAR), age, sex, concomitant diseases and readmissions . Analysis were also performed in age subgroups in patients above 70 years (70+) or younger (70-). Propensity score analysis was performed by matching EVAR to OAR patients controlling for age, gender and concomitant diseases. P values of less than 0.05 were considered significant. The statistical analysis of the data was performed using R . EVAR was performed in 5469 of patients (70.1%) who were significantly older, with a higher incidence of concomitant disorders, as compared with 2336 patients treated with OAR. The demographic data are presented in Short-term  among OAR group was significantly higher as compared to EVAR group . EVAR patients had higher long-term all-cause mortality . Thirty-day and long-term mortality in all patients are presented in All-cause mortality trends over time are presented in Cox analysis revealed that the type of procedure influenced both long-term and short-term survival independently Tables  and 4. TKaplan-Meier curves for short and long-term mortality remained statistically different between OAR and EVAR patients after propensity score matching .Readmissions occurred more frequently in EVAR than OAR group  . AccordiComparison of demographic data between 70+ and 70- patients as well as between EVAR and OAR in each age subgroup separately is presented in In adjusted, Cox analysis the type of procedure significantly affected all-cause long-term mortality only in the 70- group , but 30-Our analysis of 7805 patients treated for AAA with a median follow up 27.5 months revealed survival benefit of EVAR over OAR disappeared early during the first year after the procedure.In randomized trials and observational studies both in-hospital and 30-day mortality was significantly higher in OAR than EVAR patients ,6,9, andRecent meta-analyses of randomized trials ,17 showeObservational studies may have some advantages as compared with randomized trials, since they represent current, every-day practice and better reflects the patients population allocated for different types of treatment . In our Lower long-term survival rates in randomized and earlier observational studies, as compared to our cohort, may be explained by younger age of our studied population ,8,16,18,Also, it may be speculated, that lower short-term and long-term mortality in our group may result from the differences in aneurysmal diameters and referral of many patients with small AAA  with lesInternational guidelines recommend that intervention should be considered once the aneurysm diameter exceeds 55 mm in men or 50 mm in women , but thePredicting late survival before elective AAA repair remains the Achilles’ heel of AAA management . RandomiThe recent systematic review and meta-analysis of relevant articles reporting risk factors influencing long-term survival following OAR and EVAR revealed that patients with end stage renal disease and advanced COPD had the worst long-term survival with HR 3.15 and HR 3.05 for death, respectively . An incrSimilar to other studies , repeateOur study presents the typical limitations of a retrospective analysis of reimbursement data. However, the data were collected systematically and prospectively by the single insurer all over the country. Due to the limitations of the NHF database we were unable to assess many other important parameters such as smoking, prevalence of COPD or PAD, preoperative physical status, AAA diameter and anatomy, type of endovascular device. Since a majority of deaths occurred outside hospitals and autopsies are rarely performed in Poland we were unable to establish the cause of death. There is a risk of potential errors or underreporting of certain diagnoses or procedures. Non-randomized design is another obvious limitation.Our large-population based study of patients treated for unruptured AAA revealed significantly higher late mortality after EVAR than after OAR, particularly in patients below 70 years of age. Compared to other studies we have found lower short-term and long-term mortality in our cohort, which suggests lower cardiovascular risk in treated population. However, contrary to other studies, survival benefit of EVAR disappeared early during the first year after the procedure, accompanied by an increased frequency of readmissions of EVAR patients. Taking into consideration significant variations in the management of AAA, in particular low compliance with EVAR device guidelines  our dataS1 Fig(PDF)Click here for additional data file.S1 TableAAA—abdominal aortic aneurysm.(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 TableHTN—hypertension, CRF—chronic renal failure, DM—diabetes mellitus, CAD—coronary artery disease. Data presented as means±SD or numbers (percentages).(DOC)Click here for additional data file.


Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections.Meticillin-resistant We describe MRSA colonisation/infection and bacteraemia rate trends in Dutch–German border region hospitals (NL–DE-BRH) in 2012–16.All 42 NL–DE BRH  within the cross-border network EurSafety Health-net provided surveillance data . Guidelines defining risk for MRSA colonisation/infection were reviewed. MRSA-related parameters and healthcare utilisation indicators were derived. Medians over the study period were compared between NL- and DE-BRH.Measures for MRSA cases were similar in both countries, however defining patients at risk for MRSA differed. The rate of nasopharyngeal MRSA screening swabs was 14 times higher in DE-BRH than in NL-BRH . The MRSA incidence was over seven times higher in DE-BRH than in NL-BRH . The nosocomial MRSA incidence-density was higher in DE-BRH than in NL-BRH  and decreased significantly in DE-BRH (p = 0.0184) during the study. The rate of MRSA isolates from blood per 100,000 patient days was almost six times higher in DE-BRH than in NL-BRH . The patients had longer hospital stays in DE-BRH than in NL-BRH . DE-BRH catchment area inhabitants appeared to be more frequently hospitalised than their Dutch counterparts.Ongoing IPC efforts allowed MRSA reduction in DE-BRH. Besides IPC, other local factors, including healthcare systems, could influence MRSA epidemiology. Cross-border patient mobility is a priority in the European Union (EU), because the most accessible or appropriate care for citizens living in border regions may be available abroad. When, in 2013, the directive 2011/24/EU came into force, patients’ right to access healthcare in other Member States including reimbursement and medical follow-up in their respective home countries was entitled in an EU law for the first time. With this, cross-border cooperation in infection prevention and control (IPC) using comprehensive strategies is important [Staphylococcus aureus (MRSA) is still one of the most important causes of healthcare-associated infections due to AMR pathogens [Antimicrobial resistant (AMR) pathogens are a serious threat to public health in Europe, leading to increased healthcare costs, treatment failure and deaths. For invasive bacterial infections, prompt treatment with effective antimicrobial agents is essential and is one of the most effective interventions to reduce the risk of fatal outcomes . Currentathogens .S. aureus infections was proposed as an indicator for surveillance of AMR pathogens in humans [S. aureus infections in Europe reached its lowest level (13.7%) since the ECDC first presented population-weighted data for the EU in 2009, large inter-country variations (1.2 to 50.5%) remain in Europe [In 2017 in a consensus report of the European Centre for Disease Prevention and Control (ECDC), the European Food Safety Authority (EFSA) and the European Medicines Agency (EMA), the proportion of MRSA in invasive n humans . Althougt level 1.7% sinces 1.2 to 0.5% remaThe occurrence of MRSA still necessitates continuous surveillance and preparedness to optimise IPC to further decrease MRSA rates -9. SinceThe cross-border IPC network cooperation, i.e. the Dutch−German web-based communication portal for handling MRSA problems for healthcare workers, patients and the public was continued from 2009 to 2015 within the INTERREG IVA funded project EurSafety Health-net. This enabled hospitals and nursing homes to acquire Euregional Quality and Transparency certificates. Moreover, since 2016, the collaboration was further prolonged within the INTERREG VA funded project EurHealth-1Health inter alia. Within this, the Dutch signaling meeting of the Hospital-acquired Infection and Antimicrobial Resistance Monitoring Group (SO-ZI/AMR) occurs in the German study region. Here, we analysed 2012 to 2016 MRSA surveillance data from Dutch and German border region hospitals (NL-BRH and DE-BRH) in the network in order to describe temporal and spatial trends of MRSA rates and find differences between these groups of hospitals. We also used the data to calculate the MRSA rates per inpatient and per patient days in both groups of hospitals and the MRSA rates per inhabitants in the patient catchment areas of NL-BRH and DE-BRH respectively in order to compare the two groups in relation to these parameters.http://www.eursafety.eu/) the German part of the project region