Despite a substantial reduction in virological failures following introduction of new potent antiretroviral therapies in the latest years, drug resistance remains a limitation for the control of HIV-1 infection. We evaluated trends and correlates of resistance in treatment failing patients in a comprehensive database over a time period of relevant changes in prescription attitudes and treatment guidelines.pol sequences from 49 centres stored in the Italian ARCA database during the 2003–2012 period. Patients  with viremia > 200 copies/mL received a genotypic test while on treatment. Mutations were identified from IAS-USA 2013 tables. Class resistance was evaluated according to antiretroviral regimens in use at failure. Time trends and correlates of resistance were analyzed by Cochran-Armitage test and logistic regression models.We analyzed 6,796 HIV-1 The use of NRTI backbone regimens slightly decreased from 99.7% in 2003–2004 to 97.4% in 2010–2012. NNRTI-based combinations dropped from 46.7% to 24.1%. PI-containing regimens rose from 56.6% to 81.7%, with an increase of boosted PI from 36.5% to 68.9% overtime. In the same reference periods, Resistance to NRTIs, NNRTIs and PIs declined from 79.1% to 40.8%, from 77.8% to 53.8% and from 59.8% to 18.9%, respectively . Dual NRTI + NNRTI and NRTI + PI resistance decreased from 56.4% to 33.3% and from 36.1% to 10.5%, respectively. Reduced risk of resistance over time periods was confirmed by a multivariate analysis.Mutations associated with NRTIs, NNRTIs and PIs at treatment failure declined overtime regardless of specific class combinations and epidemiological characteristics of treated population. This is likely due to the improvement of HIV treatment, including both last generation drug combinations and prescription guidelines. Despite substantial reductions in AIDS-related morbidity and mortality ,2, advanpol genotype, control viral replication even in patients with primary resistance.Treatment failing patients appear to be the major cause of the transmission of resistant strains  which maAvailable results on the prevalence of drug resistance are often difficult to compare as they differ in resistance associated mutations taken into account, timing of samples and selection of study populations. The latter could vary from all subjects on antiretroviral treatment, to patients failing ART, to subjects with available resistance test results. Moreover, these studies have estimated the prevalence of HIV drug resistance using a variety of analytical methods resulting in a wide range of estimates, ranging from 50% to 80% of subjects failing an antiretroviral treatment -16.A reduction of acquired resistance has been recently reported in Italy until 2009 ,18. ThisThe aim of this study was to monitor acquired resistance to understand present trends and correlates of class resistance in subjects failing cART regimens in a multicenter Italian network based over the 2003–2012 period. Previous reports considered resistance prevalence among the whole studied population, regardless of the regimen administered at the time of resistance testing -18. Sinchttp://www.hivarca.net) database, a nationwide repository used for non-profit research purposes and stored on a central server. Written informed consents had been obtained by patients. The research did not require approval from the Ethics Committees, according to the Italian law at the time when the study was conducted, since it was performed as an observational study in the context of clinical routines .Patients included in the study were adult HIV-infected individuals enrolled in 49 Italian clinical centres during the 2003–2012 period. All the clinical centres contributed data to the Antiretroviral Resistance Cohort Analysis  mutation list [Genotyping was based on a partial HIV-1 ion list . Any thyNRTI, NNRTI and PI class resistance was evaluated according to NRTI, NNRTI or PI based treatment used at time of virological failure.Four periods of study were evaluated encompassing years 2003–2004, 2005–2006, 2007–2009 and 2010–2012.Subtype was assigned using the NCBI HIV-1 subtyping tool.χ2 and logistic regression. A subsequent multivariate analysis was done on all variables, using the same tests with a full model. Analyses were done with SAS Software version 9.1.The Cochrane-Armitage test was used to evaluate temporal trends. The crude and Mantel-Haenszel adjusted odds ratios (OR) of class resistance detection with 95% confidence interval (CI) were calculated. Univariate analysis was performed using pol sequence was available for the analysis. Two, 3, 4 and more than 4 sequences were collected for 800, 227, 65, 24 subjects .The sequences were obtained from 5,246 HIV-1 positive individuals. Overall, male to female ratio was 2.1 , the route of transmission was injection drug use (IDU) for 35.4% , heterosexual sex in 33.5% , and men having sex with men (MSM) were 16.7% (n = 876) of the study population. Non-B subtypes were detected in 9.1% of the subjects included in the study (n = 477). For 4,130 (78.7%) patients a single pol sequences from subjects with viral load above 200 copies/ml while on treatment in the 2003–2012 period. Their distribution was as follows: 2,450 in 2003–2004 (36.1%); 2,050 in 2005–2006 (30.2%); 1,641 in 2007–2009 (24.2%); 655 in 2010–2012 (9.6%). Median age of patients at time of genotyping was 42 years . Median HIV-RNA viral load was 4.06 Log copies/ml (IQR 3.36-4.74) and median CD4 number was 282 cells/μl (IQR 168–429 cells/μl). The median number of previous treatments experienced by enrolled subjects was 5 for each patient (IQR 2–9).The study included 6,796 At genotypic testing 99.4% , 37.4%  and 67.5%  sequences were obtained from NRTI-, NNRTI- or PI-treated patients, respectively. HAART combination included NRTIs + NNRTI in 32.5% , NRTIs + PI(s) in 62.6% , NRTI(s) + PI + NNRTI in 4.3% (n = 293), an NNRTI + PI(s) in 0.6% (n = 39).NRTI usage significantly decreased overtime, ranging from 99.7% in 2003–2004 to 97.4% in 2010–2012 (p < .0001). NNRTI-based regimens decreased from 46.7% to 24.1% (p < .0001), while an increase of cART regimens including PIs was observed  overtime. The same increasing trend was confirmed for ritonavir boosted PI regimens .Antiretroviral classes were administered in several combinations during the study period: the prevalence of the association of NRTIs + NNRTI decreased from 40.4% to 18.3% (p < .0001), while combinations of NRTIs + PI(s) increased from 53.4%, to 75.9% (p < .0001). The combination of all the three classes showed a significant declining trend from 6.0% to 3.2% (p < .0001) , didanosine (ddI) and lamivudine (3TC); their use went from 63.0% to 16.0% (p < .0001), from 36.6% to 1.7% (p < .0001) and from 63.0%, to 35.6%, (p < .0001), respectively. An opposite trend was observed for tenofovir (TDF) from 28.7%, to 63.4% (p < .0001), abacavir (ABC) from 8.9% to 16.6% (p < .0001) and emtricitabine (FTC) from 0% to 57.7% (p < .0001). Regarding NNRTIs, a decrease of both nevirapine (NVP) and efavirenz (EFV) use was detected at failure; their use went from 24.1% to 7.0% (p < .0001) and from 22.6% to 12.5% (p < .0001), respectively. Among subjects assuming an NNRTI, the rate of subjects in treatment regimens including NVP decreased from 51.6% to 29.1% (p < .0001). Among PIs, significant reductions in usage frequency at failure were observed for indinavir , nelfinavir , and saquinavir . In contrast, an increasing trend was observed for lopinavir , fosamprenavir , atazanavir , darunavir , and tipranavir .Resistance to NRTIs declined from 79.1% to 40.8%, from 2003–2004 to 2010–2012 (p < .0001). A decreasing trend was also found for NNRTI mutations, which dropped from 77.8% to 53.8% (p < .0001). PI mutations decreased from 59.8% to 18.9% (p < .0001). Among subjects assuming NRTIs combined with an NNRTI, resistance to both classes went from 56.4% to 33.3% (p < .0001); resistance to NRTI + PI went from 36.1% to 10.5% (p < .0001). Triple class resistance did not significantly vary overtime in subjects assuming NRTI(s) + NNRTI + PI, ranging from 54.8% to 42.9% in the study period  to 7.62% (8/105) (p = <.0001). A significant decline was observed for both TAM1 and TAM2 profiles, ranging from 30.4% (469/1543) to 3.8% (4/105) (p < .0001) and from 19.6% (302/1543) to 1.9% (2/105) (p < .0001), respectively. The K65R mutation went from 4.1% (101/2438) to 2.2% (14/637) (p = 0.0071) in regimens based on TDF, ddI, stavudine and/or ABC. The M184I/V mutations dropped from 67.3% (1131/1680) to 34.1% (211/618) (p < .0001) in patients on 3TC-, FTC,- or ABC-containing regimens. This mutation was more common in subjects assuming 3TC rather than FTC . Among subjects failing ABC- or ddI-including cART, the L74V mutation decreased from 16.5% (165/1000) to 3.3% (4/120) (p < .0001).Frequencies of specific mutations and resistance patterns are shown in Table Table Independent predictors of NNRTI resistance in NNRTI-assuming subjects were gender, viral load at failure, previous virological failures.Predictors of PI resistance were gender, risk factor, viral load at failure, previous virological failures, number of previous antiretroviral regimens, prior exposure to suboptimal NRTI therapy, use of TA and period of study. The usage of boosted rather than unboosted PIs was not associated with the risk of evolution on PI resistance.The risk of resistance to any antiretroviral class was not influenced by the use of TA-including or sparing regimens or by the use of FTC rather than 3TC in the multivariate model, even though a lower prevalence of any NNRTI resistance was found in subjects assuming FTC compared to 3TC .Our data provide a solid evidence of a marked decrease in the prevalence of class resistance among treated individuals in Italy, as observed from a retrospective analysis including 49 clinical centres participating in the ARCA cohort during the last decade. Studies exploring resistance trends in European countries have observed an initial decrease over the 2005–2008 period -18,21. HDifferently from previous works ,15 the dThis analysis allowed to detect a twofold and threefold decrease in resistance prevalence to NRTIs and PIs, respectively during the 2003–2012 decade. The study periods included two biennia and two triennia to balance the distribution of subjects overtime. NNRTI resistance declined less markedly, from 77.8% to 53.8%. Nevertheless, the reduction of NNRTI resistance overtime is more relevant than that observed in previous studies ,22 even Several epidemiological correlates were found to have an impact over the selection of resistance mutations only for NRTIs and PIs. An increased risk of resistance was found for sexual routes of transmission  compared to IDU, in agreement with other Italian studies ,18. ThisLower drug resistance rates were associated with plasma HIV-1 viremia above 100,000 copies/ml. This finding is different from previous observations obtained from the ARCA database  and it iAs expected, the occurrence of a previous virological failure increased the risk of resistance to any antiretroviral class. Other treatment history features, such as the number