A); 0.05% potassium sorbate+0.05% sodium benzoate+1% citric acid (B); 0.1% potassium sorbate+0.1% sodium benzoate+1% citric acid (C); 0.2% potassium sorbate+0.2% sodium benzoate+1% citric acid (D); 1% citric acid (E). Samples were inoculated with Escherichia coli and Staphylococcus aureus at levels of 5.2 x 109 cfu/100g and 1.5 x 109 cfu/100g, respectively, before being evaluated during storage for 10 weeks at prevailing ambient temperature (15-25°C) and refrigeration temperature (4°C). Total aerobic counts, yeasts, and molds were also evaluated. E. coli and S. aureus counts declined significantly (p<0.05) by 4 log cycles in all puree treatments except for control and puree with only citric acid. Total viable count, yeasts, and molds were completely inhibited except for puree with only citric acid. Combination of chemical preservatives and acidification is effective in inhibiting pathogens and spoilage microorganisms in sweet potato puree.Orange Fleshed Sweet Potato (OFSP) puree, a versatile food ingredient, is highly perishable limiting its use in resource constrained environments. It is therefore important to develop shelf-stable puree. A challenge test study was carried out to determine the effect of combinations of chemical preservatives and acidification on microbial growth in stored puree. Puree was prepared and treated as follows: control ( Ipomoea batatas) is an important crop for food security and income generation in Sub-Saharan Africa (SSA) [β-carotene, an important pro-vitamin A carotenoid [Sweet potato (ca (SSA) . Sweet protenoid . Biofortrotenoid  that canrotenoid .mandazi), chapatti, and porridges for children. OFSP puree is being used as a partial substitute for wheat flour in bakery products in Rwanda and Kenya. There are several economic advantages of using OFSP puree compared to OFSP flour. According to research, 1.25 kg of OFSP fresh roots makes 1 kg of puree while 4-5 kg of OFSP fresh roots is needed to make 1 kg of flour [In east Africa, OFSP roots are processed into puree (boiled and mashed) that is used at household level to make food products such as fried doughnuts  also increases the acidity of the food product thus increasing the effectiveness of sodium benzoate and potassium sorbate since a greater proportion of the acids is in undissociated form . Therefo S. aureus and E. coli in salad vegetables have been examined [ S. aureus could take place during postprocess handling of the puree by operatives [ E. coli through the use of nonpotable water for cleaning surfaces and equipment [ E. coli has been known to survive and persist on surfaces for months [The potential growth of spoilage and pathogenic microorganisms in OFSP puree could affect its quality and shelf-life, as well as its safety. In order to determine the growth of pathogens in OFSP puree, a microbial challenge test (MCT) is important. This involves the inoculation of a food product with pathogenic microorganisms, storing the food under controlled conditions while evaluating the growth potential of the pathogens in the food product over time . MCT is examined . One imperatives . This paquipment . E. colir months . These a S. aureus and E. coli in stored OFSP puree with preservatives. Conducting the challenge test in OFSP puree will help in establishing the ability of potassium sorbate, sodium benzoate, and citric acid to inhibit the growth of S. aureus, E.coli, total counts, yeasts, and molds in OFSP puree held under ambient (15-25°C) and refrigeration (4°C) conditions and therefore facilitate more extensive use of the puree. Results from this study will determine the feasibility of developing shelf-stable OFSP puree which can be stored without refrigeration. Shelf-stable OFSP puree will bridge the OFSP puree shortage during off seasons and will also help reduce postharvest losses for OFSP farmers.Therefore, the current study was designed to examine survival and growth potential of E. coli counts, S. aureus counts, total aerobic counts, and yeast and mold counts.The current study employed an experimental study design by preparing orange fleshed sweet potato (OFSP) puree and carrying out microbial evaluation. The experimental study design included two independent variables: use of different combinations of preservatives and two different storage temperatures. The dependent variables wereAbout 25 kg of root samples was randomly collected from a pool of five batches from farmers in Homabay-Kenya. Kabode variety which is the most adopted and widely grown OFSP variety was used for the current study. The processed puree was packaged in 5-kilogram polyethylene bags of 300 micron and vacuum sealed before being transported overnight to the Department of Food Science, Nutrition and Technology (University of Nairobi) laboratory for evaluation.  