To investigate neovascular age-related macular degeneration (AMD) in patients aged ≥90 years from several perspectives for a comprehensive overview: prevalence, presenting characteristics, treatment adherence, reasons for discontinuation, and efficacy of antivascular endothelial growth factor (VEGF) treatment comparing Ranibizumab and Aflibercept.  In this retrospective chart review, we determined the prevalence and presenting characteristics by reviewing all data for patients referred to our department with treatment-naïve neovascular AMD. By looking at historical cohorts, we determined adherence to treatment, reasons for discontinuation, and treatment outcomes after loading dose, 12 months, and 24 months.  Patients aged ≥90 years constituted 7% of the patients. Treatment was discontinued in 51%, primarily because of death and treatment burden. Mean change in best-corrected visual acuity was 3.2, 1.5, and −2.2 ETDRS letters at 4, 12, and 24 months, respectively. Aflibercept was superior to Ranibizumab in visual and anatomic outcomes. After two years of treatment, patients losing ≥15 ETDRS letters made up 19% in the Aflibercept group and 26% in the Ranibizumab group.  We propose that the very old patients with neovascular AMD may constitute a distinctive group warranting special attention and possibilities for individualized therapy. Possible differences between anti-VEGF agents need further investigations. Age-related macular degeneration (AMD) is the most common reason for irreversible vision loss in the developed world . Neovascage. Several studies find that the change in best-corrected visual acuity (BCVA) is negatively correlated with age [The pivotal phase 3 trials MARINA and ANCHOR studies demonstrated that monthly Ranizbiumab  improved mean visual acuity for all subtypes of neovascular AMD , 7. Its with age –22. Subgwith age . Similarwith age . ConsideAgeing is an inevitable biological process that significantly affects the cells and organs of the body. Macula undergoes a range of changes . The numThe average life expectancy is 80 years in developed countries such as Denmark, but diving into the statistics reveals that although 60+ years old individuals constitute 25%—every fourth—of the population, only <1% reaches 90+ years of age . These vIn this study, we investigated neovascular AMD in patients aged ≥90 years from several perspectives to give a comprehensive overview. First, we looked at their overall prevalence in a large tertiary retinal center and their presenting characteristics. Then, we looked at their adherence to anti-VEGF treatment and reasons for discontinuation. Finally, we evaluated results from anti-VEGF treatment and compared efficacy of Ranibizumab and Aflibercept using historical cohorts where each was the primary choice of treatment.This study is a retrospective review of patients attending the Department of Ophthalmology at Zealand University Hospital, Roskilde, Denmark. All aspects of this study were conducted in accordance with the ethical principles stated in the Declaration of Helsinki. According to the national law and local hospital research guidelines, no institutional review board approval is required for retrospective observational clinical studies that review routine clinical practice.We first determined the epidemiological aspects of patients with neovascular AMD aged ≥90 years: the prevalence and their presenting characteristics. For this part of the study, we reviewed all data for patients referred to our department in the year 2014. Eligible for analyses were patients with treatment-naïve neovascular AMD regardless of the status of their lesion  or enrollment to anti-VEGF treatment. Diseases that share features with neovascular AMD  were not included.Then, we looked at adherence to treatment, reasons for discontinuation, and treatment outcomes for a 2-year period comparing Aflibercept and Ranibizumab. For this part of the study, we included treatment results from all our patients aged ≥90 years diagnosed with treatment-naïve neovascular AMD from 2009 to 2012 (where Ranibizumab was the primary choice of treatment) and from 2014 to 2015 (where Aflibercept was the primary choice of treatment). In 2013 where Aflibercept was introduced in our clinic, we were concerned about the ongoing discussions about Aflibercept increasing the risk of cerebrovascular events  and alloIn Denmark, primary sector units  and hospital departments refer all patients suspected of neovascular AMD to ophthalmology departments since this is the only access to free-of-charge anti-VEGF treatment. Within 1-2 workdays after receiving an electronic referral, the patient is invited and booked for detailed retinal diagnosis. From the time of booking, the patient is in most cases seen within two weeks, depending on the patient's availability and the availability of the department. Patients are offered free-of-charge transportation between their home and the hospital department, including patients that may need wheelchair or special support.All patients referred for retinal diagnosis undergo a comprehensive ophthalmic examination including dilated fundus examination, measurement of best-corrected visual acuity (BCVA) in each eye using the Early Treatment of Diabetic Retinopathy Study (ETDRS) chart , retinalTreatment was initiated in eyes with neovascular AMD as defined in the Clinical Age-Related Maculopathy Staging System (grade 5)  and in tAll eyes received three consecutive monthly intravitreal injections with either Aflibercept (0.05 mL) or Ranibizumab (0.05 mL). Choice of Aflibercept or Ranibizumab was based on our local guidelines which were Ranibizumab prior to widescale Aflibercept introduction in 2013 and Aflibercept as first line of treatment starting from 2014 based on national guidelines for the treatment of neovascular AMD. Injections were given by physicians or specially trained injection nurses . After tThe prevalence of patients with neovascular AMD with an age ≥ 90 was calculated including a confidence interval for the prevalence estimate with a continuity correction . We reviχ2-test and Fisher's exact test in case any subcategory had n < 5. Continuous variables were checked for normal distribution using the Kolmogorov-Smirnov test. Where normal distribution was present, data was presented using mean and standard deviation (SD) and tested using parametric tests. Age did not fit normal distribution and was right tailed, so this parameter was presented in median and interquartile range (IQR) and tested using Mann–Whitney U test.Categorical variables are presented using numbers and percentages and compared using the Using data from eligible patients enrolled between 2009–2012 and 2014-2015, we determined which patients discontinued treatment and noted the reason. Based on different reasons for discontinuation, we explored adherence to treatment over time using a time-to-event curve. We defined the start point as date of treatment start and followed the patient until either event or censoring. Time-to-event curves were made specifically for each reason for discontinuation by censoring the other reasons.Visual and anatomical treatment outcomes were measured as change in BCVA and average central retinal thickness (CRT) after approximately 4 months (first reevaulation after treatment commencement), 12 months, and 24 months. The CRT was defined as the average thickness of an area with a diameter of 1 mm around the fovea including any subretinal hyperreflective material. Due to different reasons for treatment discontinuation, we analyzed data using the last observation carried forward (LOCF) method to account for missing data for all patients that had at least one reevaluation after treatment commencement. LOCF was not made for 24 months follow-up for patients started in treatment in 2015 since most of these patients did not have their follow-up at time of analysis (March 2017). We assumed that using the LOCF on these patients would bias the results towards better outcomes in the Aflibercept groups because 12 months outcomes in general are better than 24 months outcomes.t-test with a test value of 0. Changes in BCVA and average CRT were then compared between patients receiving Aflibercept with patients receiving Ranibizumab using a two-tailed independent samples t-test. Effect size was calculated using Cohen's d. Cohen defined the following interpretation as a rule of thumb: 0.2 small, 0.5 moderate, and 0.8 large [When a patient's both eyes were eligible for our analyses, we only included data from one eye (the first eye diagnosed with neovascular AMD or the right eye in case both eyes were diagnosed simultaneously) to avoid statistical problems with assumptions of independent sampling. In the analyses, we first looked at whether the change in BCVA and average CRT was significant using a two-tailed one sample .8 large .P values below 0.05 were interpreted as sign of statistical significance.Statistical analyses were made in SPSS version 23 . Figures were made using Prism 7 . A total of 282 patients with neovascular AMD were referred to our clinic for retinal diagnosis during the year 2014. Twenty of these patients were ≥90 years, corresponding to a prevalence of 7.1% (CI 95%: 4.5 to 10.9%). Age ranged from 90 to 99 years with median 92 years and IQR 90 to 95 years. Eleven (55%) were females and nine (45%) males.All patients presented with new neovascular AMD in one eye only, and all patients presented with a macular condition in the contralateral eye: early AMD in seven patients (35%), geographic atrophy in five patients (25%), old fibrotic AMD in five patients (25%), macular hole in one patient (5%), vitreomacular traction in one patient (5%), and subretinal drusen in one patient (5%).μm (SD: 125 μm). Mean lesion size was 3563 μm (SD: 1081 μm) measured using the greatest linear dimension. Eleven eyes (55%) had predominantly classic lesion, seven eyes (35%) had predominantly occult lesion, and two eyes (10%) had RAP. In 15 eyes (75%), lesions were hemorrhagic in their appearance. All 20 patients were deemed eligible for treatment.Mean BCVA was 48 ETDRS letters (SD: 15 ETDRS letters). Average CRT was mean 454 For this part of the study, we included treatment results from all our patients aged ≥90 years diagnosed with treatment-naïve neovascular AMD from 2009 to 2012 (where Ranibizumab was the primary choice of treatment) and from 2014 to 2015 (where Aflibercept was the primary choice of treatment) and which during the follow-up period of 2 years was not switched from one anti-VEGF to another.n = 54) or Ranibizumab (n = 62). During the 2 years follow-up, 59 patients (51%) discontinued treatment. Reasons for discontinuation are presented in We identified a total of 116 patients treated with either Aflibercept  remained in treatment for follow-up after the loading dose phase. These patients were included for a comparison of Aflibercept and Ranibizumab efficacy in terms of visual and anatomical outcomes. Patient characteristics are summarized in P = 0.036; one sample t-test) and stabilized at 12 and 24 months (mean change 1.5 (SD: 16.5) ETDRS letters, P = 0.342; mean change −2.2 (SD: 20.1) ETDRS letters, P = 0.288; one sample t-test, resp., for 12 and 24 months). The average CRT decreased significantly and remained decreased at all follow-ups (mean change −130 (SD: 143) μm, P < 0.001; mean change −117 (SD: 150) μm, P < 0.001; mean change −115 (SD: 158) μm, P < 0.001).Overall, anti-VEGF therapy improved the BCVA at 4 months (after the loading dose phase) (mean change 3.2 (SD: 15.5) ETDRS letters, The mean number of treatments during 2 years was 5.7 (SD: 3.0). For Aflibercept and Ranibizumab, the mean number of treatments was 5.6 (SD: 2.9) and 5.8 (SD: 3.1), respectively. These numbers were influenced by 51% of the patients discontinuing treatment during the follow-up. Looking only at patients that continued treatment during the 2 years, the