of cART regimens, have shown a role in the emergence of class resistance to NRTIs and PIs, but not to NNRTIs. The fact that NNRTI resistance shown no association with the temporal length of antiretroviral treatment can be explained by the characteristic pattern of resistance evolution of this antiretroviral class, whose use can be compromised by a single substitution leading to cross resistance .The risk of resistance to NRTIs was higher when the patients failed NNRTI rather than PI based treatments. This is in agreement with previous findings and confirms that a higher protection against NRTI resistance is obtained by cART regimens including antiretrovirals with a higher genetic barrier ,18,20. HInterestingly, M184V and NNRTI resistance were less common in subjects failing regimens including emtricitabine rather than lamivudine, as observed by previous studies . This fiSome limitations of our work should be acknowledged. First of all, Italian data on precise extent of HIV-1- treated population, specific treatment prescriptions and related overall achievement of virological suppression on cART are not available at present. In fact, an increase of treatment effectiveness could explain both the reduction of the available resistance testing and the extent of resistance to antiretrovirals. Moreover, we were not able to consider the impact of self-reported adherence in our study population, due to the lack of information regarding this issue for a large number of patients, referring to several clinical centers in Italy. Furthermore, patients with resistant strains in early years of the study could have been lost at follow-up, due to several reasons including death, contributing to the decline of overall resistance. As guidelines prescribe to perform a genotypic resistance testing at failure, almost all patients failing an antiretroviral regimens have been included in the analysis. Nevertheless, the full information about the number of subjects failing antiretroviral regimens is not available, and we cannot exclude a partial loss of information regarding subjects who were not tested for antiretroviral resistance.Even though it was not possible to evaluate all the data regarding the antiretroviral history of patients, our analysis included main covariates such as previous virological failures, number of previous antiretroviral regimens, prior exposure to suboptimal NRTI therapy. Importantly, this work includes an evaluation of resistance according to the antiretroviral regimen in use at failure, representing an achievement in the analysis of acquired resistance not performed in previous works ,18,21.In spite of the limitations mainly due to its retrospective and multicenter design, our study shows a substantial decrease in drug resistance after stratification for antiretroviral class in use at failure. This finding is encouraging for the goal of a more effective treatment of the HIV infected population, in order to achieve a stable reduction of the potential virus transmitters. Moreover, the reduction we observed in resistance prevalence among treated individuals is probably the main driving factor leading to the observed reduction in primary resistance in our country .In conclusion, our analysis shows a reduction of drug resistance overtime, even when correcting for several other or covariates or confounders. This is likely the consequence of time-associated factors including the availability of new potent drugs, updated guidelines for the treatment of HIV infection, the availability and frequent use of resistance testing to select for the optimal antiretroviral treatment at baseline and whenever a virological failure occurs.A.D.L. has received funds for research from ViiV and he is a member of the Gilead speakers bureau and Siemens Diagnostics, ViiV, Gilead, Janssen-Cilag and Abbvie Advisory boards. He also received travel grants from Bristol-Myers Squibb and ViiV. F. C-S. has received funds for attending symposia, speaking, organizing educational activities, grant research support, consultancy and advisory board from Abbott, Merck Sharp & Dohme, Gilead, Janssen Cilag, Roche, Bristol Myers Squibb, and ViiV. M.Z. has received research funds from ViiV, has received speaker fees from Merck Sharp and Dohme, Janssen-Cilag and Gilead, has been a memeber of the advisory board of Abbott Molecular.MF, MV, MZ and CB participated in the design of the study. MF and MV drafted the manuscript. MV performed the statistical analysis. AA, ADL, FCS, NG, CT and SB provided the clinical and virological data stored in the ARCA database. MZ and CB coordinated the study and supervised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/398/prepub


This study aims at describing the epidemiological and clinical characteristics, severity, reversibility testing and response to treatment using simple spirometry in asthmatic patients attending a model specialized Asthma Care Center.Eligible subjects must have a suggestive clinical picture and confirmed by spirometry to have a 12% plus 200ml absolute increase in FEV1 either by reversibility testing or after a therapeutic trial with inhaled and/or oral steroid therapy. Budesonide-Formoterol Turbohaler was used for reversibility testing and for maintenance therapy with or without the addition of oral prednisolone.One hundred and nineteen patients were eligible for the study. Age ranged between 10 -70 years. One hundred and thirteen patients (95.0%) had an FEV1 less than 80% of predicted. One hundred and five patients (88.2%) had reversibility testing of whom 72 (68.6%) had a significant reversibility. Sixty two patients (52.1%) were prescribed Budesonide-Formoterol Turbohaler only whilst 57 were prescribed both Budesonide-Formoterol Turbohaler and oral prednisolone. Patients were reviewed after a mean of 14.9 days (range 6.0-28.0). Seventy two patients (60.5%) had increased their FEV1 to more than 80% of their predicted value. By logistic regression analysis, predicted FEV1 at baseline was a significant negative predictor of a complete response.Most patients had abnormal spirometry with more than half having an FEV1 that is 60% or less of their predicted normal reading. Reversibility testing using Budesonide-Formoterol Turbohaler confirmed the fast onset of action of its Formoterol component and helped in cutting the cost of this test. The majority improved with treatment with 60% normalizing their spirometry highlighting the feasibility and applicability of specialized asthma care centers in resource-poor countries. The global burden of Bronchial Asthma (BA) particularly in developing countries is on the increase , 2. ApprA retrospective chart review of patients with Bronchial Asthma. Patient's consent was not sought as this was a retrospective chart review study and there was no disclosure of confidential patient information.This study was undertaken in a dedicated Bronchial Asthma Care Center in Khartoum, Sudan. The center is manned by a Consultant Pulmonologist, Asthma Educator/Spirometry technician and a Clerk.Patients attending the center with a clinical picture suggestive of Bronchial Asthma who were prescribed BFT and who were confirmed by spirometry to have a 12% increase plus 200 ml absolute increase in FEV1 after reversibility testing or after a steroid trial were eligible for inclusion in the study. A steroid trial was with inhaled steroid  for two weeks with or without oral prednisolone 30 mg per day for 5 days. Patients unable to do spirometry or have poor technique were excluded. Patients with a diagnosis of bronchiectasis, COPD or other respiratory diseases were also excluded. Patients were seen by one Pulmonologist. A detailed history is taken along a pre-specified and unified format detailing all the essential historical data pertaining to Bronchial Asthma. Patients were then seen by the Asthma Educator/Spirometry technician who performed baseline spirometry (hand-held spirometer -Spirodoc-GIMA) and educated the patients on their illness and their inhaler technique. Spirometry was performed with the patient sitting. The first two puffs of their BFT were then administered and spirometry repeated 15 minutes later. Patients must be able to have three attempts at spirometry with variations of less than 0.1 liter in FEV1 between the two best readings for a reliable trace. Most patients were requested to re-attend clinic in 2 weeks for clinical reassessment and spirometry. However, those attending up to 4 weeks post first visit were included. As normative spirometric data for Sudanese subjects are not available and these are generally believed to be 10 percent lower than those of Caucasian, Caucasian FEV1 data corrected by deducting 10% were utilized. Data was entered on a Minitab 15 spreadsheet for analysis. Simple descriptive statistical tests were used together with the 2-Sample t-test, Chi square test and logistic regression analysis as appropriate to test for significance and associations. A p-value of less than 0.05 is considered significant and the actual value quoted.One hundred and nineteen patients were eligible for the study. Sixty two were male and 57 were female with an average age of 30.5 (range 10-70) and 30.3 (range 10-68) years respectively. Mean body mass index was 23.2 (range 11.8-33.2) for males and 27.4 (range 14.3-44.1) for females. A BMI of more than 30 was more frequent in females (20) versus (6) in males . Ninety patients (75.6%) were previously diagnosed as having bronchial asthma. Mean duration of asthma for male patients was 156.9 months (range 6-720 months) and for females was 191.4 months (range 1.2-600 months). Twenty four of the known asthmatic patients (26.7%) have never been prescribed inhaler therapy before. The mean duration of asthma for this group was 156 months (range 1.2-540 months). Family history in first degree relatives for asthma was positive in 73.9% of patients. Of 66 patients who could describe their inhalers, thirty nine (59.1%) were on a steroid inhaler. Twenty patients (16.9%) were active or previous smokers. Eleven patients reported an allergic reaction to food of whom 6 implicated Aubergines as a precipitant Cough and shortness of breath were the commonest symptoms and were reported by 112 patients (94.1%). Wheeze was reported by 98 patients (85.2%). Eighty patients (72.1%) had productive cough.Forty patients (35.7%) admitted to have frequent heartburn and 64 patients (54.7%) reported symptoms suggestive of allergic rhinitis and/or postnasal drip. Those with gastro-esophageal reflux disease (GERD) symptoms had a lower percent predicted FEV1 than those without, but this did not reach statistical significance: 52.0% vs 57.7% (Two-sample t-test p= 0.10). Percent FEV1 readings were comparable in those with and those without upper respiratory symptoms: 54.8% versus 56.0% (Two-sample t-test p= 0.72).On auscultation 84 (70.6%) patients had audible wheeze.Mean FEV1 at baseline was 1.8 L (range 0.6-3.2L) for males and 1.5 L (range 0.5-2.4L) for females. Expressed as a percent of the predicted normal values, mean FEV1 in males was 52.9% (range 18.7-87.7%) and for females was 57.9% (range 14.5-85.6%). Wheezy