E. coli and S. aureus and then microwaved and assessed again. The initial enumeration of E. coli and S. aureus in the puree was carried out as described in previous studies [−6. A volume of 0.1 mL from each dilution was spread in triplicate onto Brilliance E. coli/Coliform agar  and incubated at 37°C for 24 hours for enumeration of E. coli. Similarly, 0.1 mL of each dilution was spread in triplicate onto Baird parker agar  and incubated at 37°C for 48 hours for the enumeration of S. aureus. Enumeration was done for plates with 30-300 colonies. All microbial counts were expressed as mean base-10 logarithms of colony forming units per gram (log cfu/g). Data points were expressed as means from the triplicate analysis. The results indicated high levels of E. coli and S. aureus in puree before treatment with preservatives. E. coli and S. aureus were not detected after microwaving OFSP puree as shown in  E. coli and S. aureus into the puree.Before conducting the challenge test, OFSP puree was assessed microbiologically for studies . Puree sEscherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 pellets were obtained from the American Type Culture Collection . The pellets were activated by suspending a single pellet of each microorganism in phosphate buffer (0.1 M) and incubating it at 38°C for 30 minutes. From the buffer, 1mL was transferred to nutrient broth and incubated at 35°C for 24 hours to allow for growth of the bacteria. The levels of inoculum obtained after plating were 5.2x109 cfu/mL for E. coli and 1.5x109 cfu/mL of S. aureus. The inoculum was then stored at -80°C to avoid changes that may affect growth [t growth .OFSP puree was first sterilized for 3 minutes in a microwave. OFSP puree samples (100 g) were then dosed with combinations of selected chemical preservatives as shown in μL of bacterial suspension containing 5.2 x 109 cfu of E. coli and 1.5 x 109 cfu of S. aureus resulting in a load of 5.2x107 cfu (7.8 log cfu) of E. coli/g of puree and 1.5x107 cfu (7.2 log cfu) of S. aureus/g of puree and vacuum sealed. Some inoculated samples were incubated at ambient (15-25°C) and others at refrigeration temperatures (4°C) with enumeration of bacterial load at weekly intervals. Serial dilutions of all samples were prepared up to 10−6 and each dilution was plated in triplicate for the different tested microorganisms. The experiments were performed independently three times.Puree samples (100 g) treated with preservative combinations and that without preservatives were inoculated with 1000 −7. TVC was determined by transferring 1 mL of each sample dilution to sterile Petri dishes in triplicate to which approximately 20 mL of Plate Count Agar  was added. The plates were swirled and allowed to solidify before being incubated at 30°C for 72 hours [OFSP puree was prepared as shown in 72 hours . Yeasts 72 hours . EnumeraOne gram of OFSP puree sample was homogenized in 1mL of distilled water in a test tube. The pH values of the samples were measured using pH meter  by immersing the electrode directly into the sample in the test tube. Before the measurements, pH meter was calibrated using pH 4.0 and 7.0 buffers.Experiments were carried out in triplicate and quality control measures were taken into account. Data was analyzed by analysis of variance (ANOVA) using SPSS software (Version 20.0 SPSS Inc). Tukey test was used to determine the significant difference of mean values. The significance level was expressed at 5 % level. Microsoft Excel was used to plot line graphs.The initial pH of OFSP puree was 5.23 before treatment with preservatives. Addition of different combinations of sodium benzoate and potassium sorbate and citric acid led to a decline in pH of the puree kept at ambient (15-25°C) and refrigeration (4°C) conditions as shown in Immediately after treatment of puree with preservatives, the highest pH values of 5.19 and 