overall mean number of treatments increased to 7.2 (SD: 2.9). For Aflibercept and Ranibizumab, these numbers increased to a mean of 6.7 (SD: 3.0) and 7.9 (SD: 2.9), respectively.P = 0.014; one sample t-test)  and remained significantly decreased at 12  and 24 months  (Aflibercept treatment significantly improved BCVA at 4 months at mean 5.5 ETDRS letters ( t-test) . Althoug t-test) . The Δ a t-test) .P = 0.028; one sample t-test)  and remained significantly decreased at 12  and 24 months  (Ranibizumab treatment was not associated with a significant improvement or worsening of BCVA at 4 months or 12 months but lead to a significant decrease of 5.8 ETDRS letters after 24 months ( t-test) . The Δ a t-test) .P = 0.149), after 12 months (4.5 (CI 95%: −1.9 to 10.8) ETDRS letters, P = 0.164), and after 24 months (9.2 (0.8 to 17.5) ETDRS letters P = 0.031)  ETDRS letters, = 0.031) . This cover time . After 2μm, 47 μm, and 43 μm, resp., for 4, 12, and 24, months)  . These cPatients aged ≥90 years constitute 7% of patients with neovascular AMD—approximately one out of every 14. These patients have a high rate of treatment discontinuation, where death and burden of treatment play a considerable role. We also found that in these patients, Aflibercept therapy was superior to Ranibizumab, although the effect sizes were only small to moderate. Difference between groups in the CRT was small and not statistically significant.Different aspects of anti-VEGF treatment may explain our results. Klettner et al. investigated the efficacy of Aflibercept, Ranibizumab, and Bevacizumab in an experimental setting using a RPE/choroid organ culture, where VEGF in the supernatant was measured throughout 7 days and using different concentration of anti-VEGFs . First, Another explanation to our findings is that Aflibercept allows eight weeks treatment intervals, which gives more flexibility from a clinical point-of-view. It is our experience that flexibility is much needed in the very old for a number of practical reasons, such as localized infections around the eye, systemic infections, fall trauma, or other conditions that may shift the priorities and the focus of the patient. For such cases, the eight weeks coverage of Aflibercept is less likely to undertreat the patients compared with the four weeks coverage of Ranibizumab. Indeed, when looking at the number of treatments, we only found that on average, ~1 additional treatment was given with Ranibizumab, underscoring our speculations of undertreatment of the Ranibizumab group.We used a PRN regime, which some studies suggest may be inferior to the treat and extend (T&E) regimen in terms of visual outcomes . In a syA considerable number of our patients discontinued treatment due to death or inacceptable burden of treatment. These aspects reflect the specific difficulties when dealing with patients aged ≥90 years . In our Although our health system is based on a free-of-charge concept and even offers free-of-charge transportation to hospitals, actually utilizing that system may not be as straightforward in this group of very old as in others. Some of these patients informed that they did not seek help after several months, which is particularly problematic since timely treatment can be important factor for neovascular AMD , 40. It Limitations should be noted when interpreting our results. This was retrospective observational study where historic cohorts were compared. Although we tried to minimize selection bias by not including participants from 2013, where Ranibizumab was used on patients with cardiovascular comorbidities; a better study design would include a randomized allocation of patients to Aflibercept and Ranibizumab. We used LOCF handle missing data due to treatment discontinuation, and in that regard, it should be noted that a considerable number of our patients discontinued treatment for different reasons. However, we do not suspect that this results in skewed data between Aflibercept and Ranibizumab since the groups did not differ in treatment discontinuation. Finally, it should be noted that the mean difference between Aflibercept and Ranibizumab was at ~2 ETDRS lines at most, and as a rule of thumb, a clinically significant difference is 3 ETDRS lines .Patients aged ≥90 years constitute a small but important proportion of those referred for treatment of neovascular AMD. These very old patients have a high rate of treatment discontinuation, where death and burden of treatment play a considerable role. Although both Aflibercept and Ranibizumab decreased the average CRT, Aflibercept seemed superior to Ranibizumab in terms of change in BCVA after the loading dose and after 12 and 24 months in patients aged ≥90 years. Important reasons may be pharmacokinetic differences between the two drugs or the relative more flexibility of treatment every 8 weeks versus 4 weeks with Aflibercept versus Ranibizumab; however, studies with prospective and randomized design are needed for more conclusive results. We propose that the very old patients constitute a distinctive group that may warrant special attention.


We also found that agg is significantly enriched in isolates with medium and strong biofilm formation ability (P = 0.0026). Additionally, vancomycin, applied at sub minimal inhibitory concentrations, inhibited biofilm in four out of five strong biofilm-forming isolates. In conclusion, we suggest using agg and fsrB genes, together with the previously established gelE, for better prediction of biofilm strength and gelatinase activity, respectively. Future studies should explore the mechanism of biofilm inhibition by vancomycin and its possible use for antivirulence therapy.Enterococci are nosocomial pathogens that can form biofilms, which contribute to their virulence and antibiotic resistance. Although many genes involved in biofilm formation have been defined, their distribution among enterococci has not been comprehensively studied on a genome scale, and their diagnostic ability to predict biofilm phenotypes is not fully established. Here, we assessed the biofilm-forming ability of 90 enterococcal clinical isolates. Major patterns of virulence gene distribution in enterococcal genomes were identified, and the differentiating virulence genes were screened by polymerase chain reaction (PCR) in 31 of the clinical isolates. We found that detection of  Enterococci are known for their ability to form biofilms, which are populations of cells irreversibly attached to various biotic and abiotic surfaces and encased in a hydrated matrix of exopolymeric substances, proteins, polysaccharides and nucleic acids2. Biofilms contribute to bacterial virulence in several ways. For example, adherence, an early step in biofilm formation, allows the bacteria to bind to catheters , biliary stents and silicone gastrostomy devices3. Additionally, biofilms contribute to bacterial resistance to antibiotics4 and to phagocytosis, making their eradication extremely difficult5. In a mature biofilm, the bacterial cells can tolerate antibiotics at concentrations 10–1000 times higher than those required to kill planktonic cells5.Enterococci are Gram-positive bacteria that cause serious nosocomial infections, including urinary tract, bloodstream infections and endocarditis6. These include the aggregation substance (Agg), Enterococcus faecalis endocarditis-associated antigen A (EfaA), adhesion of collagen of E. faecalis (Ace) and biofilm on plastic operon (Bop)7. Moreover, expression of pili on the cell surface, which facilitates cell adhesion, is considered the trigger of biofilm formation8, and pili components are encoded by the endocarditis- and biofilm-associated pili genetic locus (EbpABC) and an adjacent downstream sortase-encoding gene, srt9.Several enterococcal virulence proteins have been studied for their important roles in biofilm development10. In E. faecalis, biofilm formation is regulated by the well-defined quorum sensing system, fsr  locus11. This locus consists of three genes, fsrA, fsrB and fsrC, immediately located next to two virulence factor-encoding genes: one encoding a gelatinase (gelE) and the other a serine protease (sprE). The expression of gelE is regulated by the fsr locus12.In general, biofilm formation is associated with quorum sensing, which is the regulation of bacterial gene expression in response to large cell population densities. This regulation is usually driven by molecules known as autoinducers13. Some of these plasmids carry genes that regulate or promote biofilm formation, such as the plasmid-encoded aggregation substance genes14. A direct link between pheromones and biofilm formation was demonstrated in Candida albicans15. Examples of secreted enterococcal pheromones are Cpd, Cob, and Ccf16.Enterococcal cells do not only communicate through Fsr quorum signaling, but are also capable of communicating by peptide pheromones, secreted by recipient cells to induce the conjugative apparatus of donor cell, which mediate the transfer of pheromone-responsive plasmids17. One such method is the use of sub-inhibitory concentrations of antibiotics, which was shown to modify the physicochemical properties and the architecture of the outer surface of enterococcal cells, thus affecting their overall virulence1. Sub-inhibitory antibiotic concentrations have also been used to inhibit bacterial initial adhesion to abiotic substrates18.Because of the role of biofilms in virulence and antimicrobial resistance, different methods have been developed for biofilm prevention and removalgelE19); yet, no study has systematically investigated whether the complete or partial presence of this gene set is a predictor of the biofilm formation phenotype. Such correlation would be of great diagnostic value, but is hindered by poor metadata available about published enterococcal genomes, which impede attempts for comparative genomics between biofilm and non-biofilm formers.Additionally, despite a growing body of literature about the aforementioned genetic determinants involved in biofilm formation and regulation in enterococci, no clear genotype–phenotype correlation has been established for these determinants. Associations between some of these genes and biofilm formation have been recognized , with a good number of E. faecium (22 isolates or 24.4%). Two of the remaining isolates belonged to the species E. casseliflavus and only one was identified as E. gallinarum , which showed a wide variety of patterns, most of which representing distinct enterococcal clades  eighty-one isolates that varied in their biofilm formation strength between moderate and weak; and (iii) four non-biofilm-producing isolates. This assay could not persuasively differentiate between moderate and weak biofilm formation ability. On the other hand, the Crystal Violet biofilm assay differentiated isolates into strong, moderate, weak, and non-biofilm-forming according to the O.D. values at 545 nm. Again, only five of the isolates (5.5%) were classified as strong biofilm-formers; 38 isolates (42%) were moderate; 43 were weak biofilm-formers (48%); and four (4.5%) could not form any detectable biofilm.Congo-Red agar biofilm assay differentiated the 90 enterococcal isolates into (i) 11. Here, all isolates were screened for their in vitro gelatinase activity, but only 27% were gelatinase positive.Gelatinase activity has been described as one of the first steps in the process of biofilm formationE. faecalis and E. faecium), strain-to-strain variations remained important to determine by ERIC.Of note, neither biofilm formation strength nor gelatinase activity correlated with ERIC-PCR types, except that three strong biofilm-forming strains fell in the same clade , but there was obvious association  with the sample source—urine and stool samples being enriched with biofilm-forming strains with higher average strength .E. faecalis genomes . The genomes represented different biofilm