geographically comprised six districts in the Münsterland region  and wascs, NUTS . The Dutcs, NUTS . InitialBoth NL-BRH and DE-BRH implemented MRSA-related IPC measures according to their national guidelines and recommendations, issued by the Dutch Working Group on Infection Prevention (WIP) and the German Commission for Hospital Hygiene and Infection Prevention (KRINKO) at the Robert Koch-Institute, respectively ,18. Of nAn MRSA case was defined as an inpatient who was colonised or infected with MRSA at admission or for nosocomial MRSA cases, after admission. A blood culture positive for MRSA, from a single inpatient and from a single hospital stay was qualified as MRSAB case. If an MRSA case, or MRSAB case, had several stays in a year, each hospital stay was counted as an MRSA case, or MRSAB case, in the surveillance. S. aureus in blood cultures (one isolate per patient case) were assessed. The MRSA-surveillance data as described above were collected in all DE-BRH using a protocol adapted from the national German Nosocomial Infections Surveillance System (MRSA-KISS [Supplement Table S1). For cross-border analysis, the laboratories serving for all NL-BRH provided retrospectively collected data for the period 2012 to 2016, according to the same protocol. On both sides of the border, the collected surveillance data of inpatients (i.e. excluding outpatients) included the number of nasopharyngeal swabs performed for MRSA screening before or at admission, the numbers of MRSA cases  − in DE-BRH and in several NL-BRH MRSA cases were additionally classified as imported or nosocomial , the number of cases and the number of patient days. Additionally, in DE-BRH and in several NL-BRH the patient days of MRSA cases  were also recorded. Moreover, the number of inpatients with a blood culture positive for MRSA  and the number of RSA-KISS ); see SuEthical approval was asked from ethical committee at the University Medical Center Groningen (UMCG) and approval was not necessary for this study.S. aureus isolates detected in blood cultures, (iv) incidence density of MRSA isolates detected from blood cultures , (v) nosocomial MRSA incidence density , (vi) length of stay in hospital  length of stay in the hospital of MRSA cases (number of patient days of MRSA cases/MRSA cases). We calculated the mean annual numbers of inpatients per 100 inhabitants and of patient days per 100 inhabitants of the patient catchment area of NL-BRH and DE-BRH. Furthermore, we calculated the mean annual number of nasopharyngeal swabs performed for MRSA screening before or at admission to hospital per 100 inhabitants in the patient catchment area of the regional hospitals (DE-BR and NL-BR) as well as of inpatient MRSA cases per 1,000 inhabitants and the MRSAB/1,000,000 inhabitants using our surveillance data of inpatients (i.e. excluding outpatients). The number of inhabitants were assessed from the official statistical database [We analysed the surveillance data of 5 years (2012–16) and calculated the following parameters: (i) screening rate , (ii) MRSA incidence (MRSA cases/100 inpatients), (iii) percentage of MRSA isolates per all database .Time trends of MRSA parameters were analysed by Friedman tests. The percentage of nosocomial MRSA cases on all MRSA cases was assessed by Cochran–Armitage test of linear trend. The cross-border regional comparisons were analysed using Wilcoxon rank sum test. All statistical analyses were done using SAS 9.4 software ; p < 0.05 was considered significant. Results of significance tests were discarded if the software displayed an alert due to more than 10% of missing values in the respective dataset. The map was made using RegioGraph10 .The total numbers of MRSA cases (detected in DE-BRH and NL-BRH are shown in S. aureus blood culture isolates decreased from 12.5% in 2012 to 5.0% in 2016 in DE-BRH (p = 0.0959), while it remained stable in NL-BRH (p = 0.1679)  , but was 0.1679) . The med 0.1679)  and was  0.1679) . The per 0.1679)  decrease 0.1679) , but did 0.1679) .We compared the available data on healthcare utilisation in DE-BRH and NL-BRH. The median length of stay (LOS) in the hospital was 6.8 days in DE-BRH compared with 4.9 days in NL-BRH (p < 0.0001) ; LOS of As patients in the EU have the right to healthcare across the borders of Member States (EU directive 2011/24/EU), it is of interest to compare the quality of care, safety