5.18 were obtained in the control sample at ambient and refrigeration temperatures, respectively, while least values of 4.60 and 4.61 were recorded in the sample with 1 % citric acid at ambient and refrigeration temperatures, respectively. At the end of the storage period (10 weeks), the highest pH values of 4.99 and 4.63 were recorded in the control sample, while least values of 3.94 and 3.98 were obtained in samples with 1 % citric acid at ambient and refrigeration temperatures, respectively. E. coli in OFSP puree treated with a combination of selected chemical preservatives and that without preservatives and stored at ambient (15-25°C) and refrigeration temperatures (4°C) for 10 weeks. E. coli counts increased significantly after inoculation by 2 logs with subsequent increase in storage from 7 log cfu/g to 9 log cfu/g. All combinations of potassium sorbate, sodium benzoate, and 1 % citric acid led to a significant (p<0.05) 4-log reduction in E. coli counts in puree kept at ambient temperature from 7 log cfu/g to 3 log cfu/g while 1 % citric acid gave a 3-log reduction from 7 log cfu/g to 4 log cfu/g.In nonsupplemented puree, E. coli by 4 log cycles (from 7 log cfu/g to 3 log cfu/g) in OFSP puree stored at refrigeration temperature. 1 % citric acid also resulted in a 4-log reduction in the numbers of E. coli. E. coli counts in nonsupplemented puree increased significantly by 2 logs immediately after inoculation but declined after 3 weeks storage from 7 log cfu/g to 6 log cfu/g. There was no significant difference (p>0.05) in E. coli populations in OFSP puree with different combinations of sodium benzoate, potassium sorbate, and citric acid in the two storage conditions.Similarly, preservative treatment with all combinations of potassium sorbate, sodium benzoate, and citric acid led to a significant (p<0.05) reduction of S. aureus in OFSP puree treated with a combination of selected chemical preservatives and that without preservatives and stored at ambient (15-25°C) and refrigeration temperatures (4°C) for a period of 10 weeks. S. aureus counts in OFSP puree kept at ambient and refrigeration temperatures from 7 logcfu/g to 3 logcfu/g. S. aureus counts in nonsupplemented puree kept at ambient temperature recorded a 2-log increase immediately after inoculation with subsequent increase in counts during storage from 7 log cfu/g to 9 log cfu/g while that at refrigeration recorded 2-log increase after inoculation with subsequent decline in counts during storage from 7 log cfu/g to 6 log cfu/g.Combined use of sodium benzoate and potassium sorbate at different concentrations with 1 % citric acid led to a 4-log reduction in S. aureus population by 1 log cycle at ambient temperature from 7 log cfu/g to 6 log cfu/g and by 2 log cycles at refrigeration temperature from 7 log cfu/g to 5 log cfu/g. Treatment of OFSP puree with potassium sorbate and sodium benzoate at different concentrations together with 1 % citric acid had a slightly greater effect on S. aureus growth compared to 1 % citric acid when used alone.Treatment with 1 % citric acid recorded a reduction inOFSP puree was found to contain high levels of bacteria (9.0 log cfu/g) immediately after preparation. The counts declined significantly (p<0.05) from 9 log cfu/g to nondetectable levels at the end of the storage period in OFSP puree treated with different combinations of potassium sorbate and sodium benzoate together with 1 % citric acid both at ambient and refrigeration conditions. However, there was a decline in aerobic microorganisms from 9 log cfu/g to 3 log cfu/g in puree with 1 % citric acid at ambient temperature at week 10 of puree storage.The level of yeast and molds counts in OFSP puree after preparation was 7.92 log cfu/g. The counts declined significantly (p<0.05) with storage from 7.92 log cfu/g to nondetectable levels at week 7 of puree storage in all the treatments kept at ambient temperature except for the puree with 1 % citric acid in which yeast and mold counts declined from 7.92 log cfu/g to 5 log cfu/g at week 10 of puree storage. For the puree kept at refrigeration temperature, yeast and mold counts declined significantly (p<0.05) from 7.92 log cfu/g to nondetectable levels at week 3 of puree storage. Pack distention and alcoholic odors were noted in OFSP puree treated with citric acid only after one