phenotypes ranging from strong biofilm formers (V583) to the non-biofilm-forming probiotic strain Symbioflor1 .First, we screened for the presence/absence pattern of these 17 protein-coding genes in five fully sequenced or1 Fig. . Apart fene Fig. . A more fsr/gelE operon, and that strain 62 lacked fsrA and fsrB but had fsrC.We confirmed the aforementioned results by in silico PCR primer screening, using BLASTN searches for the primer pairs of the 17 genes mentioned above Table  in the fE. faecalis in an unbiased way, we conducted genome-wide comparative analysis between biofilm-forming strains and the non-biofilm-forming Symbioflor 1. Genome-scale SEED comparative analysis confirmed BLAST results and, in addition, identified several prophage and pathogenicity island genes that differentiated the different strains   Table . Bidirec 62 Fig. .agg and ace sequences are too variable to serve as good PCR biomarkers; on the other hand, gelE and the fsr locus genes could be more promising, notably fsrA and fsrB. Yet, the actual correlation of these candidate genes with biofilm phenotypes remained to be tested on clinical isolates.Taken together, the above results indicate that srt, ccf, bop, efaA, and cpd genes were present in 94% of the isolates. gelE, sprE, and fsrC genes in 90% of the isolates, ebpC and pil in 84%, agg in 81%, ebpA in 77%, fsrB in 65%, ebpB in 55%, cob in 50%, and ace and fsrA in 45% of the isolates.Out of the 90 enterococcal isolates, 31 were screened by PCR for the 17 genes listed above Table , and thegelE/fsrA locus genes in diagnosis and gelatinase activity prediction. Additionally, patterns of agg, ace combined with gelE/fsrA could distinguish weak biofilm phenotypes  and tigecycline  in comparison to untreated cells. The adherence of another three isolates was only significantly decreased by vancomycin . The adherence of one isolate was neither affected by vancomycin nor tigecycline  instead of screening a full panel of virulence genes. To this end, a major step was to first establish a good genotype–phenotype correlation relying on genomic analysis to define good biomarker genes for biofilm formation and gelatinase activity, as two separate—even if interdependent—phenotypes. Once the genes were defined, they were validated experimentally.The main focus of this study was to determine the biofilm-forming ability of clinical enterococcal isolates, its correlation with gelatinase activity and et al. reported that 68% of isolates in Tamilnadu, India were biofilm producers21 and 64.40% of bacterial isolates from urinary tract infection in a hospital in Dhaka, Bangladesh were biofilm producers22.Our work demonstrates a higher than usual frequency (~96%) of biofilm production by enterococcal clinical isolates collected from Egyptian hospitals, regardless of the strength of the biofilm formed. This frequency is much higher than other reports from developing countries. For instance, Sindhanai 23. Sindhanai and coworkers21 reported that 34% of their isolates formed strong biofilms; 49% formed moderate biofilms; and 17% formed weak biofilms. As determined in this study, differentiation between weak and moderate biofilm formation may be associated with the aggregation substance (Agg), as the gene encoding it is enriched in moderate and strong isolates . There are mixed reports about the effect of glucose on biofilm formation: Kristich et al. reported an inhibitory effect of increasing glucose concentrations25, yet with a reasonable biofilm formation at 0.5% glucose, whereas Baldassarri et al.26 and Pillai et al.27 reported potentiating effect at concentrations <1%. Using 0.5% seemed reasonable. Likewise, we consistently performed the assays after 48 hours incubation based on prior reports29; this consistency was important to guarantee that the incubation time would not be a confounding factor.Using already-established Crystal Violet assays, we differentiated the clinical isolates into strong (5.5%), moderate 42%), weak (48%), and non-biofilm-forming (4.5%) strains. Congo-Red agar assay could not differentiate moderate from weak biofilm forming strains, so the results of the Crystal Violet assay were considered. The superiority of Crystal Violet assays has been previously described2%, weak E. faecalis, first starting by screening five well-defined genomes for a set of 17 biofilm-associated genes, and then by extending the screen to ~190 genomes for agg, ace and fsr locus genes.To better understand the genotype–phenotype correlations for a complex phenotype such as biofilm formation in enterococci, we performed extensive genomic analysis of gelE, ccf, sprE, bop, efaA, cpd, and fsrC (Table gelE (previously determined as a biomarker for biofilm formation), agg was found to be a predictor of weak vs. strong/medium biofilm formation  the gelatinase phenotype. Because the genomic survey we performed on ~190 genomes .Thus, we conclude that the absence of gelatinase activity in fsr in Enterococcus, which was shown to have a pronounced effect on biofilms11. In the current study the strong biofilm-forming strains contained the entire quorum sensing locus, and loss of any of the quorum sensing genes was correlated with decrease in biofilm strength. Mohamed et al.4 reported that loss of fsr and/or gelatinase function resulted in diminished biofilm formation, in comparison with biofilm production by progenitor, fsr-positive, gelatinase-positive E. faecalis isolate. Carniol and Gilmore discussed the role of signal transduction, quorum sensing and extracellular protease activity in biofilm formation by E. faecalis36. Another research team found that biofilm formation was reduced in all three fsr mutants  by ~28 to 32% compared to wild-type E. faecalis OG1RF4.In several pathogens, biofilm production is regulated by quorum sensing systems, including Enterococcus isolates to azithromycin, ciprofloxacin, vancomycin, gentamycin and tigecycline was estimated through determination of each antibiotic’s MIC by the broth microdilution method. Although studies on the antimicrobial susceptibility patterns of enterococci have affirmed the worldwide emergence of multiple-drug resistant enterococci, particularly to vancomycin37, 96.7% of our isolates were actually sensitive to vancomycin. This is not unprecedented, e.g., Karmarkar et al. reported that 76% of the isolates they screened were sensitive to vancomycin38. Additionally, all the isolates were sensitive to tigecycline, while 60.8% and 46.7% were sensitive to ciprofloxacin and gentamycin, respectively, and only 22.8% were sensitive to azithromycin. Strong biofilm-forming strains were only sensitive to vancomycin and tigecycline.Another focus of this study was the mutual effects between biofilm formation and antibiotics/antibiotic resistance. Here, the antimicrobial susceptibility of 39. To the best of our knowledge, there are no published reports of biofilm inhibition by vancomycin in enterococci, but there is a precedent case reported for a clinical staphylococcal isolates16. Further studies should explore the mechanism of biofilm inhibition by vancomycin and whether it is through direct interaction with the gelatinase enzyme or with any of its regulators. Other possibilities for such inhibition could be related to alteration of gene regulator circuits in response to vancomycin-induced stress or to selection of a bacterial subpopulation that has higher tolerance for low doses of vancomycin.By evaluating the effect of sub-MIC of vancomycin and tigecycline on adherence of strong biofilm-forming isolates, we found that vancomycin decreased the adherence significantly in four isolates while tigecycline only decreased the adherence in one isolate. Tigecycline has previously been reported to inhibit biofilm formation in enterococci. Maestre and colleagues demonstrated that tigecycline decreased the adherence of 55% of the isolates that had strong biofilm-formation abilityin vitro PCR screens and phenotypic assays, are gelE, agg, and fsrB. Finally, we show that vancomycin, and to a lesser extent tigecycline, could inhibit biofilm formation when used at concentrations below their measured MICs. This work highlights the importance of integrating phenotypic and genotypic assays of biofilm-forming determinants in clinical enterococcal isolates. In addition, it calls for further understanding of the mechanism by which some antibiotics inhibit biofilm formation, thus affecting enterococcal virulence and possibly resistance to other antibiotics.We conclude that the biofilm-forming ability of enterococcal isolates sampled in this study, and representing different Egyptian hospitals, does not particularly correlate with their overall genetic make-up, which was highly divergent (as reflected by ERIC-PCR patterns). Several genetic determinants, including the quorum sensing locus, which encodes proteins that regulate biofilm formation, were detected in these biofilm-forming isolates, but none of them was detected in non-biofilm-formers. The genes with highest diagnostic values, as determined by in silico genome analysis and validated by Enterococcus colonies were isolated by surface streaking of clinical specimens on Enterococcosel agar  and identified by Gram stain followed by catalase test. The identity was further confirmed by cultivation on Chromogenic UTI agar , currently known as Brilliance UTI agar. This differential culture medium provides presumptive identification of several urinary tract pathogens. Unlike other species, Enterococcus species express beta-glucosidase but not beta-galactosidase or tryptophan deaminase. The beta-glucosidase activity targets the chromogen, x-glucoside, and produces blue colonies. Confirmed Enterococcus strains were streaked on Brain Heart agar slants and kept at 4 °C for the period of experimentation. Duplicate glycerol stocks of each isolate were stored at −80 °C.Isolates were collected from Egyptian hospitals in the period from 2009 to 2015. A number of isolated colonies (5–10) from a fresh overnight pure culture were resuspended in 3.0 mL of sterile saline solution. The turbidity was adjusted to 0.5 McFarland standard. GP (Gram Positive) identification cards were inoculated with the suspended microorganism by an integrated vacuum apparatus, and test tubes containing the bacterial suspension were placed into a special rack while the identification cards were placed in the neighboring slot. Cards were sealed and inserted into the VITEK 2 reader-incubator, and then subjected to fluorescence measurement every 15 min. Results were obtained automatically and interpreted by the ID-GPC database.Enterococcus strains was extracted as described by Soumet et al.40. Specific primer pairs were designed and used for amplification of the different genes  and 0.25 µg extracted enterococcal genomic DNA (or non-template control of nuclease-free water). PCR products were analyzed in 1% agarose gels by electrophoresis, stained with ethidium bromide and visualized under UV light  has been developed for typing enterococci41. This method was used as previously described, with the primers ERIC-1: ATGTAAGCTCCTGGGGATTCAC and ERIC-2: AAGTAAGTGACTGGG GTGAGCG. PCR conditions were as follows: For ERIC-1, an initial denaturation at 94 °C for 5 min was followed by five cycles of 94 °C for 5 min., 35 °C for 5 min. and 72 °C for 5 min; then 30 cycles of 94 °C for 1 min., 35 °C for 1 min., 72 °C for 2 min and a final extension of 72 °C for 10 min. For ERIC-2, the initial denaturation step was followed by 35 cycles of 94 °C for 1 min., 25 °C for 1 min. then, 72 °C for 4 min, and a final extension of 72 °C for 10 min.A modified http://genomes.urv.cat/UPGMA/) was used for ERIC-PCR gel electrophoretic pattern analysis and dendrogram generation. Trees were rendered, colored, and annotated by FigTree v.1.2.3 (obtained from http://tree.bio.ed.ac.uk/software/figtree).Unweighted pair group method using arithmetic overage algorithm (UPGMA), available at  containing 0.25% glucose. The culture density was spectrophotometrically adjusted to approximately 0.5 McFarland standard (at absorbance = 600 nm). Each broth culture was diluted 1:100 in fresh broth, and 200 µl of each diluted culture was distributed in three wells of a 96-well microtiter plate and incubated for 48 hours at 37 °C. Negative control wells were included. Planktonic cells were removed, and wells were (i) washed with sterile phosphate-buffered saline for removal of non-adherent cells, (ii) decanted, and then (iii) left to dry. Adherent cells were stained with 200 µl of 2% (W/V) crystal violet for 15 minutes. Excess stain was gently rinsed off with water. Plates were allowed to air-dry. The dye bound to the adherent cells was re-solubilized with 200 µl of 33% (v/v) glacial acetic acid. The optical density (OD) was measured at 545 nm in a plate reader . Readings from triplicate wells were averaged , strains were classified as weakly adherent; (2x O.D.c) < O.D. ≤ (4x O.D.c) = moderately adherent; and (4x O.D.c) < O.D. = strongly adherent.Strains were classified into categories according to the O.D. measurement of their biofilms as follows: Those with O.D. ≤ O.D.c (O.D. of the negative control) were considered 44  gelatin and incubated for 48 hours. After incubation, plates were flooded with Frazier solution. A positive gelatinase activity was exhibited as transparent halo zones surrounding colonies44 Fig. .45.The MICs of vancomycin, tigecycline, ciprofloxacin, gentamycin and azithromycin were determined by broth microdilution performed in 96-well plates containing Mueller Hinton broth, in accordance with CLSI guidelinesE. faecalis isolates were sensitive were selected for studying the effect of their sub-MIC on biofilm production and gelatinase activity. Bacterial suspensions of the strong biofilm-forming isolates were prepared as described under the Crystal Violet assay: Wells (200 µl) were inoculated with bacterial suspensions, TSB containing 0.25% glucose and the corresponding antibiotic sub-MIC concentrations or TSB without antibiotic (antibiotic-free controls). Plates were incubated for 48 hours at 37 °C. The O.D. of isolates with and without antibiotics at sub-MIC was measured at 545 nm using the plate reader as detailed above. Results were taken as the average of triplicate reads.MICs of different antibiotics were determined by the broth microdilution method, and the antibiotics to which strong biofilm-forming The evaluation of sub-MIC antibiotic concentrations on gelatinase activity was performed in a similar way to their effect on biofilm formation. Bacterial suspensions were mixed with TSB containing the corresponding antibiotic (at sub-MICs) or supplemented TSB without antibiotic (antibiotic-free controls). A loopful of this mixture was grown on agar plates containing 3% (w/v) gelatin and incubated for 48 hr, then halos were recorded as detailed above.E. faecalis V583 (Accession # AE016830.1), E. faecalis OG1RF (Accession # CP002621.1) and E. faecalis D32 (Accession # CP003726.1). The other two strains were E. faecalis Str. Symbioflor1 (Accession # HF558530.1), a probiotic strain with no biofilm-forming capacity, and E. faecalis 62 (Accession # CP002491.1), a commensal strain isolated from a healthy child46.Five representative strains whose genomes have been previously sequenced were used in this analysis. Three of the five strains have known biofilm formation ability: 47 (http://blast.ncbi.nlm.nih.gov/) and the SEED subsystems analysis and database comparative genomics tool20 (http://pubseed.theseed.org/?page = MultiGenomeComparehttp://pubseed.theseed.org/comp_genomes.cgi) with reference organism set to V583 as follows: organism = 226185.1 and comparison organisms set to: 936153.3 (62), 1206105.3 (D32), 474186.5 (OG1RF) and 1261557.3 (Symbioflor 1).Amino acid and DNA sequences of the selected proteins and their coding genes, respectively, were obtained from NCBI based on the standard strain V583. Analysis tools for genome screens and comparative genomics were BLASTX, BLASTNAll statistical analyses, including descriptive statistics and hypothesis tests  and GraphPad Prism .Supplementary material


To examine the association between elder abuse and poor sleep using a Malay validated version of Pittsburgh Sleep Quality Index (PSQI).This study was divided into two phases. Phase I tested the construct validity and reliability of the Malay version of PSQI. Phase II was a population-based, cross-sectional study with a multi-stage cluster sampling method. Home-based interviews were conducted by trained personnel using a structured questionnaire, to determine exposure and outcome.Kuala Pilah, a district in Negeri Sembilan which is one of the fourteen states in Malaysia.1648 community-dwelling older Malaysians.The Malay version of PSQI had significant test re-test reliability with intra-class correlation coefficients of 0.62. Confirmatory factor analyses revealed that one factor PSQI scale with three components  was most suitable. Cronbach’s Alpha was 0.60 and composite reliability was 0.63. PSQI scores were highest among neglect (4.11), followed by physical (4.10), psychological (3.96) and financial abuse (3.60). There was a dose-response relationship between clustering of abuse and PSQI scores; 3.41, 3.50 and 3.84 for “no abuse”, “1 type of abuse” and “2 types or more”. Generalized linear models revealed six variables as significant determinants of sleep quality–abuse, co-morbidities, self-rated health, income, social support and gait speed. Among abuse subtypes, only neglect was significantly associated with poor sleep.The Malay PSQI was valid and reliable. Abuse was significantly associated with poor sleep. As sleep is essential for health and is a good predictor for mortality among older adults, management of abuse victims should entail sleep assessment. Interventions or treatment modalities which focus on improving sleep quality among abuse victims should be designed. Elder abuse and neglect (EAN) is increasingly becoming a global health concern. The World Health Organization (WHO) has defined EAN as ‘a single or repeated act, or lack of appropriate action, occurring within any relationship where there is an expectation of trust which causes harm or distress to an older person’. There aNumerous adverse effects of abuse in late life–across different health domains—have been documented. These include premature mortality , 7, deprQuality sleep is one of the key determinants of good health. Its importance is more prominent among older adults, as sleep disorders and sleeping difficulties become more pervasive with aging . Poor slSleep can be measured either objectively or subjectively. Objective measurement of sleep often employs polysomnography (PSG) and the Multiple Sleep Latency Test (MSLT), but these methods have been considered impractical for clinical screening and research . The morThe PSQI is a nineteen-item questionnaire measuring seven components of sleep–subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction–yielding one global score . Most stWe thus aim to: 1) validate the Malay version of PSQI among older Malaysians through confirmatory factor analyses (CFA) and reliability tests, and; 2) examine the association between EAN and sleep quality.This study consisted of two phases. In Phase 1, we validated the Malay version of PSQI and in Phase 2, we examined the relationship between EAN and sleep quality. Assessment was carried out in a span of six months, from November 2013 to May 2014.Phase 1: Validation of Malay PSQI.Eight low-cost, subsidized government flats were randomly chosen from a list of all low-cost flats in the Klang Valley. A total of 239 residents aged 60 or more were then randomly selected and invited to join the study. The Malay version of PSQI was administered face-to-face by trained personnel two times, in a two-week interval.The PSQI is a self-rated questionnaire comprising nineteen questions with an additional five questions rated by another person such as bed-partners or roommates. The five questions were not administered since they are usually used for clinical purposes and not included in the scoring system. According to the guidelines of Buysse et al.  the ninePhase 2: EAN and sleep quality.This was a cross-sectional study, involving community-dwelling older Malaysians aged 60 or more. It was part of the Malaysian Elder Mistreatment Project (MAESTRO) initiated in early 2013 .The district of Kuala Pilah in Negeri Sembilan, a state in Peninsular Malaysia was chosen. Largely rural, Kuala Pilah is located roughly 100 km away from the capital city, Kuala Lumpur.A multi-stage cluster sampling strategy was employed. Kuala Pilah was first randomly selected from Negeri Sembilan’s seven districts. Out 254 enumeration blocks (EB) in the district, 156 EBs were randomly chosen, followed by another random selection of sixteen to twenty households from every EB based on a computer-generated list. A house-to-house visit was then performed to identify selected participants.Our study respondents were restricted to those aged 60 and older, Malaysian nationals, had taken up residence in Kuala Pilah for at least 12 months, and those who were able to communicate independently. We excluded residents of long-term care institutions, those unable to communicate for reasons such as severe cognitive impairment, deafness or severe hearing impairment and post-stroke complications, and those who scored 4 or less in the Mini Mental State Examination (MMSE).Respondents were interviewed at home by trained personnel using a structured questionnaire. A number of 1648 older respondents successfully completed the interviews. Further details on the study methodology have been previously published .Elder abuse and neglect (EAN) was captured using a questionnaire derived from the modified Conflict Tactic Scales (CTS) and revised by Naughton and colleagues . Each reSleep quality in this study was assessed using the Malay validated Pittsburgh Sleep Quality Index (PSQI) as described above. Higher scores indicate poorer sleep, as suggested by Buysse et al. . Scores Other variables measured were: 1) socio-demographics ; 2) health-related variables ; 3) psychosocial variables  and; 4) physical function (2.4-meter gait speed).Monthly household income was categorized into “low” (less than RM1000), “medium” (RM1000-RM2499) and “high” (RM2500 and above). Education level was grouped into “low” , “medium” (primary to secondary) and “high” (college and above). Depression was determined using the Geriatric Depression Scale (GDS-15) , while sData was analyzed with the IBM Statistical Package for Social Science (SPSS) software version 20.0 for Windows . Validatp-value of less than 0.05 was considered statistically significant and 95% CIs were reported.To study the relationships between independent and dependent variables, Generalized Linear Models (GLiM) were employed; using Maximum Likelihood Estimation (MLE) as the method of parameter estimation, distribution of independent variable (PSQI) set as Gamma , and ‘Power’ as the link function. Multi-collinearity diagnostics was run prior to analyses, and interactions between EAN and other predictor variables  were examined. Ethnicity was collapsed into two groups; “Malay” and “Non-Malay”, given the very small sample size of each of the non-Malay ethnic groups . A This study was approved by the Medical Ethics Committee of the University of Malaya Medical Centre (MEC Ref 902.2) and the Medical Research and Ethics Committee, Ministry of Health Malaysia (NMRR-12-1444-11726). Thorough explanation was given to each respondent and written consent was taken prior to interview.Phase 1: Validation of Malay PSQI.Out of 239 older adults invited, 194 agreed to participate in the validation study. Response rate was 81.2%. Those found to have severe depression (GDS scores 12 or more) and cognitive