patients had a significantly lower predicted FEV1 than those with no audible wheeze: 50.4% versus 67.1% . One hundred and thirteen patients (95.0%) had an FEV1 less than 80% of predicted and 68 (57.1%) had a reading that is less than 60% of predicted. Eight (11.8%) of the latter group did not have audible wheeze on auscultation.One hundred and five patients (88.2%) had reversibility testing of whom 72 (68.6%) had a significant change of 12% plus 200 ml increase in FEV1. The average percent increase in FEV1 in this group was 30.1% (range 12.1-71.8%) with a mean absolute increase of 442.6 mls (range 200-1130 mls). All of those who did not have an initial reversibility testing (14) and those who did not attain a significant change after Formoterol (33) did achieve a 12% plus 200 ml increase following their course of treatment. Mean percentage increase in FEV1 was 65.5% (range 13.4-297.0%) with an absolute mean increase of 746.6 mls (range 230.0-2200mls). Prescribed Medications and Symptomatic Response to Therapy Sixty two patients (52.1%) were prescribed BFT only whilst 57 were prescribed both BFT and oral prednisolone. Of the latter group, 48 patients (84.2%) had an FEV1 of less than 60% of predicted. Patients were reviewed after a mean of 14.9 days (range 6.0-28.0). On their second visit, 118 reported that their symptoms improved (99.2%) of whom 74 (62.2%) said their symptoms have disappeared or much improved.Spirometric Response to Therapy  The averThe mean percent increase in FEV1 in those who normalized their FEV1 as opposed to those who did not were 62.5% (range 13.4-300) and 63.0% (range -1.7-292.5) respectively (Two-sample t-test p = 0.82). By logistic regression analysis, predicted FEV1 at baseline was a significant negative predictor of a complete response with age, duration of asthma and initial FEV1 change to reversibility testing having no such correlation. Forty six patients who were prescribed BFT normalized their spirometry compared to 26 patients who were prescribed BFT plus oral prednisolone (Chi Square test p= 0.001). Mean percent FEV1 of predicted at baseline was 66.6% for the former and 43.0% for the latter (Two-sample t-test p <0.0001).An initial striking feature in this study is the extremely low baseline spirometric readings of this cohort of patients. This highlights the deficiency of care this cohort of patients was receiving. Anti-inflammatory therapy is the mainstay of treatment of Bronchial Asthma . More thThe presence of audible wheeze on examination was clearly a marker of severity as these patients on the whole, had a significantly lower predicted FEV1 readings. However, almost a third of this cohort of asthmatic patients had no wheeze on examination including no less than 10% of those with the more severe spirometry readings. Spirometric assessment of suspected patients with Bronchial Asthma is thus extremely vital for quality care. Obesity is seen in a proportion of our patients specially females. As is known, obesity is a risk factor for asthma and is also associated with poor asthma control and needs to be properly managed by dieting and weight reduction . Almost Almost three-quarters of our patients had significant increases in FEV1 after reversibility testing. The use of the prescribed medication (BFT) for reversibility testing assisted us in realizing many goals of our service. The need of using salbutamol MDI via spacer or nebulizer for testing was no longer required. This meant that we were able to save on reagent cost  and spacer devices or nebulizer machine use (unit price plus sterilizing between patients). We had more time to educate these patients on their inhaler device use instead of training on MDI via spacer etc. use for reversibility testing. We were very pleased to see very symptomatic and breathless patients feeling already improved following the use of their new medication and before leaving clinic. We hoped that this will translate into better patient compliance.The outcome of our interventions was similarly very satisfactory. Almost all of our patients reported improvements in their symptoms and a good proportion had become asymptomatic or remarked that they were much improved. This was mirrored by the large improvements in the patient's FEV1. More than 60% of patients normalized their FEV1. The most important (negative) predictor of FEV1 normalization was the baseline reading. Patients with very poor spirometric figures were less likely to achieve their target normal reading. Patients with incomplete responses had, albeit non-significant difference, a longer duration of Bronchial Asthma. This emphasizes the importance of early diagnosis and proper asthma care interventions , 17. AddIn summary, our objectives of providing a satisfactory dedicated service to asthma patients were fulfilled. The use of BFT for an average of two-weeks with or without oral prednisolone was effective in improving the symptoms in almost all of our patients who were prescribed this inhaler. Furthermore, a good proportion (60%) normalized their spirometry. Those with very poor FEV1 readings at the outset (more than 50% of our cohort) were less likely to normalize their spirometry. The magnitude of improvement in FEV1 was appreciable in all grades of severity provided that oral prednisolone is added to BFT in those with FEV1 of less than 60% of predicted. The importance of routine spirometry in Bronchial Asthma care particularly in severity assessment and choice of therapy cannot be overemphasized.


The gastric endocrine cells in patients with irritable bowel syndrome (IBS) tend to normalize following dietary guidance. The aim of the present study was to identify the gastric endocrine cell types that are changed following such dietary guidance.Fourteen IBS patients and 14 healthy subjects were included in the study. Patients received three sessions of individual dietary management guidance. Gastroscopy was performed on both the controls and the patients at baseline and then again for the patients at 3–9 months after dietary guidance. Biopsy samples from the corpus and antrum were immunostained for all gastric endocrine cell types. Endocrine cells were quantified by computerized image analysis.2, respectively. The densities of the gastrin cells in these groups were 155.8±21.0 (95% CI 110.3–201.2), 159.4±24.3 and 211.6±28.0 cells/mm2, respectively; the corresponding densities of serotonin cells in the corpus were 18.2±3.9 (95% CI 9.8–26.6), 10.6±3.4 and 14±2.0 cells/mm2 and in the antrum were 44.6±12.2 (95% CI 18.1–71.1), 1.7±0.5 and 14.7±6.3 cells/mm2. The densities of the somatostatin cells in the corpus were 40.0±7.7 (95% CI 23.5–56.5), 23.0±3.0 and 37.3±4.2 cells/mm2, respectively, and in the antrum were 138.9±22.0 (95% CI 91.4–186.3), 95.6±15.9 and 86.0±16.9 cells/mm2, respectively.The densities of the ghrelin cells for the controls and IBS patients before and after dietary guidance were 149.6±36.2  71.3–227.8), 114.5±32.7 and 161.8±37.8 cells/mmThe densities of all of the gastric endocrine cell types changed towards the healthy control values in the IBS patients following a change in food intake. These patients were told not to take any kind proton pump inhibitors during the study.The inclusion criteria for this study were that patients fulfilled Rome-III criteria for IBS diagnosis and were aged 18–70 years. The following exclusion criteria were applied: women who were pregnant or lactating, or who had a Caesarean section or hysterectomy, and patients with organic gastrointestinal or other systemic diseases, drug abuse, serious psychiatric disturbances or previous abdominal surgery (with the exception of appendectomy). Eight patients did not consume any kind of medications. Six patients used one or a combination of the following: proton pump inhibitors (n=3) or angiodysplasia (n=1) or because of health worries in healthy subjects due to family member(s) having been diagnosed with gastrointestinal cancer (n=10) were used as controls. The control group comprised nine females and five males with a mean age of 54 years (range 26–70 years).Patients who underwent gastroscopy to investigate gastrointestinal bleeding, where the source of bleeding was identified as haemorrhoids . Patients were examined physically, and blood tests were taken to exclude inflammation, infection or other organic disease. These patients also underwent colonoscopy with segmental biopsy sampling to exclude microscopic colitis. Each patient was scheduled for three sessions of individual dietary management guidance of about 45 min, each with a nurse with education and long experience in dietary guidance in IBS. These sessions were timed at intervals of at least 2 weeks. The patients underwent gastroscopy, before the first session and 3–9 months (median 4 months) following the third dietary management guidance session.The information was delivered orally with the help of charts, as well as written illustrated information. The first session included general information about IBS and the importance of regular and healthy eating habits, the diets that worsen IBS symptoms, such as insoluble dietary fibre and FODMAPs . The patients were encouraged to consume dairy products daily and were informed that milk and dairy products do not provoke IBS symptoms. The patients were required to keep a diary for 2 weeks, in which they registered their daily food and drink intake as well as the frequency and degree of abdominal pain, abdominal distension and stool frequency and consistency. In addition, the patients were asked to test a protein-, fat- or carbohydrate-rich/poor diet. The patients were also instructed to avoid the intake of food items supplemented with probiotics, antibiotics and other medications (if not otherwise advised) during the course of the study. In the second session, the information given at the first session was briefly repeated. The nurse and the patient determined the symptom-triggering items based on the information noted in the patient's diary. The patients were then advised to alter the proportions of protein, fat and carbohydrate, avoid items rich in FODMAPs and insoluble fibre, as well as consume vegetables and fruits containing less FODMAPs and insoluble fibre. During the final session, patients described their experience of dietary management to the nurse. The nurse and patient then together designed a suitable diet for the patient, who followed it strictly until the end point of the study.8 It enquires about the intake of 225 food items and captures the dietary habits of the participant, including the intake of any oral supplements, according to typical Norwegian meal patterns. The participants received two sets of MoBa food frequency questionnaire, which were filled out and delivered before and at least 3 months after dietary management guidance.9Dietary intake was assessed using a semi-quantitative, self-administered food frequency questionnaire (MoBa food frequency questionnaire). The questionnaire reported the frequency of consumption and portion size of a series of foods and beverages over a defined period of time. The data were then analysed by software for nutrient