standards and risks of nosocomial infection by AMR pathogens between EU countries. In this respect, the cross-border systematic and continuous MRSA surveillance is one of the cornerstones to ensure equal quality of healthcare . S. aureus detected in blood cultures was more than 34 times higher in DE-BRH than in NL-BRH . While antimicrobial consumption was not the focus of the current study, NRW has been reported as the region in Germany with the highest antimicrobial consumption in outpatients  . In thisFor MRSA IPC, the recommendations in Germany and the corresponding guidelines in the Netherlands were comparable regarding the measures performed for MRSA carriers ,18. HoweThe higher screening rates on the German side of the border may be ascribed to the fact that in German IPC recommendations, previous hospitalisation in Germany is a risk factor for MRSA carriage. This constitutes a main difference in defined risk factors between Dutch- and German MRSA IPC guidelines, whereby Dutch guidelines mostly consider screening for patients previously hospitalised outside the Netherlands  and 3 1,24. In tAnother explanation for screening rate differences between the two countries may be distinct underlying epidemiological situations regarding MRSA. For example, the MRSA prevalence is higher in the population in Germany than that in patients at hospital admission in the Netherlands (0.7% vs. 0.13%) ,26. MoreThe screening and IPC measures in the DE-BRH appeared to be nevertheless appropriate. In 2006, in the project region excluding Groningen and Drenthe , investiAbout 1% of all patients admitted in DE-BRH carried MRSA, which corresponds well to results of investigations evaluating the prevalence of MRSA carriage in the regional general, non-hospitalised population in 2012 . In termFrom 2012 to 2016 however, the nosocomial MRSA incidence density in DE-BRH decreased significantly, a trend already observed from 2009 to 2011 . MoreoveWe also observed for that the difference of the incidence of MRSA bacteraemia per inhabitants between the German and Dutch border region  was apparently smaller than calculated in a previous study, which used 2009 Dutch and 2010 German data respectively to derive the difference between NRW and the Netherlands  , which iS. aureus from blood cultures was lower in DE-BRH than in other laboratories in western Germany (DE-BRH/ARS-region west (NRW), 2012–16: 12.5%/19.0%, 14.3%/15.0%, 10.5%/13.5%, 9.8%/13.3%, 5.0%/12.0%)  , reveale%/12.0%) , as wellS. aureus detected in blood culture during 2012–16 was higher (1.5% vs 1.3%) in NL-BRH compared with Dutch national data of Infectious Disease Surveillance Information System for Antibiotic Resistance, (ISIS-AR) covering data of 52% of diagnostic laboratories [In contrast, the mean MRSA percentage of ratories .S. aureus blood culture isolates between different countries [As typical for all passive surveillance systems, bias due to differences in reporting behaviour cannot be excluded and is a limitation of this study. However, as MRSA surveillance in DE-BRH started in 2007, a stabilised compliance in reporting can be assumed for the period from 2012–16. The higher number of MRSA cases per inhabitants on the German side compared with the Netherlands is biased if there is more than one episode of MRSA detection per year for one individual patient among the number of cases. Also the inclusion of three psychiatric hospitals and one rehabilitation clinic, which have usually longer average lengths of stay, may have prolonged hospital stay in the DE-BRH. However, the data are in accordance with German-wide assessment systems. The clinical relevance of MRSA isolates detected in blood cultures is undisputable, but variations in blood culture diagnostics  may result in bias when comparing MRSA percentages of ountries . A limitEnterobacteriaceae in the future. This study on MRSA covering all hospitals across part of a European border as well as hospitals of all three care-categories demonstrated that routine MRSA surveillance may be helpful to monitor trends of MRSA parameters, to compare the MRSA rates and to indicate needs for further improvement to reach low MRSA rates EU-wide. Our results supplement the European and national surveillance systems. Ongoing efforts in MRSA prevention are recommended, including all healthcare sectors, especially with focus on One Health -42. More