week of storage.Effectiveness of preservative is dependent on pH of the product  and pH i E. coli and S. aureus would occur, the data presented indicate that these pathogens would grow extensively in the puree under ambient temperature assuming a 10-week storage period. The increase in E. coli and S. aureus counts in nonsupplemented puree (control) can be attributed to nutrient availability and favorable environment for their growth [Information on the survival potential of pathogenic microorganisms in orange fleshed sweet potato (OFSP) puree is limited and such knowledge would be of significance in OFSP puree storage period by the consumer further contributing to food safety and quality. If postprocessing contamination of OFSP puree byr growth  such as  E. coli and S. aureus populations in stored OFSP puree with preservatives can be attributed to various factors. Sodium benzoate and potassium sorbate activity is largely dependent on the pH of a food product. The optimum inhibitory activity of these preservatives takes place at low pH which favors the undissociated form of the molecule that freely moves across the plasma membrane into the cytoplasm [ E. coli and S. aureus in the puree. The results are similar to those reported by Chikthimmah [ E. coli counts in apple cider stored under ambient and refrigeration conditions.The decline inytoplasm . The lowytoplasm , 32. In kthimmah , who dem E. coli and S. aureus growth which is 6-7 or due to the disruption of the pathogens' membrane permeability thus preventing entry of essential nutrients for its growth. Other researchers have demonstrated the effectiveness of citric acid in inhibiting the growth of S. aureus. For instance, Seo [ S. aureus in chicken meat in 5 days. Abu-ghazaleh [ S. aureus growth in growth medium after 24 hours of incubation. Similarly, the use of 2 % citric acid alone on chicken meat led to the decline in E. coli counts by 4 log cycles in 12 hours [The antimicrobial activity of citric acid was due to reduction of pH of OFSP puree below the optimal range of pH values fornce, Seo  found thghazaleh  showed t12 hours . E. coli and S. aureus populations in OFSP puree with different combinations of sodium benzoate, potassium sorbate, and citric acid in the two storage conditions. This suggests that even the lowest concentration of preservatives used in combinations was effective in inhibiting the growth of E. coli and S. aureus in OFSP puree during the storage period. There was a slightly better inhibition of E. coli and S. aureus in treatments with different combinations of potassium sorbate, sodium benzoate, and citric acid as compared to citric acid alone at ambient and refrigeration temperatures. This suggests that combination of a number of hurdles (preservative factors) gives higher or multiple inhibitory effects against microorganisms compared to a single hurdle. According to Lotte Dock [ E. coli was enhanced through the combined use of 0.1 % potassium sorbate and 0.1 % sodium benzoate at 8°C with survival time being reduced by 50 % compared with the one with 0.1 % sodium benzoate alone [There were no significant differences (p>0.05) intte Dock , the effte alone . S. aureus and E. coli was more rapid at 25°C compared to 4°C. This is because, at low temperatures, the fluidity of the cytoplasmic membrane of microorganisms is reduced, thus interfering with transport mechanisms [ E. coli is able to grow at a temperature range of 4-45°C with an optimum of 37°C but can survive refrigeration and freezing temperatures. A study carried out on the effect of temperature on the growth of E. coli revealed that it can grow and survive well on a range of temperatures but can grow well at 37°C compared to other temperatures [ S. aureus on the other hand grows on a temperature range of 7-48°C with an optimum of 37°C [Temperature is also known to be one of the significant factors affecting microbial growth in food products . The enzchanisms . Therefochanisms . E. colieratures . S. aure of 37°C .The high levels of aerobic microorganisms in OFSP puree before treatment with preservatives would be attributed to poor handling during preparation. The reduction in levels of aerobic counts in OFSP puree samples treated with different combinations of potassium sorbate, sodium benzoate, and citric acid indicates the benefits of the