calculations. The MoBa food frequency questionnaire used in the current study was developed and validated by the Norwegian Institute of Public Health in Oslo, Norway.After an overnight fast, patients and control subjects underwent standard gastroscopy. Four biopsy samples were taken from the corpus (major curvature) and another four biopsy samples from the antrum of the stomach. A further four biopsy samples were taken from the duodenum to exclude coeliac disease.10 Briefly, the sections were incubated for 2 h at room temperature with a primary antibody/antiserum, after which they were washed in phosphate-buffered saline (pH 7.4) and incubated for 30 min at room temperature with biotinylated swine antimouse immunoglobulin G diluted to 1:200. The slides were washed with phosphate-buffered saline and then incubated for 30 min with avidin–biotin–peroxidase complex diluted to 1:100. The slides were again washed with phosphate-buffered saline, submerged in 3,3′-diaminobenzidine peroxidase substrate and then counterstained with haematoxylin–eosin. The primary antibodies/antisera used were monoclonal mouse anti-N-terminal of human ghrelin , monoclonal mouse antihuman serotonin , polyclonal rabbit antisynthetic, cyclic somatostatin , monoclonal mouse antihistamine (serotonin)-hexamethylene, diisocyanate-bovine serum albumin  and polyclonal rabbit antihuman gastrin .The biopsy samples were fixed in 4% buffered paraformaldehyde overnight, embedded in paraffin wax and then cut into 5-μm-thick sections. Biopsy samples from the stomach and duodenum were examined histopathologically. Biopsy samples from the corpus and antrum were stained with haematoxylin–eosin and immunostained using the avidin–biotin complex method using the Vectastain ABC kit  and the 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories) as described previously.2. The endocrine cell density is expressed as the number of cells per square millimetre of epithelium. Quantification was conducted by the same person (TM), who was blinded to the identity of the sections.The densities of the various types of endocrine cells in the corpus and antrum in patients with IBS and controls were evaluated using the software Cell ⁁D . The number of endocrine cells and the area of epithelial cells were measured in 10 randomly chosen fields, at a magnification of × 40, for which each field represented a tissue area of 0.14 mmt-test was used to compare patients before and after dietary guidance and healthy controls. The data are presented as mean±s.e.m. values. The cutoff for statistical significance was set at P<0.05.The paired Twenty-five patients withdrew their consent, 2 before and 23 after receiving dietary guidance and with consequent improvement in their symptoms. Two patients were excluded because of non-compliance, two because they were diagnosed with coeliac disease and one who had been diagnosed with lupus. In addition, two patients were excluded because of pregnancy during the study and moving abroad. Thus, 14 of the initial 46 patients completed the entire study. These patients comprised 9 females and 5 males with a mean age of 34 years (range 20–45 years). Biopsy samples were not obtained from the corpus of one of the female patients.P=0.016).9 The daily consumption of fibre (mean±s.e.m.) before and after dietary guidance were 27.4±2.5 and 23.1±2.2 g, respectively, (P=0.093).9The daily total consumption (mean±s.e.m.) of FODMAP-rich fruits and vegetables decreased significantly after dietary guidance compared with before receiving dietary guidance  71.3–227.8). The density of ghrelin cells in the IBS patients did not differ significantly between before (114.5±32.7 cells/mm2) and after (161.8±37.8 cells/mm2) receiving dietary guidance .The densities of the gastrin cells in the antrum were 155.8±21.0 (95% CI 110.3–201.2), 159.4±24.3 and 211.6±28.0 cells/mmectively . The cel2 , while those in IBS patients before and after receiving dietary guidance were 10.6±3.4 and 14.0±2.0 cells/mm2, respectively (P=0.4). In the antrum, the corresponding values were 44.6±12.2 cells/mm2 (95% CI 18.1–71.1), 1.7±0.5 and 14.7±6.3 cells/mm2, respectively. There was no significant difference between the findings before and after dietary guidance in the IBS patients (P=0.06).In the corpus, the density of serotonin cells in the control group was 18.2±3.9 cells/mmectively . The cel2 (95% CI 23.5–56.5) and 138.9±22.0 cells/mm2 (95% CI 91.4–186.3), respectively. The somatostatin cell density in the antrum in the IBS patients did not differ significantly between before and after receiving dietary guidance . However, the cell density was significantly higher in the corpus compared with before the intervention  and the expectation that the patient follow the recommended diet strictly for at least 3 months. It is noteworthy that the dropouts occurred mostly after receiving dietary guidance and the associated improvements in the patients' symptoms. There were also other factors that decreased the number of IBS patients who completed this study. Despite the small number of patients in the studied sample, a clear change in the densities of all the gastric endocrine cells occurred following changes in the food intake in the IBS patients towards the values measured for the healthy control subjects.Studies of IBS patients have experienced high dropout rates, ranging from 44% to 48%.16 Furthermore, ghrelin accelerates gastric and small- and large-intestinal motility and stimulates the secretion of gastric acid.28 Serotonin stimulates large-intestinal motility and accelerates transit through the small and large intestines.37 It also activates the submucosal sensory branch of the enteric nerve, which conveys sensation from the gut to the central nervous system.35 Somatostatin inhibits intestinal contraction and gastrointestinal exocrine and endocrine secretion.39 Finally, gastrin is the main hormonal stimulant of acid secretion in the stomach.44 Given the collective functions of these four types of gastric endocrine cells, it is possible to speculate that the changes in the densities of these cells towards normal levels following a change in food intake are responsible for the simultaneous improvements in IBS symptoms associated with disturbed gut motility, visceral hypersensitivity and abnormal gastrointestinal secretion.The endocrine cells in the oxyntic mucosa of the stomach are the main source of ghrelin in the body. Ghrelin has several functions, including the release of growth hormone and regulating appetite and energy metabolism.2 Stem cells rapidly differentiate into endocrine cells in the gut, over a period of just a few days.43 It is tempting to speculate that the change in diet and the consequent change in the contents of the gut lumen are responsible for the changes in the gastric endocrine cells observed here. If this assumption is proven to be true, this would open new frontiers with important clinical implications for IBS patients.The gut luminal contents, and especially the nutrients, are the main stimuli that trigger gut endocrine cells' activity.


To compare the use of a generic molecular assay to ‘standard’ investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants .VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests  and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).Sixty-five babies with suspected clinical sepsis were prospectively included. The performance indicators are presented with 95% confidence limits. For the detection of bacteria, blood culture had sensitivity of 0.57 (0.34–0.78), specificity of 0.45 (0.30–0.61); +LR of 1.05 (0.66–1.66) and—LR of 0.94 (0.52–1.7); PPV of 33.3 (18.56–50.97) and NPV of 68.97 (49.17–87.72). Serum CRP had sensitivity of 0.92 (0.64–1) and specificity of 0.36 (0.17–0.59); +LR of 1.45 (1–2.1) and-LR of 0.21 (0.03–1.5); PPV of 44.46 (26.6–66.6) and NPV of 88.9 (51.8–99.7). The universal molecular assay had sensitivity of 0.76 (0.53–0.92), specificity of 0.95 (0.85–0.99); +LR of 16.8 (4.2–66.3) and-LR of 0.25 (0.1–0.5); PPV of 88.9 (65.3–98.6) and NPV of 89.4 (76.9–96.5).In VLBW infants this universal molecular assay performed better in the diagnosis of late onset sepsis (LOS) than blood culture and CRP. Further development is required to explore and improve the performance of the assay in real-time diagnosis. Late onset infection in the preterm infant is commonly defined as a bacteraemia with an inflammatory response occurring greater than 72 hours of age. Very low birth weight (VLBW) infants are at greater risk of infection because of their developing immune system and the additonal risk factors associated with neonatal intensive care such as the insertion of central long lines –3 IncideThe current ‘gold standard’ for the diagnosis of neonatal blood stream infection is the recovery of organisms by blood culture. There are few published data on the performance of blood cultures in VLBW infants and blood cultures can yield false positive results due to contaminating bacteria from skin. In a post-mortem study involving extremely low birth weight (ELBW) infants, 56 of 111 infants were diagnosed with sepsis after death but 61% of these infants were not considered to be septic during life . In a fiBacterial detection techniques have evolved and to encompass modern molecular methods, which potentially represent a more rapid, sensitive and specific means of identifying bacteria. Molecular assays that have been studied in neonates include conventional and real time polymerase chain reaction (PCR) assays targeted at universal and specific gene sites . Only a We present a molecular method for the detection of bacteria in VLBW infants presenting with late onset infection. This method is based on reverse transcriptase and probe-based real time PCR amplification of the 16S ribosomal RNA (rRNA) transcript region of the bacterial genome. This technique has not been reported exclusively in VLBW infants before. The aim of this study was to compare the diagnostic ability of this universal assay with CRP and blood culture for the diagnosis of late onset sepsis in VLBW infants.We prospectively studied VLBW infants who had been admitted to the neonatal intensive care unit (NICU) of the Royal Jubilee Maternity Hospital, Belfast. The entry point into the study was the clinical suspicion of sepsis, when the infant was more than 48 hours old and was still less than 1500 g in weight. Suspicion of clinical sepsis was determined by the NICU team . No specific criteria were given to clinical staff as guidance. The attending medical team would us internal judgement and reasoning to determine whether an individual infant required investigation for sepsis. We excluded infants with known sepsis-like inflammatory conditions such as necrotizing enterocolitis . This was to ensure a ‘pure sepsis’ population without overlap with NEC.Clinical and laboratory information were collected from each infant at the time of sample collection. This included patient demographic information, cardiovascular, respiratory, gastrointestinal and neurobehavioral parameters. Laboratory data included total and differential white blood cell count , platelet count  and C reactive protein concentrations . Other information included the results of any radiological investigation and whether a central line was present. Written informed parental consent was obtained. Ethics approval was granted by the Office of Research Ethic Committees, Northern Ireland (07/NIR01/71).Blood was collected by venepuncture or from an indwelling vascular catheter following skin decontamination with an alcohol swab or 0.05% aqueous chlorhexidine solution. Blood sampling was undertaken at the time each infant was reviewed for possible infection. Sampling took place before the administration of antibiotics.Paediatric BACTEC bottles were used for blood cultures . A blood volume of 0.5 ml was collected in potassium EDTA-coated Vacutainer tubes  for the molecular assay. Samples were stored at 2°C until further processing. Extraction of nuclear material took place within 48 hours of collection. Total nucleic acids were extracted from clinical specimens using the QIAamp DNA Blood Mini Kit .The 16S rRNA assay was designed for the ABI 7000 sequence detection system . The reaction master mix was produced in batches, under conditions designed to reduce the potential for nucleic acid contamination. The mastermix was divided into 1.25 ml aliquots and frozen for use as required throughout the study. The assay was based on a commercial amplification ‘kit’ with the addition of manufactured primers and probes  with combined Primers and Probe ).S. aureus 16S rRNA gene. The primer and probe sequences were P891F–TGGAGCATGTGGTTTAATTCGA and P1033R TGCGGGACTTAACCCAACA—FAM Universal Probe—FAM-CAGGAGCTGACGACARCCATGCA-TAMRA.The primers and probes used were based on those as described by Yang et al (2002)  and amplT) values, i.e. the number of cycles required for the fluorescent signal to cross the detection threshold. CT values are inversely proportional to the amount of target nucleic acid in the sample. The assay had been validated and tested on a panel of gram positive and negative organisms commonly associated with late onset neonatal infection and had been shown to be capable of detection of the common expected organisms.The standard volume of master mix used in each reaction was 18μl, with 2μl of extracted clinical sample. The cycling conditions for the described primers were 50°C for 30 minutes, 95°C for 10 minutes followed by 35 cycles of 95°C for 15 seconds and completed at 60°C for one minute. These conditions amplify RNA and DNA, enabling detection of ribosomal RNA molecules, which are present in multiple copies per bacterial cell. No quantification or identification of specific bacteria was undertaken at this stage of the study. In real time PCR a positive reaction is determined by accumulation of fluorescent signal. Positive samples were quoted as cycle threshold . The lowest CWe used a non-culture based clinical indicator of sepsis in preterm infants to compare the performance of the new test. This was a combination scoring system based on a German national surveillance scoring system, The Surveillance System nosokomialer Infecktionen fur Fruhgeborene auf Intenivstationen (NEO-KISS) . This syNEO-KISS sepsis was defined in this study, as the following: The attending physician instituted antibiotic treatment for 5 days and two of the following clinical criteria were present;Fever (>38°C) and/or temperature instability with hypothermia,Tachycardia (>200/min) or bradycardia (<80/min),Capillary refill time >2 seconds,New or increased apnoea >20 seconds,Unexplained metabolic acidosis (base excess < -10),Hyperglycaemia (serum glucose > 7.5) orThere were other signs of sepsis including increasing oxygen demand, need for intubation or lethargy.In addition, one of the following laboratory criteria was required to be present;CRP > 2.0 mg/dl,Immature to total neutrophil count >0.2,Platelets <100/nl or leucocytes <5/nl (18).These criteria were applied retrospectively without knowledge of the blood culture or 16S rRNA results. A NEO-KISS septic episode was determined to have concluded once the antibiotics had stopped and provided the infant was not re-cultured within 5 days. Only the first sample in the septic episode was studied.The results are presented as sensitivity/specificity, positive and negative likelihood ratios (+/-LR) and positive/negative predictive values (PPV/NPV) for discrete data with the addition of receiver operating characteristic (ROC) curves for the continuous variables CRP and 16S rRNA. Comparison of CRP between septic and non-septic patients was undertaken with the Mann Whitney U test. Statistical analysis used SPSS 19.0  and MedCalc . The sensitivity, specificity, likelihood ratios and predictive values were calculated from the ROC analysis. These data are calculated from the optimum sensitivity and specificity (performance) for the test in question. The Medcalc software package automatically calculated optimum sensitivity and specificity using the Youden index [The study was conducted over an 18-month period beginning November 2007. In total, 86 episodes of clinical sepsis in 60 babies  were recorded and sampled. Nine babies with definite necrotizing enterocolitis (n = 9) were excluded. After excluding repeat testing, 65 samples were analyzed for 16S rRNA. The median gestational age was 26.7 weeks (Interquartile range (IQR): 26–29 weeks) with a median weight 0.98 kg (IQR: 0.77–1.19 kg) and the median age since birth at the time of entry into the study was 15 days (IQR: 6–28 days).Staphylococcus . Other microbes detected included 4 Staphylococcus aureus, 1 Streptococcus agalactiae, 1 Candida albicans and 2 unspecified mixed growths. Twelve positive blood cultures were classified as ‘NEO-KISS sepsis positive’, 24 as ‘NEO-KISS sepsis negative’ using the described scoring. The bacteria recovered in these groups are described in Thirty-six blood cultures were positive and of these 28 were positive for coagulase negative Positive and negative blood cultures are presented in The median CRP at presentation in the infants with NEO-KISS sepsis was 19.7 (IQR: 8.35–84.6) and 11.05 (IQR: 3–43.2) in the non NEO-KISS septic infants (p = 0.16). The optimum sensitivity and specificity was determined at a CRP of 5.5 mg/dl. Serum CRP had a sensitivity of 0.92 (0.64–1), specificity of 0.36 (0.17–0.59); +LR of 1.45 (1.0–2.1) and-LR of 0.21 (0.03–1.5); PPV 46.46 (26.6–66.6) and NPV 88.9 (51.8–99.7). The area under the ROC curve for CRP was 0.645 (standard error (SE) 0.1, 0.47–0.8, p = 0.13).T value was obtained. The CT values for NEO-KISS sepsis positive samples had a median of 21.97 . The universal RT-PCR 16S rRNA assay had a sensitivity of 0.76 (0.53–0.92), specificity of 0.95 (0.85–0.99), + LR of 16.8 (4.2–66.3) and-LR of 0.25 (0.1–0.5); PPV 88.9 (65.3–98.6) and NPV 89.4 (76.9–96.5). The area under the ROC curve for the molecular assay was 0.86 . Receiver operator characteristic curves are presented for the 16S rRNA molecular assay and CRP .In this study we describe the performance of a real time 16S rRNA assay for the detection of late onset sepsis in a population of VLBW infants. The diagnosis of sepsis in preterm babies is challenging. Blood cultures currently have a central role in the detection of sepsis and are the reference standard for all new detection methods. There aBlood cultures were positive in 24 babies who had negative NEO-KISS sepsis scores. These infants did not meet the criteria for sepsis. These blood cultures are considered to be false positives. The NEO-KISS score allows for diagnostic determination and comparison of the tests without bias being given to a specific method. We believe this scoring system is the most accurate means of determining the true state of sepsis given the flawed nature of all current methods of determination.Other tests, such as CRP, have limitations if taken at the time of presentation. CRP was of only moderate value in predicting sepsis . CRP rises more slowly than required for immediate diagnosis but is useful in monitoring the inflammatory response.The positive likelihood ratio (positive LR) is the ratio between the probability of a positive test result given the presence of the disease and the probability of a positive test result given the absence of the disease. In this study the positive LR of 16SrRNA testing was 16.8. This demonstrates that the use of 16S rRNA in this population significantly increases the probability of disease (‘rules in’). The positive likelihood ratios of blood culture and CRP reflect the poor performance of these tests in the VLBW and confirm the superiority of 16S RT-PCR to diagnose sepsis in these infants.A discussion of the criteria used to determine the presence of sepsis is required. The NEO-KISS is a well-established national surveillance system for nosocomial infection in German NICUs. The surveillance system was developed following an extensive period of validation period. Between 2005 and 2009 the NEO-KISS surveillance group included over 14,321 patients and collected data on neonatal infection, pneumonia and NEC  A methodOther means of determining neonatal sepsis also exist and as in the NEO-KISS system are used in population surveillance frameworks. These include definitions used in the U.K for the National Neonatal Audit Project  and in tStaphylococcus-targeted assay in a preterm, VLBW population and found a sensitivity and specificity of 0.57 and 0.95, respectively.The 16S rRNA molecular assay described in this study demonstrated better performance compared to the other methods of detection with an area under the ROC curve of 0.86 (sensitivity 0.76 and specificity 0.95). The test performance is compared to that described in a recently published systematic review of 23 studies of molecular methods in neonates (sensitivity 0.9 (0.78–0.95) and specificity 0.96 (0.94–0.99). This reAn advantage of a molecular assay is rapidity, as sufficient information is available to potentially stop antibiotics after 3 hours  from presentation. This woLimitations of this study revolve around that of 16S molecular technology and the relatively poor test sensitivity. The main disadvantage of 16S technology is the potential for reagent contamination with bacterial DNA and the painstaking methods required to prevent this. ContamiThe disappointing test sensitivity is probably related to the known difficulties in the extraction of nuclear material from gram positive bacteria . To furtThis molecular assay is more sensitive and specific than blood culture and so provides more definitive information. Smaller test volumes would reduce phlebotomy losses and so aid the preservation of circulating blood volumes in VLBW infants. This assay also has the ability to quantify the degree of bacteraemia and potentially monitor this over time and thus antibiotic response.We describe the comparative test performance of two routine and a new RT-PCR 16s based test in the diagnosis of sepsis in VLBW infants. The 16S rRNA assay demonstrated superior performance than blood culture and CRP and may provide a faster, more accurate diagnosis of sepsis in this population of infants.S1 Database(XLS)Click here for additional data file.


Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5’ untranslated region (5’UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5'UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS. Characterization of hepatitis C virus (HCV) transmission patterns remains challenging due to the long incubation periods, asymptomatic course of infection and the scarcity of samples from donor and recipient around the time of transmission , 2. In uHorizontal transmission of HCV from spouse to spouse has been shown in a number of publications –8. Such Next-generation sequencing (NGS) techniques, which allow for the evaluation of a wide spectrum of quasispecies including minor variants, is uniquely suited for the investigation of transmission . This apRecently, several studies approached in-depth analysis of hepatitis C virus transmission patterns, using single-genome or next-generation sequencing , 17. HowThe aim of the current study was to use NGS to analyze transmission and selection of HCV variants from chronically infected female spouse (donor) to her male spouse (recipient) who subsequently developed acute infection and chronic hepatitis C. We studied two different HCV genomic regions: HVR1 (hypervariable region 1) and 5’UTR (5’ untranslated region). HVR1 is a highly exposed fragment of envelope 2 glycoprotein and a major target for specific antiviral response and its variability facilitates immune evasion and reflects the immune pressure of the host . In contThe study involved serum samples from a 53-year old female (donor) who had documented chronic hepatitis C for 10 years (most likely due to iatrogenic infection) and her 58-year old male spouse who developed an acute infection evolving to chronic hepatitis (recipient). Both were infected by the same subtype 1b and samples were collected at baseline (October 2012), which was the time of acute infection in the male (month 0) and after 9 and 13 months. Some clinical and virological data on both spouses are presented in Following recommendations the male spouse was treated for 6 months with interferon alfa-2b  3 mln IU given daily for the first 4 weeks and three times per week thereafter for 20 weeks, but without success . Except Sera from 10 randomly selected, chronic hepatitis C patients were used as controls for phylogenetic and genetic distance analysis. These patients were of similar age, were infected with same HCV subtype, and were recruited at the same time as the study subjects. The study was approved by the Bioethical Committee of the Medical University of Warsaw  and all patients provided written informed consent.5′-CATTGCAGTTCAGGGCCGTGCTA-3′ (nt 1632–1610) and 5′-GGTGCTCACTGGGGAGTCCT-3′ (nt 1389–1408). Primers used for reverse transcription and first round HCV 5’UTR amplification were as follows: 5’-TGRTGCACGGTCTACGAGACCTC-3’ (nt 342–320) and 5’-RAYCACTCCCCTGTGAGGAAC-3’ (nt 33–55). Primers employed in the second round PCR contained tags recognized by GS Junior sequencing platform, standard 10-nucleotide multiplex identifiers and target-complementary sequence [5′- TCCATGGTGGGGAACTGGGC-3′ (positions 1428–1447) and 5′-TGCCAACTGCCA TTGGTGTT-3′ (nt 1603–1584). Target-complementary sequences of primers for the second round PCR for 5’UTR amplification were as follows: 5’-ACTGTCTTCACGCAGAAAGCGTC-3’ (nt 57–79) and 5’-CAAGCACCCTATCAGGCAGTACC-3’ (nt 307–285).HVR1 and 5’UTR amplifications were performed as described previously , 25. In sequence . Target-7 amplicons was subjected to emulsion PCR using GS Junior Titanium emPCR Lib-A Kit . Pyrosequencing was carried out according to the manufacturer’s protocol for amplicons using GS Junior System (454 Life Sciences).The amount of DNA equivalent to 3×10https://www1.ethz.ch/bsse/cbg/software/shorah) [https://github.com/ozagordi/LocalVariants/blob/master/src/LStructure.py).Sequencing errors  were corrected and haplotypes inferred using the program diri_sampler from the Shorah software (/shorah) . HaplotyMolecular Evolutionary Genetics Analysis), version 5.0 (http://www.megasoftware.net/) [http://www.ub.edu/dnasp/) and MEGA 5.0. Sequence similarity was compared using Clustal 2.1 Percent Identity Matrix  [http://weblogo.berkeley.edu/) [Additionally, although error correction allows for a reliable estimation of variants at a lower frequency, we applied a 1.5% frequency cut-off to improve the specificity of our analysis . As demore.net/) . Phylogere.net/)  using ME/omega/) . Amino aey.edu/) .Altogether, 71,923 reads were obtained from 6 analyzed samples, 13,410 for HVR1 and 58,513 for 5’UTR region. After reconstruction, 7 to 49 variants were inferred per sample for HVR1 and from 6 to 10 for 5’UTR. Application of the cut-off lowered the number of inferred variants as there were now 4 to 20 variants per sample for the HVR1 and from 1 to 2 for the 5’UTR. For the unrelated 10 chronic hepatitis C patients, 33,810 HVR1 reads were obtained (4 to 16 variants per sample).No identical HVR1 variants were identified to be present in both spouses , but a minor baseline donor variant (1.7% frequency) was found to be closely related to all recipient variants present at baseline (97.1%–98.3% sequence similarity). Furthermore, recipient consensus sequence at baseline differed only by two nucleotide substitutions and one insertion when compared to the putative infecting variant of 1.7% frequency. Only one amino acid difference was present between the infecting minor variant and the recipient major variant .When employing the 1.5% cut-off, the number of variants in the male recipient increased from four (baseline) to five at month 9 and to seven at month 13. At baseline, the HVR1 population was composed of one predominant variant (62.5%), one variant of 24.7% frequency and two minor (defined as <10% frequency) variants. Sequence similarity of the predominant variant to other baseline variants was from 97.7%, to 98.9%. None of the variants present in the initial sample were found in the two follow-up samples, and only one variant was present both at 9 and 13 months . At 9 months, the frequency of predominant variant was 76.0% but at month 13 the population became more dispersed, with two most abundant variants constituting 52.8% and 23.0% of the population, and five minor variants. Nucleotide diversities per site within HVR1 populations are shown in Without the cut-off, the number of variants in the recipient increased from seven (baseline) to 11 at month 9 and 17 at month 13. At baseline, HVR1 population was composed of one predominant variant (62.5%), one variant of 24.7% frequency and five minor variants (defined as <10% frequency). Sequence similarity of the predominant variant to other baseline variants ranged from 97.1%, to 99.4%. None of the variants present in the initial sample was found in the follow-up samples, and only one variant was present both at month 9 and 13 . At 9 months the frequency of predominant variant was 76.0% but at month 13 the population became more dispersed, with the two most abundant variants constituting 52.8% and 23.0% of the population, and 15 minor variants. These populations gave rise to a steep curve on the cumulative distribution plots . Nucleotvs month 9), 0.048 (month 9 vs month 13) and 0.050 (baseline vs month 13).The number of variants above the cut-off value of 1.5% increased in the donor during the follow-up from 14 (baseline) to 19 (month 9) and 20 (month 13). The baseline HCV population consisted of one predominant variant (33.2%), followed by a variant representing 13.6% and 12 minor variants. At 9 months, the contribution of minor variants increased to 17, while the frequency of the major variant declined to 20.9%. At 13 month, the population was even more dispersed with the most abundant variant constituting only 12.8% of the population and 18 different variants of lower frequency. Seven of the baseline variants were also present at month 9 (36.8% of population) and six baseline variants were still detectable at month 13 sample (30.0% of variants). The frequency of variants found in at least two donor samples ranged from 1.5% to 33.2%. Nucleotide diversity per site within HVR1 in the donor are presented in Genetic distance between donor’s and recipient’s baseline populations (interhost) was 0.054, remained the same at month 9 and increased to 0.057 at month 13.vs month 9), 0.041 (month 9 vs month 13) and 0.040 (baseline vs month 13).When conducting the analysis without the cut-off, the number of variants in the donor fluctuated during the follow-up from 41 (baseline) to 38 (month 9) and 49 (month 13). The baseline HCV population consisted of one predominant variant (33.2%), followed by a variant representing 13.6% and 39 minor variants (< 10% each). At 9 months, the number of minor variants was 37, while the proportion of the major variant decreased to 20.9%. At 13 months the population was even more dispersed with the most abundant variant constituting only 12.8% of the population and the presence of 48 different minor variants. These populations resulted in a flatter curves on the cumulative distribution plots when compared to the recipient spouse . EighteeGenetic distance between donor’s and recipient’s populations (interhost) were 0.037 at baseline, 0.041 at 9 months, and increased to 0.053 at 13 months.When conducting the analysis with 1.5% cut-off, one predominant HCV variant, which was identical to one of donor baseline variants, was present in all samples .Conducting the analysis without the cut-off did not change the frequency of the predominant variant, but there were now nine minor variants at baseline, five at 9 months and six at 13 months. Their frequency ranged from 0.1% to 1.4% of the total population.Two variants of similar frequency (46.5% and 51.0%) were present in the donor spouse serum at baseline and during the follow-up period (49.4% and 47.2% at month 9); (54.4% and 41.7% at month 13). Interhost genetic distance between baseline populations was 0.002, and remained the same at month 9 and 13.When conducting the analysis without the cut-off, the above two dominant variants were present at identical frequency at baseline (46.5% and 51.0%), at 9 months (49.4% and 47.2%), and at 13 months (54.4% and 41.7%). However, there were now also minority variants present: seven at baseline, seven at 9 months, and six at 13 months, ranging in frequence from 0.1% to 1.1%. Genetic distance between baseline populations (interhost) was 0.007, 0.006 at month 9 and 0.004 at month 13.Minor variants in both spouses mostly differed by deletions and/or insertions in the homopolymeric cytosine region within 5’UTR (positions 127–129 of the AJ242654 reference genome) and at positions 66–71 (AJ242654 reference genome).−2 substitutions per site per year, [Phylogenetic analysis revealed that HVR1 recipient variants were highly similar to one minor donor variant of 1.7% frequency . Based oer year, ), molecuBaseline viral variants from both spouses were compared to sequences from 10 non-related chronic hepatitis C subjects . As seenClose to the C-terminus of HVR1 amino acid sequence, two overlapping epitopes for neutralizing antibodies were described, encompassing amino acid positions 394–404 (Epitope 1) and 397–407 (Epitope 2) , 34. In In the donor, 40.5% of positions were homogenous. Nine positions were heterogeneous at baseline , eight at month 9  and eight at month 13 . Over the time of follow up, seven positions were unstable .The increased risk of transmission from HCV-positive patients to household members, including siblings, parents, offspring as well as homo- and heterosexual partners, has been well documented –40. It iThe actual prevalence of HCV among family members of infected patients was found to be very diverse ranging from 1.3% to 36.4% and depends on the studied population , 42. In In our study phylogenetic analysis has demonstrated much more similarity between HCV strains in spouses than between unrelated subjects, supporting the occurence of intrafamilial transmission . To the Importantly, this minor donor variant would have been overlooked with the use of classical Sanger sequencing approach as it would require sequencing of 175 clones in order to detect a variant of 1.7% frequency with 95% probability. This observation is of special importance for transmission studies, because relationship traits between viral populations may be rapidly lost, especially when using Sanger-based techniques with the lowest detectable variants typically having ≥ 10–20% of frequency , 54, 55.It must be noted that the frequency of the variant putatively transmitted to a new host (1.7%) was close to the applied 1.5% cut-off. This particular cut-off value corresponds to the error rate of the amplification and sequencing procedures determined previously by analysing of cloned HVR1 . Of noteThe baseline HVR1 population found in the recipient was very narrow which is compatible with the bottleneck phenomenon and is consistent with some other studies in which only a single variant established infection , 18, 19.Despite the high sensitivity of NGS, it is impossible to determine the exact transmission route, but both sexual and household transmissions seem plausible. We collected cervical-vaginal lavage (CVL) to verify the presence of viral RNA, but neither 5’UTR nor HVR1 could be amplified, which is in line with some previous finding of low prevalence of HCV RNA in genital tract of HCV-monoinfected women . InteresMolecular evolution of HCV HVR1 was strikingly different in the chronically infected donor and acutely infected recipient. Recipient's population showed increased complexity (number of variants) over time as well as change in variants composition, with only a few variants dominating the population. This change, called selective sweep, could be the result of selection pressures and is common for such pathogens as influenza , HCV 6363, 64 anvs 9 months). While interferon activates cellular rather than humoral response and thus would be expected to have a limited impact on the variability of the HVR1, rapid evolution of this region during therapy is common and predictive with respect to outcome [After an unsuccessful antiviral treatment was attempted, the composition of variants was entirely changed (baseline  outcome –69. HoweIn contrast, the majority of female spouse variants were present in subsequent samples, most of them at a higher frequency. This could be due to virus adaptation and/or limited immune pressure possibly due to immune exhaustion, which is common in chronic HCV infection . SimilarThe 5’UTR sequence remained relatively stable, probably due to lack of selective pressures.In conclusion, it seems that intrafamilial HCV transmission may be established by a very minor variant and thus the investigation of this phenomenon requires high-sensitivity assays, such as NGS.