impairment (MMSE scores 17 or less) were excluded. The final sample size was 183. Descriptive statistics of all PSQI components are shown in Appendix 1 . CFA wasThe overall model fit indices for the original models  were acceptable but the item loadings were poor. Components 3,4,6 and 7 were then dropped one by one and finally PSQI with three components  was found appropriate, demonstrating good convergent validity. The final scale had acceptable Cronbach’s alpha and composite reliability, and significant ICC of 0.61 . The finPhase II: EAN and sleep quality.A total of 2118 individuals agreed to participate. Response rate was 84.9%. There was no difference between respondents and non-respondents with regards to socio-demographic characteristics. Screening of cognitive impairment using MMSE excluded 191 individuals, and 279 did not show up on the day of PSQI interview (unit non-response), yielding a final sample size of 1648. Those who did not undergo PSQI interview were older, while other characteristics did not differ. The sample comprised of 97.4% Malay, 60.2% women, and slightly more than half belonged to the younger age group (60–69). Missing data was found to be MAR (missing at random), and this was addressed by multiple imputation method.The prevalence of EAN overall was 8.1% (n = 133). Financial abuse was the most common subtype, comprising 4.9% while psychological, physical, neglect and sexual abuse were 3.3%, 1.2%, 1.2% and 0.2% respectively. Unadjusted analyses showed that abuse had significant relationships with low income, depression and poor sleep quality. Mean PSQI scores for EAN victims were 3.80 (SD = 1.55), whereas for those not abused, mean scores were 3.41 (SD = 1.38). Mean PSQI scores were highest in neglect , followed by physical , psychological , and financial abuse . Sexual abuse had to be excluded from analysis due to the extremely small number of cases (n = 5). A dose-response relationship was evident between clustering of abuse and PSQI scores; 3.41 (SD = 1.3), 3.50 (SD = 1.4) and 3.84 (SD = 1.3) for “no abuse”, “1 type of abuse” and “2 types or more”, respectively. Analyses using Generalized Linear Models were then performed to determine the relationship between independent variables and PSQI scores. There was no multi-collinearity between the any of the predictor variables. Six were found to be significantly associated with poor sleep (higher PSQI scores); 1) EAN–abuse victims were more likely to have poorer sleep than those not abused; 2) co-morbidities–those with multiple chronic diseases were more likely to have poor sleep than those with no or few diseases; 3) self-rated health; those who rated their health as “good” were more likely to have better sleep than those who rated their health as “poor”; 4) income; those with lower income had poorer sleep; 5) social support; those with lower social support were more likely to have higher PSQI scores, and; 6) gait speed; those with lower gait speed had poorer sleep. No interaction was found between abuse and other variables in affecting sleep quality. When similar analyses were run with overall EAN being replaced by the four subtypes–physical, psychological, financial and neglect–only neglect was associated significantly with decline in sleep quality.Our study showed that, overall, respondents with abuse experiences had significantly poorer sleep than those who were not abused. Unadjusted analyses showed associations between EAN and low income and depression . This coThe graded relationship between clustering of abuse and PSQI scores strengthened the association between EAN and poor sleep–as abuse experience is multiplied, sleep quality declines further as demonstrated by the steady increase in PSQI scores. This can be explained by the link between stress–with EAN being a form of stressor–and sleep problems as reported by a number of studies: 1) high levels of emotional arousal as a result of adverse events were said to cause sleep difficulties ; 2) expoAmong the subtypes of abuse, neglect had the strongest association with poor sleep. Similarly, mean PSQI scores were highest in neglect. The operational definition of neglect in this study included: 1) not being provided with support for basic activities of daily living , and; 2) being deprived of safe and clean living conditions, medical attention or treatment, clean clothing, and adequate, nutritional food . The dynOther variables found to be significantly associated with sleep quality were self-rated health, co-morbidities, gait speed, income and social support. These findings have been substantiated by previous studies; older adults who rated their health positively, or had no or less co-morbidities reported better sleep in comparison with those with poor self-rated health and more chronic diseases , 54. LowOn the other hand, depression was not associated with poor sleep in our study, in contrast with prior findings which suggested a relationship between the two , 61. HowGiven that our findings supported an association between EAN and poor sleep, current approach to managing abuse victims needs to be tailored accordingly. Healthcare providers should always be alerted to the possibility of sleeping problems when dealing with EAN victims, especially when there are elements of neglect. On top of that, detection of co-existing multiple chronic diseases, poor self-rated health and low social support–all of which can be easily identified–may be a simple yet effective technique to screen for sleeping problems among abuse victims, prior to confirmation with more sophisticated methods. Improvement of sleep quality therefore should be one of the aims while managing EAN victims. This can be done either through designation of specific treatment modalities by clinicians or a more general form of intervention by public health experts and policy-makers.This study has several limitations. First, its cross-sectional design disallows causality. The possibility of the relationship between EAN and poor sleep in the opposite direction cannot be ruled out. Second, PSQI is a subjective measurement of sleep. Its accuracy may differ from objective methods such as polysomnography. It was reported that differences in results were found between the two methods, and that the correlation between self-reported and objectively-measured sleep could be biased , 64. ThiS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 File(SAV)Click here for additional data file.


ZRANB1, FAM110A, PREP, ANKRD9 and LAMB2 were also differentially expressed in an existing database of hypertensive mouse model (GSE19817). A total of 16 pathways were identified by the GSEA. ZRANB1 and six pathways identified are related to TNF-α. Three pathways are related to interleukin, one to metabolic syndrome, and one to Hedgehog signaling. Identification of these genes and pathways suggest the importance of 1. inflammation, 2. visceral fat metabolism, and 3. adipocytes and osteocytes homeostasis in hypertension mechanisms and complications.Hypertension is an important public health problem in the world. Since the intermediate position of the gene expression between genotype and phenotype makes it suitable to link genotype to phenotype, we carried out a two-stage whole-genome gene expression association study to find differentially expressed genes and pathways for hypertension. In the first stage, 126 cases and 149 controls were used to find out the differentially expressed genes. In the second stage, an independent set of samples (127 cases and 150 controls) was used to validate the results. Additionally, we conducted a gene set enrichment analysis (GSEA) to search for differentially affected pathways. A total of nine genes were implicated in the first stage . Among these genes,  Findings on hypertension genes can be used not only for screening high-risk individuals, preventing disease development, but also for elucidating disease mechanisms. Most of the previous hypertension genetic study focused on hypertension with relatively older ages. We focused on young-onset hypertension which has a stronger genetic component than hypertension in general5. In this study, we followed our previous studies which used age 50 as the cut-off to screen young-onset hypertension5.Hypertension is an important public health problem in the world. Essential hypertension is one of the most predictive or associated risk factors of cerebral hemorrhage and infarction, coronary heart disease, heart failure, diabetes mellitus, and kidney disease. The substantial heritability (30–60%)6. Recent advances in molecular biology and technology have made it possible to monitor the expression levels of all genes simultaneously. Several gene expression association studies on human hypertension have been conducted10.Gene expression level may represent the phenotype most immediately connected to DNA sequence variation. The intermediate position of the gene expression makes it suitable to bridge between genotype and phenotype11, but these tissues are not accessible in human. Human lymphoblastoid cell lines (LCLs) are gaining popularity as a research tool for studying genome-wide individual differences13. LCL presents some advantages not only in its tractability and availability, but also in its potential of negating environmental influence. Lymphoblast cells are transformed and grown in the same conditions, which presumably minimize the environmental sources of variation15.The mechanism of essential hypertension is complex. The target tissues of essential hypertension are likely multiple. Animal studies on hypertension gene expression have used the aorta, heart, liver and kidney tissues16 database was used to further support our findings. Moreover, we further conducted a gene set enrichment analysis (GSEA)17 to investigate whether the transcripts were enriched for the various known pathways.In this study, we conducted a two-stage whole-genome gene expressions association study. Afterwards, a hypertensive mouse data obtained from Gene Expression Omnibus (GEO)Written informed consent was obtained from each participant at his/her initial clinic visit. The study was approved by the Internal Review Board of Academia Sinica (Permit Number: AS-IRB01-08012). All methods were performed in accordance with the relevant ethical guidelines and regulations.Figure In the first stage, a total of 126 young-onset hypertensive patients and 149 normotensive controls were included. In the second stage, another 127 young-onset hypertensive patients and 150 normotensive controls were used to validate the finding in the first stage. Finally, we combined 253 case and 299 control samples together in the combination analysis. The characteristics of these samples are presented in the Tables 5. Age and sex matched normotensive controls were selected from “Han-Chinese Cell and Genome Bank in Taiwan”18 which has established by Institute of Biomedical Sciences of Academia Sinica.Young-onset hypertensive patients were selected from “Academia Sinica Multi-Center Young-Onset Hypertension Genetic Study”, which had recruited 1023 non-aboriginal Taiwanese individuals with essential hypertension aged 20 to 50. Details regarding this young-onset hypertension genetic study has been described elsewhere2; (g) both parents and all grandparents are Han-Chinese; and (h) have been genotyped by Illumina 550 K beadchip.Inclusion criteria for young-onset hypertensive patients are described as follows: (a) systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg at least twice in the previous 2–6 months, or SBP/DBP ≥ 120/80 mmHg at least twice for those who were on anti-hypertensive medication for two months or more; (b) participant initially diagnosed with hypertension between the ages of 20 and 51years; (c) no secondary hypertension such as chronic renal disease, renal arterial stenosis, primary aldosteronism, coarctation of the aorta, thyroid disorders, Cushing’s