combination of antimicrobial chemicals having multiple effects against bacterial growth in OFSP puree. Vacuum packaging of the puree eliminates oxygen which is essential for the growth of aerobic microorganisms thus eliminating them even in the nonsupplemented puree. Other researchers showed the effect of preservatives on the growth of aerobic microorganisms. For instance, Ogiehor & Ikenebomeh  recordedThe complete inhibition of yeast and molds in OFSP puree with and without preservatives would be attributed to the vacuum packaging of the puree that eliminates oxygen and therefore prevents the growth of molds and oxidative yeasts since they do not grow in the absence of oxygen . However Escherichia coli and Staphylococcus aureus in OFSP puree as indicated by the microbial challenge test. Use of citric acid alone was less effective in controlling the growth of these pathogens.The study sought to investigate the effect of different combinations of preservatives on microbial growth in OFSP puree at different storage conditions and time. Different combinations of sodium benzoate, potassium sorbate, and citric acid improved microbial keeping quality and inhibited the growth of


Analyzed by presence of esophageal varices, the SVR12 rates were 98.4% (95% CI 91.4–99.7) and 97.1% (95% CI 92.9–98.9) in patients without and with varices, respectively (p = 1.0). In real life, SOF/VEL GT3 cirrhotic patients with evidence of portal hypertension can achieve SVR12 levels comparable to those of patients without portal hypertension. These SVR12 rates are similar to what is reported in compensated cirrhosis treated within clinical trials.In clinical trials, a sofosbuvir/velpatasvir (SOF/VEL) pangenotypic single-tablet regimen was associated with high sustained virological response (SVR) rates at 12 weeks (SVR12) after the end of treatment, regardless of genotype and fibrosis stage. No real-life data on genotype 3 (GT3) cirrhotic patients with portal hypertension are available. The aim of this study was to assess the effectiveness of SOF/VEL in GT3 cirrhotics with portal hypertension. Patients with GT3 and advanced cirrhosis were treated for 12 weeks with SOF/VEL without ribavirin at five different centers in Italy from June 2017 to August 2018 and their SVR12 was assessed. Of the 227 GT3 cirrhotics evaluated, 205 met the inclusion criteria and 111 had transient elastography results ≥20 KPa. SVR12 was 97.6% (95% CI 94.4–98.9), rates were 99.1% (95% CI 95.7–99.8) in patients with ≥20 KPa and 95.8% (95% CI 89.5–98.3) in those with <20 KPa ( In the era of directly acting antivirals (DAA) treatment, despite advances in global sustained virological response at 12 weeks (SVR12) rates, patients with genotype 3 (GT3) infection and cirrhosis have emerged as a difficult-to-cure population [The single-tablet regimen (STR) of sofosbuvir (SOF)  inhibitor) and velpatasvir (VEL)  inhibitor) for 12 weeks demonstrated high efficacy across all genotypes in clinical trials . These rA large real-world cohort of GT3 compensated cirrhotic patients from different countries around the world was recently analyzed . This poThe use of ribavirin with the SOF/VEL STR combination has been largely debated. In patients with decompensated cirrhosis, based on the ASTRAL-4 results , all intDiscordant advice is provided by European Association of Sinological Librarians (EASL) and American Association for the Study of Liver Diseases (AASLD) in patients with compensated cirrhosis. Indeed, European guidelines recommend the addition of ribavirin in GT3 cirrhotic patients with prior treatment experience ,14. ThisPatients with compensated cirrhosis and grade 1 and 2 never bleeding esophageal varices represent a subgroup of patients with a better prognosis compared to patients in Child–Pugh–Turcotte (CTP) class B despite having an expected higher mortality than uncomplicated CTP class A. As shown by a recent analysis, identifying five prognostic stages of cirrhosis, patients with compensated cirrhosis and varices have a 5-year mortality rate of 10% as compared to the 1.5% observed in patients with compensated cirrhosis and an absence of varices .Analyses of SVR12 after DAA treatment in patients with cirrhosis and varices have not yet been performed and might provide further insights helping with the individualization of the treatment