Concentrations of glycine (Gly) in embryo culture media are often lower (~0.1 mM) than those in oviductal or uterine fluids (≥1.2 mM). The objective of this study was to determine direct and osmolarity-dependent effects of physiological concentrations of Gly on blastocyst formation and hatching, cell allocation to the trophectoderm (TE) and inner cell mass (ICM), and metabolic activity of bovine embryos. In experiment 1, zygotes were cultured with 100 or 120 mM NaCl and 0 or 1 mM Gly for the first 72 h of culture. Blastocyst formation and hatching were improved (P<0.05) when embryos were cultured with 100 compared to 120 mM NaCl. Inclusion of 1 mM Gly improved (P<0.05) blastocyst formation compared to 0 mM Gly, but this effect was only significant (P<0.05) for embryos cultured with 120 mM NaCl, suggesting bovine embryos can utilize Gly as an osmolyte. In experiment 2, embryos were cultured with 0.1, 1.1, 2.1, or 4.1 mM Gly (100 mM NaCl) for the final 96 h of culture. Blastocyst development was not affected (P>0.05) by Gly, but hatching  was improved (P<0.05) when embryos were cultured with 1.1 (31.4%) or 2.1 (29.4%) mM Gly. Blastocyst, TE, and ICM cell numbers were not affected (P>0.05) by Gly in either experiment. Blastocysts produced alanine, glutamine, pyruvate, and urea and consumed aspartate, but this metabolic profile was not affected (P>0.05) by Gly. In conclusion, Gly (1.0 mM) improves the development of both early and late stage embryos, but beneficial effects are more pronounced for early embryos exposed to elevated osmolarity. Amino acids are present in oviductal and uterine fluids –3 and seA comparison of the formulations of embryo culture media and reports on the composition of oviductal and uterine fluids indicates that cultured embryos are being exposed to non-physiological concentrations of some amino acids. Glycine (Gly) is present at ~0.05 to 0.1 mM in many embryo culture media based on the composition of Minimum Essential Medium ,16,17,20The most widely studied role of Gly during preimplantation development is its role in the maintenance of cell volume and intracellular osmolarity in hypertonic environments . TranspoSeveral studies have confirmed the importance of Gly for development of bovine embryos by showing improved development when this amino acid is added to the culture medium –33. Howe2 in air and maintained at ~38.5°C in a portable incubator during overnight shipment to our laboratory.Bovine cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries by a commercial supplier . Unless specified otherwise, all reagents were purchased from Sigma-Aldrich . Groups of 50 COCs were matured in 2 ml of a defined maturation medium  containi6 spermatozoa/ml. Gametes were co-incubated in 7.5% CO2 in air at 38.7°C for 20–22 h. This gas concentration is increased to compensate for the elevation of our laboratory (~1830 m above sea level) and is approximately equal to 6.0% CO2 at sea level (media pH = 7.2 to 7.3).Twenty two to twenty three hours after COCs were placed into maturation medium, COCs were recovered from the shipping tubes, washed, and transferred to 45 μl drops  of fertilization medium  containi2 and 6.5% O2  at 38.7°C.Presumptive zygotes were removed from fertilization drops and denuded of remaining cumulus cells and loosely bound spermatozoa by shaking on a vortex mixer for 2.5 min. In experiment 1, zygotes were randomly allocated to a medium designed for cleavage stage bovine embryos bOEC1,  in a 2x2Hatching and fully-hatched blastocysts were fixed for 20 min in 4% paraformaldehyde  and then stored in PBS with 0.5% BSA  until staining. Blastocysts were washed three times in PBS with 0.1% PVP and 0.1% Triton X-100 (TX100) and then permeabilized in PBS with 1.0% TX100 (30 min). After blocking (2 h) in PBS with 0.1% TX100, 0.1 M glycine, 0.5% BSA, and 10% (v/v) horse serum, blastocysts were incubated with primary antibodies  for SOX2  and CDX2  ,35. Foll2, 6.5% O2, 38.7°C) for an additional 48 h  and evaluated for blastocyst formation and hatching on day 7 (192 h post-insemination). Medium (10 μl) was collected from wells that contained embryos that developed to the hatching or fully hatched blastocyst stage. Control medium was also collected from wells in the same dish that did not contain an embryo. Immediately after collection, medium samples were frozen in liquid nitrogen and stored at -80°C until analysis.After 48 h of culture in bOEC2 , embryos were transferred to fresh medium in wells of a 60-well culture dish  covered with Ovoil. Gly concentrations in the bOEC2 were identical to those used for group culture during the previous 48 h . Embryos were then cultured (7.5% COm/z were scanned at five scans per second after electron impact ionization.Concentrations of nutrients were determined using gas chromatography (GC) and mass spectrometry (MS) as previously described . Aliquotm/z) was generated using XCMS software in R [Raw data files were converted to.cdf format of relative quantitation. A matrix of molecular features defined by retention time and mass  procedure in SAS. The proportions of embryos developing to the blastocyst or hatching blastocyst stage was based on the number of zygotes placed into culture (experiment 1) or the number of embryos placed into the second stage culture medium . Each embryo was scored as a 1 or 0 depending on whether or not it achieved the desired stage of development  and analyzed using a binomial error distribution and a probit link function. In experiment 1, the main effects of the concentrations of NaCl and Gly and the interaction between these factors were included as fixed factors for analysis of embryonic development. In experiment 2, the concentration of Gly  was the only fixed factor for analysis of both embryonic development and blastocyst cell numbers. Replicate was included as a random factor for analysis of embryonic development.Blastocyst cell numbers were analyzed using the mixed model procedures in SAS. In experiment 1, there were not enough hatching or fully-hatched blastocysts from the 120 mM NaCl treatments for inclusion in the analysis of blastocyst cell numbers, so the concentration of Gly was the only fixed factor. The concentration of Gly was also the only fixed factor in the second experiment.For blastocyst metabolism, two analyses were performed in the MIXED procedure of SAS. In the first, metabolite concentrations in medium that contained an embryo were compared to those in control medium that did not contain an embryo to determine if each metabolite was significantly consumed (lower than the control) or produced (higher than the control) by the embryo during culture. Comparisons were only made between treatment and the control. In the second, metabolite concentrations for the treatments were normalized to the concentrations in the control medium (no embryo) to determine the fold-change of each metabolite relative to the control, which facilitated comparisons between treatments.In all analyses, pairwise comparisons were made using Fisher’s protected least significant difference test and P<0.05 was considered a significant difference. All means are presented ± SEM.Four replicates were performed using a total of 1357 presumptive zygotes (337–342 per treatment). The proportion of zygotes that cleaved (≥71.1%) was not affected (P>0.05) by the concentration of NaCl, Gly, or the interaction between these two factors . HoweverEmbryos that were cultured for the first 72 hours with 100 mM NaCl and 1 mM Gly produced blastocysts that tended to have more TE cells  and total  cells than embryos cultured with 100 mM NaCl and 0 mM Gly  . In the Media from 6 blastocysts per treatment was analyzed for 20 metabolites. Concentrations of citrate, fructose, glycine, isoleucine, lactate, leucine, lysine, methionine, inositol, ornithine, phenylalanine, proline, pyruvate, serine, threonine, and urea in the medium were not altered (P>0.05) by the embryo regardless of treatment. In all treatments, blastocysts consumed (P<0.05) aspartate (Asp) and secreted (P<0.05) alanine (Ala) and pyruvate into the medium . BlastocFour replicates were performed using a total of 603 embryos (>4-cell) with 148 to 153 cultured per treatment. The proportion of embryos developing to the blastocyst stage was not affected (P>0.05) by the concentration of Gly in the medium (43.9 ± 4.1% to 51.6 ± 4.1%) . HoweverThe concentration of Gly in the medium did not affect (P>0.05) the number of TE (135.3 ± 9.1 to 147.5 ± 13.3) or ICM (37.0 ± 3.8 to 45.5 ± 5.6) cells, or total the total number of cells (173.3 ± 13.2 to 193.4 ± 16.5) in the resulting blastocysts .Medium from 5 to 6 blastocysts per treatment was analyzed for 19 metabolites. Concentrations of isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, and tyrosine in the medium were not significantly altered (P>0.05) by the embryo regardless of treatment. Blastocysts from all treatments consumed (P<0.05) Asp . BlastocThis is the first study to investigate the stage-specific effects of physiological concentrations of Gly on the development of bovine embryos. The results indicate beneficial effects of Gly on embryonic development when present in both the early (zygote to 8-cell) or late (8-cell to blastocyst) stages of culture. Glycine could partially mitigate the negative effects of elevated NaCl/osmolarity, suggesting bovine embryos can use this amino acid as an osmolyte. Despite the beneficial effects on embryonic development, Gly had surprisingly little effect on the metabolic profile of blastocysts. However, several notable aspects of embryo metabolism were evident, providing new insight on the physiology of the bovine embryo.Increased NaCl and medium osmolarity dramatically inhibited the development of bovine embryos, which is consistent with data from other species ,39,40 anBeneficial effects of increasing the concentration of Gly in the medium during the entire preimplantation culture period have been previously reported, but the concentrations of Gly required to elicit a beneficial effect on embryonic development (5 to 10 mM) in these studies –33 were Although supplementing the first or second step culture medium with Gly improved embryonic development, we did not observe significant consumption of this amino acid by the blastocysts. While this was unexpected, it is consistent with other reports of amino acid metabolism by bovine blastocysts in which limited (nonsignificant) consumption  or produ+/NADH balance and glycolytic activity within the embryo, due to its role in the Malate-Aspartate Shuttle (MAS) [The only nutrient that was consumed in significant quantities by blastocysts in all treatments in both experiments was Asp. Similar observations have been reported in studies of bovine ,24,45,48le (MAS) ,54. ConsWhile nutrient consumption was largely limited to Asp, blastocysts produced significant quantities of Ala and Gln in both experiments, and a small amount of urea in the second. Production of Ala has been reported in multiple studies of amino acid metabolism by bovine ,45,48,49Metabolic profiles also indicated significant production of pyruvate. Although this was consistent between both experiments, as well as our previous work , it is i+/NADH ratio in the cytoplasm [The results of the present study highlight the importance of Gly on bovine embryo development, and emphasize the importance of evaluating individual amino acids during specific periods of preimplantation development. Not only did an increased (1.0 mM) concentration of Gly improve blastocysts formation and hatching, but many of the changes observed in the composition of the culture medium following embryo culture suggest active amino acid metabolism. Three  of the four substrates that were produced by the embryos are involved in detoxification of ammonium that is produced when amino acids are used as a source of carbon ,8. Blastytoplasm ,54. Whilytoplasm ,46,64,65