syndrome and pheochromocytoma ; (d) no medical history regard to severe disease, including liver and renal failure; carcinoma; cardiac or pulmonary failure (e) fasting glucose level < 126 mg/dl and no previous diagnosis of diabetes mellitus; (f) body mass index (BMI) < 35 Kg/m2; (e) both parents and all grandparents are Han-Chinese; and (f) have been genotyped by Illumina 550 K beadchip. Finally, age and sex matched controls were selected to carry out the following experiments.Inclusion criteria for normotensive controls are: (a) participant with SBP < 120 mmHg and DBP < 80 mmHg and no anti-hypertensive medication (b) aged between 20 to 51 years; (c) no other disease (d) BMI < 35 Kg/mLymphoblastoid Cell Lines (LCLs) were used to profile the gene expression levels. A total of 33 ml of blood had been drawn from each eligible subject: 17.5 ml for basic clinical chemistry and plasma storage, 5 ml for DNA extraction, 10 ml for LCLs establishment, and 0.5 ml for Guthrie cards.Peripheral blood samples were collected and LCLs were generated by Epstein Barr Virus transformation of the B-lymphocyte component by the Bioresource Collection and Research Center  and transformed cells were grown in RPMI 1640  with 15% fetal bovine serum , 100 U/ml penicillin and 100 g/ml streptomycin .st stage (126 cases and 149 controls) and 2nd stage (127 cases and 150 controls) were generated in the same time period in the same laboratory.The LCLs of all cases and controls used in 1For the gene expression profiling, the case-control pair was assayed back to back to avoid differential expression level caused by batch effect.Total RNA was extracted with TRIzol (Invitrogen) and hybridized onto Human OneArray v5.1 . For the whole genome gene expression profile measurement, only the RNA which RNA Integrity Number (RIN) > 7 were used.19 and R statistical software were employed for quality control of the data. The data quality control procedures including: (1) background correction , (2) removal of 5,752 probes with detection p-value > 0.05, (3) quantile normalization for all 1,656 arrays, (4) removal of samples for which 3 replicated samples were not clustered for the remaining 25,216 probes, (5) median value of 3 replicates selected for further analysis, and (6) use of ComBat20, an empirical Bayes method (R package: SVA), to remove remaining batch effects.Phalanx Human OneArray v5.1 contains 30,968 human genome probes. Each sample was measured with 3 technical repeats . A microarray data management and analysis software, BRB Array toolsIn the first stage (126 case and 149 controls), logistic regression with age, sex and BMI adjustment was used to identify the significantly differentially expressed genes with hypertension status  as the independent variable. In the second stage, another independent sample set (127 cases and 150 controls) was used to validate the transcripts found in the first stage. The same statistical methods were used with age, sex, and BMI adjustment. Finally, in the combination analysis, we combined samples of the two stages together (253 cases and 299 controls) for the association test and calculated the fold change (average intensities in cases/average intensities in controls). The same analysis methods employed in the first and stages were used in the combination analysis. Bonferroni correction was employed in the first stage, second stage and combination analysis for handling multiple comparison issue. The commercial software SAS 9.4 was used to do the statistical analyses.We used the combined samples to conduct a sensitivity analysis which included the age, sex, BMI, liver function (s-GOT & s-GPT), total-cholesterol (TC), HDL-C, kidney function (eGFR), serum uric acid, education, smoking and alcohol use in the model to check whether the results are robust to these factors.Besides, we also performed a sensitivity analysis among the non-medicated patients to confirm that these transcripts were not due to effect of antihypertensive medications. Among the 253 young-onset hypertensive patients, 55 patients were non-medicated. We used these 55 non-medicated patients and 299 healthy controls to perform the sensitivity analysis.http://www.ncbi.nlm.nih.gov/geo/GPL9734)11 to validate these results.Furthermore, we used gene expression profile data of hypertensive mouse model downloaded from the Gene Expression Omnibus , BPL/1J (lowest blood pressure), and BPN/3J  mice were used to generate expression data. Gene expression profiles of tissues including aorta , liver , heart , and kidney  were measured. Merck/Affymetrix mouse 1.0 custom arrays monitoring 38,384 individual transcripts (25846 Entrez genes) were used. Raw intensity was normalized using the RMA algorithm19 was employed to perform the analysis. A total of 299 pathways from BioCarta database and 128 pathways from KEGG database were tested. For the GSEA, all of the 25,216 transcripts were included. Two-sample t-test was used to get the p-values from each single transcript. Least square (LS) permutation test and Kolmogorov–Smirnov (KS) permutation test were used to find significant gene sets. LS/KS permutation test finds gene sets which have more genes differentially expressed among the phenotype classes than expected by chance22. These results were further validated using hypertensive mouse data.On top of the two-stage whole-genome gene expression association study, we further conducted a gene set enrichment analysis (GSEA) to investigate the differentially expressed pathways. BRB-array toolsWe integrated the gene expression profiles with the SNP and copy number variation (CNV) data to find influential genetic polymorphisms for the differentially expressed genes. Use was made of data from 250 (out of 299) normotensive controls, who had both genotype and gene expression data.® HumanHap550 Genotyping BeadChip which contains 560,184 tag-SNPs. Genomic DNA was isolated from leukocytes using a PUREGENE® DNA Purification Kit  for genomic DNA isolation. The DNA concentration was quantified and adjusted to 60 ng/μl using a NanoDrop™ ND-1000 Spectrophotometer . All sample were genotyped by deCODE Genetics .These samples were genotyped with the Illumina’s SentrixGenotype calling was performed using the standard procedure implemented in BeadStudio  with the default parameters.Information on genotyping call rate (GCR), Hardy-Weinberg Equilibrium (HWE) and minor allele frequency (MAF) was used to evaluate Genotyping quality. SNPs were excluded, if: (1) they were nonpolymorphic in both cases and controls, (2) GCR < 0.95, (3) MAF < 0.01, or (4) SNP deviated from HWE with a -log10(pFDR) > 3 . Finally, 497,849 SNPs (~88.9% of SNPs on the HumanHap550 BeadChip) were analyzed in this study.Genotype based  regression was employed to test the association between genotype and gene expression. Only cis-SNPs (SNPs located in the gene region ± 50 Kbps) were used to test the association. We then tested the association between the discovered expression regulatory SNPs (eSNPs) and young-onset hypertension. Statistical significance was claimed under a significance threshold of p-value < 0.05.23 and QuantiSNP24, respectively, which identifies CNVs by integrating intensity data from neighboring probes using a hidden Markov model (HMM). Gene-based CNVs association analysis was used to test whether there was a significantly differed probability of a CNV intersecting a given gene between hypertension patients and normotensive controls. CNV analysis were performed in PLINK25.Furthermore, the intensity data of 497,849 SNPs were used to call the CNV regions. CNVs were identified using PennCNVTables −6, 0.05/25215), 9 transcripts  were considered significantly differentially expressed , most genes were highly expressed in case group compared to the healthy control group. Although the fold change of these up-regulated genes are not very big (ranged from 1.20 to 1.51), the p-value and OR of these genes are strong (OR = 3.29 to 10.12), which means the variance of these genes’ expression are small and the relative risk estimates are rather precise.In the second stage, another independent sample consisting set of 127 cases and 150 normotensive controls were used to confirm the nine genes that discovered by first stage. All of these nine genes were significantly associated with hypertension in the second stage. According to the Table Moreover, we have conducted a sensitivity analysis using all 253 case and 299 controls, which included the liver function (GOT & GPT), total-cholesterol, HDL-C, eGFR, SUA, smoking, alcohol consumption, education as well as age, sex and BMI in the model. After adjustment of these covariates, these nine genes still significantly associated with young-onset hypertension , normotensive “blood pressure normal” (BPN), and hypotensive “blood pressure low” (BPL) inbred mouse strains. Among these nine genes, Nrg2 cannot be found on this custom array and was not included in the following analyses.Furthermore, we used the mouse gene expression profile data downloaded from GEO (GSE19817)Zranb1 can be validated in the aorta, liver and kidney . Fam110a is significantly differentially expressed in aorta and kidney . Prep, Ankrd9 and Lamb2 can be only validated in aorta (pFDR of Prep = 0.016), liver (pFDR of Ankrd9 = 0.048) and kidney (pFDR of Lamb2 = 0.034), respectively. Although the raw p-value of Dcps in Heart (p = 0.0084), Wfdc12 in aorta (p = 0.037) and liver (p = 0.04) and Tpte in liver (p = 0.045) were smaller than 0.05, none of these genes were significant after FDR correction. The results have been shown in Table After 10,000 times permutation along with the FDR correction (pFDR < 0.05), In addition to the whole-genome gene expression association study, we also conducted a gene set enrichment analysis (GSEA) to investigate whether any known pathway maybe implicated. Table Furthermore, we also used the mouse gene expression profile data to validate these results. Five out of 16 pathways can be validated in the mouse model  were identified and replicated by our study, and also were validated in the hypertensive mouse model. NRG2, DCPS, WFDC12, and TPTE were identified and replicated by our study, but not replicated in the hypertensive mouse model. The discrepancy may be due to the different tissues used in our study (LCLs) and in the mouse model  or due to the species specificity (human and mouse).In this study, a total of nine genes were found differentially expressed in two independent sample sets. Among these genes, five genes  when the cases are restricted to individuals not on antihypertensive therapy. It may be due to the different sample size or the different disease stage. These non-medicated patients may have relatively less severe hypertension. These genes might have the different effects contribute to the different hypertension stages. Further study is needed to understand the pathogenesis of hypertension.Moreover, we have also performed a sensitivity analysis among the non-medicated individuals to confirm that these transcripts were not due to effect of antihypertensive medications. The association between the genes and diseases status are still very significant, which demonstrate that our results were not affected by the antihypertensive medication  to identify pathways with significantly greater number of genes been down- or up-regulated, compared to the controls. In the GSEA, 16 pathways from BioCarta or KEGG were identified.29. ZRANB1, which identified in the two-stage association study, is also related to the TNF-alpha, but this gene is not involved in any known pathway as yet. Pathway Bioactive peptide induced signaling pathway  is a pathway related to the glucocorticoid receptor, which will activate/inactivate the lipoprotein lipase, TNF-alpha, and insulin resistance32. The association between Insulin resistance and hypertension has been well recognized. Up to 80% patients with type 2 diabetes have hypertension33. Pathway hedgehog signaling pathway  and copy number variations (CNVs) data and gene expression profile data to investigate whether these hypertension related genes’ expression levels were regulated by SNPs or CNVs.NRG2 (rs2916092) and PREP  (Table 5 (Table We found some expression regulatory SNPs (eSNPs) that are associated with the gene expression level of the 11, but these tissues are not accessible in human. In this study, LCLs were used in all gene expression association tests. The magnitude of expression profiles shared among the different tissues is still under debate. Current estimates are range from very small37 to 70–80%38. The MuTHER study which has the relative large sample size has demonstrated that around 30% of gene expression profiles are shared among tissues, while 29% are exclusively tissue-specific39. Therefore, some tissue-specific signals may have been missed, but the findings should be valid.The mechanisms contributing to essential hypertension are complex. The target tissues of essential hypertension are likely multiple10. There are no overlapping genes were founded among our single gene analysis and previous studies. It may be due to the different sample size and the different surrogate tissues used. The sample size of the most gene expression studies on hypertension is small (n = 18~20), except a large scale gene expression integrative network analysis which used 3,679 non-medicated individuals in the Framingham Heart Study. Although the identified genes are not overlapping among these studies, all of these studies have identified the genes that related to immune or inflammation. Moreover, in our pathway analysis, we have identified the IL-10 Anti-inflammatory Signaling Pathway which is in line with the findings on IL-10R gene by Chon’s hypertension gene expression study8.Several gene expression association studies on human hypertension have been conductedOur study identified several previously unknown young-onset hypertension genes and pathways in Han Chinese. Identification of these genes and pathways suggest the importance of 1. inflammation, 2. visceral fat metabolism and 3. adipocytes and osteocytes homeostasis in either hypertension etiology or complications. These finding may broaden our understanding of hypertension etiology and major outcome development.Supplementary information


R0, i.e., the number of secondary cases that result from an individual infection). Published R0 values were determined by systematic literature research and ranged from 0.37 to 20. R0s ≥ 4 realistically reflected the critical early outbreak phases and superspreading events. Based on the R0, the herd immunity threshold (Ic) was calculated using the equation Ic = 1 − (1/R0). The critical vaccination coverage (Vc) needed to provide herd immunity was determined by including the vaccine effectiveness (E) using the equation Vc = Ic/E. At an R0 of 4, the Ic is 75% and at an E of 90%, more than 80% of a population need to be vaccinated to establish herd immunity. Such vaccination rates are currently unrealistic because of resistance against vaccinations, financial/logistical challenges, and a lack of vaccines that provide long-term protection against all human-pathogenic Ebolaviruses. Hence, outbreak management will for the foreseeable future depend on surveillance and case isolation. Clinical vaccine candidates are only available for Ebola viruses. Their use will need to be focused on health-care workers, potentially in combination with ring vaccination approaches.The recent West African Ebola virus pandemic, which affected >28,000 individuals increased interest in anti-Ebolavirus vaccination programs. Here, we systematically analyzed the requirements for a prophylactic vaccination program based on the basic reproductive number ( Ebolavirus contains five species: Zaire ebolavirus (type virus: Ebola virus), Sudan ebolavirus (type virus: Sudan virus), Bundibugyo ebolavirus (type virus: Bundibugyo virus), Taï Forest ebolavirus , Reston ebolavirus (type virus: Reston virus) (Ebolavirus family in 1976 in Sudan (today South Sudan) and Zaïre (today Democratic Republic of Congo), Ebolaviruses had until 2013 only caused small outbreaks in humans affecting up to a few 100 individuals  . Four Ebn virus) . Reston n virus) . Howevern virus) –4. Sinceividuals , 6. The f Africa , 6. Priof Africa , and twof Africa . Finallyf Africa , 13 and f Africa , 15. Duelc) describes the number of society members that need to be protected (R0 (number of secondary cases caused per primary case) of a pathogen  required to prevent Ebolavirus outbreaks by a prophylactic mass vaccination program based on the R0 associated with Ebolavirus infection in humans. The results were further critically considered in the context of (1) the status of current Ebolavirus vaccine candidates and (2) the feasibility of a large-scale prophylactic Ebolavirus vaccination program taking into account (a) the preparedness to participate in vaccination programs in the affected societies, (b) logistic challenges, and (c) costs.Here, we performed a systematic analysis to determine the critical vaccine coverage (R0) for Ebolaviruses, we performed a literature search using PubMed (www.ncbi.nlm.nih.gov/pubmed) for the search term combinations “Ebola R0,” “Ebola basic reproductive number,” and “Ebola basic reproduction number” (retrieved on 29th September 2017).To identify scientific articles that have calculated the basic reproductive number  was calculated using Eq. Based on the basic reproductive number Ic indicates the proportion of a society that needs to be protected from infection to achieve herd immunity. Next, the critical vaccination coverage (Vc) that is needed to provide herd immunity was determined by including the vaccine effectiveness (E) using Eq. R0 values . 29/35 studies analyzed data from the recent West African Ebola virus outbreak . The others reported on Ebola virus outbreaks in the Democratic Republic of Congo. Four studies also included data from the Sudan virus outbreak 2000/2001 in Gulu, Uganda. We also considered a review that summarized all available data until February 2015 . Small outbreak sizes may also limit the accuracy of the calculated R0 values. Additionally, virus transmission is influenced by socioeconomic and behavioral factors including the health-care response, society perceptions, religious practices, population density, and/or infrastructure . Across all relevant studies, R0s ranged from 0.36 to 12 for Ebola virus and from 1.34 to 3.54 for Sudan virus . 9 of the 35 studies that provided R0 values showed that Ebola viruses can spread with an R0 > 3, and five studies suggested that Ebolaviruses can spread with R0 values > 4. High reproductive numbers (≥4) are typically observed at the beginning of Ebolavirus outbreaks, prior to the implementation of control measures  and/or as consequence of superspreading events  do not currently exist , although preclinical data suggest that the development of such vaccines may be feasible . CurrentR0 of 3 and 83% of the population to prevent an outbreak that spreads with an R0 of 4 , such levels of vaccine coverage seem currently unachievable, even under the threat of an ongoing epidemic, although attitudes may change in the future if more  data becomes available. Therefore, more differentiated vaccination strategies with a focus on health-care workers and patient contacts appear more feasible.Limited acceptance of vaccinations may also limit Ebolavirus vaccination programs. In a rVSV-ZEBOV ring vaccination trial, only 5,837/11,841 patient contacts could be vaccinated. 34% of the contacts refused the vaccination . In a suwww.gavi.org) is providing $5 million for the development of rVSV-ZEBOV, which is expected to pay for 300,000 vaccine doses (about $16.70/dose)  . The int70/dose) . Within 70/dose) . Hence, 70/dose) , 61.Vc of 75% that is necessary to prevent an outbreak that spreads with an R0 of 4 with a vaccine that has an efficacy of 100% is currently unrealistic because of limited vaccine acceptance in the affected populations and because of financial and logistical challenges. In addition, concurrent diseases such as HIV and cancer, along with potential side effects of vaccination, may remove significant numbers of potential vaccines (In conclusion, the achievement of a vaccines , 62. Altvaccines . Moreovevaccines , potentivaccines . SAGE acvaccines .SM performed the calculations. MM performed the literature search. All authors analyzed the data. SM, MM, and MW wrote the manuscript. All authors gave their final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Correspondence: Dominique Pelca - mkdompe@gmail.comAnnals of Intensive Care 2018, 8(Suppl 2):Co-41Introduction: Recent research in the areas of the simulation and virtualization have encouraged us, and permits to refine our approach in the framework of chest physiotherapy for newborn infants. The Multidisciplinary Team original as we have incorporated brings together researchers in applied mathematics, physics, biomechanics, mechatronics, fluid mechanics, respiratory physiology and physiotherapists. Here we develop the approach used for the design of the simulator, and the theoretical elements which support.Patients and Methods: This study is based on three separate sub-models- one for the lung, one for the thorax and one to mimic chest physiotherapy manipulations. We build a minimal model, which includes only the core properties we identified as playing an important role in chest physiotherapy. This choice was mainly made in order to be able to interpret correctly the interactions between these core phenomena. It was also a way to limit the computation times of the numerical studies. As a consequence, our model can perform qualitative and comparative predictions, but not quantitative predictions.Results: Our results indicate that manipulations need to overcome secretions threshold in order to be able to mobilize secretions. Our model shows that manipulations main effect is to reduce the hydrodynamic resistance of the airway tree by secretions redistribution in the tree, eventually reaching a distribution that is not anymore affected by the manipulation. This effect means that patient breathing might be instantly improved by the manipulation. The second main effect is mucus expectoration, which is also another mean to improve the status of the patient. Mucus expectoration predicted by our model is nonnegligible only for pressures high enough, and also, in the case of high frequency chest wall oscillation (HFCWO), for frequencies high enough. These conclusions, essentially mechanistic, do not depend qualitatively on the initial mucus distribution.Conclusion: This collaboration allows us today to confront our practice to the theory, test our assumptions, in coherence with the scientific data updated. Finally, we proposed two numbers to measure the efficiency of the manipulations- the Shrek number and the comfort number. They are both performing well in the case of our idealized manipulations. They need however to be validated with clinical studies. The first mathematical and digital models from this collaboration are encouraging and offer avenues that we can build on as our works move forward.Correspondence: Damien Moerman - damien.moerman@uclouvain.beAnnals of Intensive Care 2018, 8(Suppl 2):Co-37Introduction: Early mobilization of adult patients admitted to the intensive care has become current practice since a few years. Indeed, many studies have reported about the benefits of early mobilization. However, early mobilization in pediatric intensive care is poorly described. The aim of this study was to evaluate the feasibility and the safety of early mobilization in critically ill children (0–2 years) and its impact on comfort scores.Patients and Methods: Patients admitted since 24 to 48 h in the Pediatric Intensive Care Unit were screened to identify eligible