Enterococcus faecalis using quantitative real-time polymerase chain reaction (qPCR) analysis and also to compare their cytocompatibility on L929 murine fibroblasts using Mossman’s tetrazolium toxicity (MTT) assay. The purpose of this study was to compare the antimicrobial efficacy of sodium hypochlorite (SH) and calcium hypochlorite (CH) against E. faecalis. Then, the root canals of 50 mature extracted human mandibular premolars were contaminated with E. faecalis and were randomly divided into three groups according to the irrigant used (n=20). Canals were irrigated with SH in group I (n=20) and CH in group II (n=20) at their obtained MIC. In group III (n=10), sterile saline was used. Microbial sampling was performed before and after biomechanical preparation. Quantitative PCR was used to quantify E. faecalis in the root canal samples. For cytocompatibility assessment, L929 murine fibroblasts were exposed to various concentrations of the irrigants. A broth micro-dilution susceptibility test was used to determine the minimum inhibitory concentration (MIC) of each irrigant against E. faecalis being 0.5% and 5%, respectively). However, the reduction in the normal saline group was not significant (P=0.203). In addition, 5% CH was more effective than 0.5% SH (P=0.006) in eliminating E. faecalis. Among the different concentrations of tested irrigants, 0.5% CH and 5% SH showed the least and the most cytotoxicity, respectively (P<0.001). The cytotoxicity of 5% CH and 0.5% SH was similar (P=0.99), and lower than 2.5% SH (P<0.001). Irrigation with test materials resulted in significant reduction in colony forming units (CFU) in post-instrumentation samples (with the MIC values of SH and CH against  CH at an MIC of 5% was effective in eliminating E. faecalis in planktonic state and also its biofilm and exhibited comparable cytocompatibility to that of 0.5% SH. The major cause of apical periodontitis is microbial infection of the root canal system . The hosEnterococcus faecalis  is considered as the most resistant to antimicrobial agents  [The primers were designed by allele ID software  to amplify a 138-base pair fragment of the gelatinase (BLASTN)] .E. faecalis total DNA template) were prepared in duplicate alongside experimental total DNA sample to normalize any background signal obtained from the following amplification. To estimate the number of E. faecalis and the total number of bacteria in the samples, DNA from E. faecalis was purified within a concentration range of 101 to 106 genomic DNA copies per reaction and then the linearity and efficiency of the PCR assay were determined. Each sample dilution (1 µL) was added to five replicas in PCR tubes and the real-time PCR machine was started. The efficiency of the gelE-based real-time PCR assay was calculated by the following formula: Efficiency=[10(−1/slope)]-1. Negative PCR controls (no DNA template) and negative qPCR amplification controls  analysis of the final PCR product was performed from 50 to 90°C at 1°C intervals. An automatic threshold setting of 0.2 Nano grams was used for all samples. A positive reaction and a non-template reaction were included in all experiments.Real-time PCR and data analysis were performed in iCycler iQ real-time detection system  using 2×SYBR premix Ex TaqTM II . The PCR conditions were as follows: 956 to 2 CFU/mL of the sample. The DNA was extracted, quantified, and amplified as described above and then the standard curves were constructed. The correlation coefficient (R2) and the efficiency of the amplification were calculated  solution  was added to each well and incubated in a humidified atmosphere for 3 h. Then, 100 μL of DMSO was added to each well and the culture plate was vibrated for 10 min. ELISA microplate reader  was used to determine the light absorbance of the experimental samples, culture medium (negative control), and 35% H2O2 (positive control). All tests were performed in three replicates and the absorbance values were read three times. The percentage of the mean optical density (OD) values of the experimental solutions relative to the OD value of the negative control was expressed as the mean cell viability.In order to compare the cytotoxicity of 0.5% and 5% CH and 0.5%, 2.5%, and 5% SH in L929 murine fibroblasts, an MTT colorimetric assay was used. Aliquots of 100 μL solution of RPMI 1640 medium comprising 1×10Statistical analysisThe Kruskal-Wallis and Mann-Whitney U tests were used to compare the CFU counts in S1 or S2 samples among the groups. The Wilcoxon signed-ranks test was also used to compare the CFU counts before and after chemomechanical preparations in each group. One-way analysis of variance (ANOVA) and Tukey’s tests were used to evaluate the differences in the mean cell viability values. The level of significance was set at 0.05.E. faecalis were 0.5% and 5%, respectively. The pH values of the experimental solutions were 11.57, 11.98, and 7.39 for 5% SH, 0.5% CH and 0.9% sterile saline, respectively.The MIC values for SH and CH against P<0.001), but no difference was observed in control group (P=0.203). +SD) CFU counts for S1 and S2 samples in each group. Statistical analyses revealed no significant differences among the groups in S1 samples (P=0.768), but the difference was significant among the groups in S2 samples (P<0.001). Moreover, 5% CH and 0.5% SH solutions were more efficient than normal saline in reducing the CFU counts in S2 samples (P<0.001). It was showed that 5% CH was significantly more effective than 0.5% SH in CFU reduction (P=0.006).Regarding the CFU counts, the Wilcoxon signed-rank test showed a significant difference between the S1 and S2 samples in groups I and II . Tukey’s post hoc test showed 0.5% CH and 5% SH to be the least and the most cytotoxic solutions (P<0.001). Furthermore, 5% CH and 0.5% SH had similar cytotoxicity (P=0.99) which was less than that of 2.5% SH (P<0.001). The results are summarized in The 35% Het al. [et al. [Despite the advances in the techniques employed in root canal treatment, more than 35% of the root canal surfaces may be left untouched by the instruments during root canal preparation . Therefoet al.  found th [et al.  tested t [et al. -17, 29. Availability of more chlorine in CH solution compared to SH predicts a promising antimicrobial activity of this material for endodontic purposes; however its cytotoxicity should also be considered. This solution can be prepared in different concentrations by mixing the powder of CH with distilled water. Compared to SH, CH is rather more stable and easily storable . Therefot al. [E. faecalis. They found no significant antibacterial differences between these two irrigants when no ultrasonic agitation was applied. However the authors did not present any reason selecting 2.5% concentration of this material. Moreover, no previous report was present in literature regarding its level of cytotoxicity. Thus, in the first phase of our experiment we determined the MICs of our experimental solutions to select their optimal concentration to be used in the root canals.The present study was initiated based on the acceptable results of a previous work on tissue-dissolving capacity of this material compared to SH . After 6t al.  comparedE. faecalis and exhibited comparable cytocompatibility to that of 0.5% SH. It was also showed that the pH value of 5% CH was more than 0.5% SH and this might be a reason for its higher antibacterial activity. A previous investigation also revealed that the alkaline property of alkaline solutions of hypochlorite can improve the bactericidal efficacy against the anaerobic bacteria [E. faecalis to CH at pH 11.1 for 30 min results in 0.4% cell survival and a relatively small increase in alkalinity (pH 11.5) which can per se increase the level of antibacterial activity [It was found that CH solution at its MIC concentration was more efficient than 0.5% SH in elimination of planktonic and biofilm forms of bacteria . In addibacteria . It alsoactivity . Furtheractivity .et al. [For contamination of the root canals, the methodology presented by Camara et al.  was emplIn the present investigation, a culture-independent molecular microbiology method was employed. The advantage of this method is its higher sensitivity which potentially targets culturable viable cells and viable but nonculturable cells . Also qPAlthough antimicrobial effectiveness is the most important property required for an irrigation solution , 35, thein vitro studies, the findings of the current study remain to be clinically confirmed. Further studies on other microorganisms other than E. faecalis can also be helpful to justify the antimicrobial activity of CH. Besides, studies on biocompatibility of this material in animal models can be recommended for future investigations.It should be emphasized that, as with most E. faecalis biofilms.The present study provided new data on biological properties of calcium hypochlorite as an endodontic irrigant. The results of this study indicated that 5% calcium hypochlorite had similar cytotoxicity to 0.5% sodium hypochlorite and was more effective than NaOCl in eliminating


Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a novel therapy for severe respiratory failure (SRF). Its Introduction has reduced mortality; however, patients require substantial blood product support and between 10 and 20% of cases develop a life-threatening haemorrhage.We contacted 336 practitioners at 135 centres, examining their haematological management.9/l whilst 21% maintained the platelet count >100 × 109/l. Scenario 2 described a patient with SRF secondary to a hospital-acquired pneumonia. The patient developed a haemothorax, with persistent blood loss of 200 ml/hour. Practitioners targeted a higher haemoglobin concentration of 100 g/l and targeted a higher platelet count of >100 × 109/l when compared with the patient in scenario 1, neither of these differences was statistically significant. Seventy-one per cent stated they would manage the patient off anticoagulation. There was no agreement as to the length of time off anticoagulation; 26% restarted anticoagulation in <12 hours, compared with 22% who advised no anticoagulation for >5 days. Scenario 3 examined the management of an incidental intracranial haemorrhage. There was a lack of consensus regarding the duration off anticoagulation; 14% of responders held anticoagulation for less than 12 hours whilst 37% held anticoagulation for >5 days and tranexamic was considered useful by 25%.In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Scenario 1 was based on a patient with H1N1. Practitioners targeted a haemoglobin (Hb) of 80 to 100 g/l; however, 20% targeted a Hb outside this range; 38% favouring a transfusion threshold of <80 g/l when the patient was improving compared with 32% when the patient had just started on ECMO. Seventeen per cent of practitioners transfused platelets when the count was <30 × 10There was wide variation in the use of blood products and the intensity of anticoagulation. This is not surprising given the current lack of evidence. Further work is required to provide a standardised approach.