children, between 0 and 2 years old, stable from a cardiorespiratory point of view. Early mobilization was performed between 24 and 48 h of admission. Respiratory and hemodynamic data were recorded before mobilization (T0), immediately after the treatment (T1) and after 10 min (T2), 30 min (T3) and 1 h (T4). The Comfort Behavior scale and EDIN scale were used for respectively the intubated and the extubated child in order to evaluate comfort during the mobilization session. The comfort scores were taken before mobilization (T0), immediately after the treatment (T1) and after 10 min (T2). Adverse events such as endotracheal tube or catheter loss were recorded. The institutional research ethics board approved the research protol. Written informed consent was obtained from parents or legal guardians. Statistical analysis was performed using SPSS, version 24 (IBM). To compare respiratory and hemodynamic parameters, repeated measures ANOVA test or Friedman Test was used. The same tests were used to compare the series of comfort scores. Statistical significance was fixed at p < 0.05.Results: From September 2016 untill February 2017, 237 children were screened, 65 were eligible, 20 were enrolled and mobilized. The heart rate, respiratory rate, systolic and diastolic blood pressure, oxygen saturation and comfort scores showed no significant change before and after mobilization. Four sessions of mobilization were stopped because of important agitation of the patient with at T1 change in respiratory and hemodynamic values as well as a change in comfort scores. No adverse events were reported.Conclusion: Mobilization is safe and probably feasible in children aged 0–2 years providing physiotherapists can adapt to the behaviour of the child and stop in case of poor tolerance.Correspondence: Frédéric Duprez - dtamedical@hotmail.comAnnals of Intensive Care 2018, 8(Suppl 2):Co-36Introduction: Boussignac system (BS) administers oxygen and generates a continuous positive airway pressure (cpap). With the BS, fraction of inspired oxygen (FiO2) value is approximatively equal to the ratio between O2 flow and inspiratory flow (IF). In some cases, FiO2 decreases due to IF increase. To limit the FiO2 decrease during IF increase, we developed a new system- The Dup-Reyg system. The aim of this study was to test the Dup-Reyg system connected to a BS during IF increases.Patients and Methods: The study was conducted on bench with a BS connected to a two-compartment adult lung model  controlled by a Maquet Servo I®ventilator. Three minute ventilations (MV 10 20 and 30 L min) with a Ti Ttot = 0.33 were investigated. FiO2 and MV measurements were made using an iWorx® GA207 gas analyzer. Three Positive End Expiratory Pressure (PEEP) were analyzed- 3, 5 and 10 cm H2O. The BS was supplied with an O2 flow. In order to increases the FiO2 during IF increases, we have evaluated the impact of the addition of the Dup-Reyg system  connected to expiratory way of BS. A Friedman test followed by a Holm-Sidak method were used to compare data.Results: Means are expressed with their SD . However, complementary studies must be carried out in order to verify its clinical application.Correspondence: Carlos Diaz Lopes - carlosdl90@hotmail.comAnnals of Intensive Care 2018, 8(Suppl 2):Co-39Introduction: Swallowing disorders (SD) are a major concern in Intensive Care Unit (ICU), with a prevalence of 15 to 87% (1). SD may be the cause of malnutrition, dehydratation and pneumonia (2). It may increase the hospitalization length, induce higher reintubation rate and delay tracheotomy removing (3) (4). Atrophy of laryngeal muscles, alertness and movement decreasing  and the use of sedative or neuromuscular blocking agents are important risk factors of dysphagia. Neuromuscular electrical stimulation (NEMS) is an interesting tool for the physiotherapist and studies provide promising result to support the use of NEMS associated to usual treatment in ICU patients with SD (5). A literature review highlights the lacks of standardized NEMS protocol for SD management in ICU. Objective—we propose a standardized NEMS protocol for SD patients in ICU for future research protocol.Patients and Methods: The protocol aims to include difficult-to-wean patients after a long ICU hospitalization period and suffering from SD. The presence of SD is revealed by the Food and Oral Intake Scale (FOIS) and clinical examination . FOIS is the main outcome to assess the protocol (6). NEMS is associated to conventional SD treatment- Shaker exercices, thermal-tactil stimulation, effortful swallowing maneuver, laryngeal and lingual exercises and compensatory maneuvers. NEMS is performed with a Cephar Physio4 and small conventional electrodes. Electrodes are placed to supra and infrahyoid muscles and above the superior limit of thyroid notch. Two-phases current is used with frequencies varying between 5 to 80 Hz in three differents periods . The total of the NEMS treatment varies between 16 to 26 min, one session per day until swallowing improvement or patient discharge.Results: NEMS associated to usual therapy may improve the SD management. Literature highlights a decrease of lenght of hospitalisation and a higher FOIS evolution with the use of NEMS. We aim to published this protocol for supporting the use of a standardized NEMS protocol in clinical research.Discussion: Additional studies are required to assess the efficacy of NEMS with a standardized protocol. Moreover, FOIS has limitations to evaluate SD and new validated tools should be described to assess more specifically the NEMS effects to the laryngeal muscle function.Conclusion: A NEMS standarized protocol may be usefull for clinical research allowing to assess its efficacy in SD management in ICU patients.ReferencesRobert D. Les troubles de la déglutition postintubation et trachéotomie. Réanimation. 2004 Sep;13(6–7):417–30.Clark H, Lazarus C, Arvedson J, Schooling T, Frymark T. Evidence-based systematic review: effects of neuromuscular electrical stimulation on swallowing and neural activation. Am J Speech Lang Pathol. 2009 Nov;18(4):361–75.Dziewas R, Mistry S, Hamdy S, Minnerup J, Van Der Tweel I, Schäbitz W, et al. Design and implementation of Pharyngeal electrical Stimulation for early de-cannulation in TRACheotomized (PHAST-TRAC) stroke patients with neurogenic dysphagia: a prospective randomized single-blinded interventional study. Int J Stroke Off J Int Stroke Soc. 2017 Jun;12(4):430–7.Malandraki GA, Markaki V, Georgopoulos VC, Psychogios L, Nanas S. Postextubation dysphagia in critical patients: a first report from the largest step-down intensive care unit in Greece. Am J Speech Lang Pathol. 2016 May 1;25(2):150–6.Lee KW, Kim SB, Lee JH, Lee SJ, Ri JW, Park JG. The effect of early neuromuscular electrical stimulation therapy in acute/subacute ischemic stroke patients with dysphagia. Ann Rehabil Med. 2014 Apr;38(2):153–9.Sallum RAA, Duarte AF, Cecconello I. Analytic review of dysphagia scales. Arq Bras Cir Dig ABCD Braz Arch Dig Surg. 2012 Dec;25(4):279–82.Correspondence: Daniel Ferreiro Carballa - dani_ferreiro@hotmail.comAnnals of Intensive Care 2018, 8(Suppl 2):Co-38Introduction: The lung ultrasound use in acute respiratory conditions may have the potential to increase the physiotherapist‘s diagnosis accuracy. This case report highlights the lung ultrasound impact on chest physiotherapy decision in patient with deteriorating respiratory conditions.Patients and Methods: A 73-years-old male is referred to the Respiratory Intensive Care Unit for respiratory deterioration. The physiotherapist’s clinical examination highlights hypoxemia, tachypnea, and right thoracic side reduced mobility associated to lung auscultation crakles. Moreover, pulmonary auscultation shows crackles bilaterally. Chest X-ray reveals basal opacity and reduced intercostal spaces on the right and heart deviation to the right. We also note a bilateral and diffuse alveolar syndrome. Three clinical hypotheses may be proposed: pneumonia, pulmonary oedema or atelectasis. As the chest physiotherapy treatment may be dramatically different depending on the clinical hypothesis, the physiotherapist performs a lung ultrasound examination. The lung ultrasound shows the presence of coalescent, homogeneous and diffuse B lines, a very small pleural effusion and right diaphragm paralysis. These ultrasound findings suggest a pulmonary oedema. So, the physiotherapist chooses to implement non-invasive ventilation in seated position.Discussion: The initial diagnosis retained by the physiotherapist and medical staff was pneumonia but level of uncertainty leads the physiotherapist to perform lung ultrasound. These findings have changed the therapeutic decision as treatment for pulmonary oedema is very different from pneumonia. Knowing the greater accuracy lung ultrasound have, this case report support the potential for lung ultrasound to improve the physiotherapist’s diagnosis and choice of treatment.Conclusion: Lung ultrasound provides relevant information about the lung and diaphragm statuses, which may allow differential diagnosis in situations where clinical examination is limited. This may also help physiotherapists to choice the more effective chest physiotherapy technique.Correspondence: Thomas Joannon - thomas.joannon@chru-lille.frAnnals of Intensive Care 2018, 8(Suppl 2):Co-40Introduction: Intensive care unit acquired weakness (ICUAW) is a frequent adverse event, associated with an increase of mortality and prolonged weaning from mechanical ventilation. Preventing ICUAW occurrence is an important part of intensive care physiotherapists activity, mainly by early mobilization (EM). Currently, early ICUAW diagnosis and EM efficiency evaluation remain difficult. Muscle measurement by ultrasound is a simple, non-invasive, non-ionizing and non-volitional assessment method at patient’s bedside, possibly utilized by physiotherapists in France since 2015. Our study focused on intra and inter-observer agreement of three measurements obtained by muscle ultrasound on critically ill patients. We hypothesized that reproducibility of measurements is independent of the qualification level of the observer.Patients and Methods: Prospective observational study in our 10 beds medical ICU. Main inclusion criteria were hospitalized in ICU, age over 18 and mechanically ventilated since more than 24 h. Main non-inclusion criteria were an instability that prevented the patient from being positioned for the examination and unavailability of one of the observers. All measures were realized by B-mode ultrasonography with a 4 MHz curvilinear array transducer. Position of the patient, sites of measures, position of the probe and pressure applied on the skin were standardized. We assessed rectus femoris cross sectional area (RFCSA), quadriceps thickness (QDT) and tibialis anterior thickness (TAT). Agreement of measures was studied in two different groups, first through measures obtained by a physiotherapist (group 1) then comparing a physiotherapist with an intensivist (group 2). Data were analyzed using Wilcoxon signed rank test. Coefficient of variation (CV) and bland–Altman analysis were used to assess agreement.Results: 15 patients (n = 5 and n = 10) of mean (SD) age 62 (16) and 66 (9) years old , mean (SD) BMI 25  and 28 (6) kg m−2  and mean (SD) IGS2 53 (26) and 52 (17)  were included. In group 1 mean CV for each muscle was less than 6% and Bland–Altman analysis showed excellent agreement between examinations. In group 2 mean CV were less than 5% for QDT measurements and for TAT. Mean CV were less than 13% for RFCSA. Bland–Altman analysis showed good agreement between observers for QDT and TAT but poor agreement for RFCSA.Conclusion: Results showed excellent intra-observer agreement for the physiotherapist and the intensivist. Inter-observer agreement was poor, mostly for RFCSA. Measurement protocol adjustments could allow better results.