of patients with cirrhosis.Since a chasm between efficacy  and effectiveness  persists, in particular in patients with unfavorable characteristics, we explored in a multicenter nationwide study the impact of SOF/VEL treatment in cirrhotic patients with or without evidence of portal hypertension treated without the addition of ribavirin.Patients with GT3 and cirrhosis enrolled at five different tertiary centers in Italy and treated from June 20, 2017 to August 20, 2018 with SOF/VEL 400/100 mg STR for 12 weeks without the addition of ribavirin. Eligible patients were males and females, 18 years of age or older with confirmed chronic GT3 infection and compensated cirrhosis. Patients with CTP class C and prior-treatment experience  were excluded. Patients with previous decompensation and patients with malignancies were included. Patients being treated in those centers with the addition of ribavirin as well as patients that were treated for longer than 24 weeks were excluded. Patients with human immunodeficiency virus (HIV) or HBs Ag were considered eligible if hepatitis B virus DNA (HBV DNA) was not reactive.Serum HCV RNA levels were measured using commercial HCV RNA quantitative assays. HCV RNA was defined unquantified when below the lower limit of quantification (LLOQ) and unquantified but detectable when 12 IU/mL or 15 IU depending on the quantitative assays used. Viremia load was defined as high when it was higher than 1,000,000 IU/mL. The aspartate/platelets ratio index, AST to platelet ratio index (APRI) score and fibrosis-4 (FIB-4) were calculated for every patient. Cut-offs of >2.0 and >3.25 were respectively used to define cirrhosis . DiagnosPortal hypertension absence or presence was estimated by transient elastography results <20 KPa or ≥20 KPa and platelet count <150,000 µL. . Upper aAdverse events and laboratory tests were conducted during this study, and at week 12 post-treatment for a safety evaluation. Monitoring and grading of adverse events (AEs) and serious AEs (SAEs) were performed. Evidence of esophageal or gastric varices and evidence of hypertensive gastropathy confirmed portal hypertension. Ultrasonographic evidence of portal hypertension was based on the combination of spleen longitudinal diameter ≥15 cm and portal vein diameter ≥14 mm.The primary efficacy endpoint was SVR12 (HCV RNA < LLOQ 12 weeks after the end of therapy). Patients with missing SVR12 data were considered a treatment failure. The primary safety endpoint was discontinuation due to AEs.Patients’ demographical and clinical characteristics were reported as mean and standard deviation or as median and range and as frequency and percentages for continuous and categorical variables, respectively. Group comparisons were performed using Mann–Whitney U-test or Fisher exact test for continuous and categorical variables, respectively.p-value <0.05 was considered as statistically significant. Statistical analyses were performed using SPSS  v. 16.0.The SVR rates were assessed overall and in subgroups as defined by demographical and clinical variables. SVR rates by treatment regimen and treatment experience were also analyzed. Confidence intervals (CIs) of 95% for SVR rates were assessed using the binomial distribution. A Of the 227 patients evaluated, only 205 with HCV GT3 and compensated cirrhosis with or without evidence of portal hypertension were included. They were treated with SOF/VEL, STR for 12 weeks without RBV. Of the 22 who were not enrolled, eight had CTP class C, five had active HBV DNA replication that required a delay in treatment start and nine were lost to follow-up .Patients’ baseline characteristics are summarized by overall and by TE results . The meaOverall, 111 patients had TE results that were >20 KPa; clinical characteristics are reported in A PLT count of <150,000 µL was observed in almost 65.3% of cases. Among patients with high TE results, 10 had hepatocellular carcinoma (HCC). Two patients had non-Hodgkin’s lymphomas and both of them had TE results <20.0 KPa. Overall, 35 patients had a spleen longitudinal diameter of 15 cm or higher and a PV diameter >15 mm. Of them, 29 had TE results >20 KPa, while the remaining were shown to have a lower value. Mild ascites was detected in eight patients.p = 0.61).After 12 weeks of treatment, SVR12 was 97.6% (95% CI 94.4–98.9) overall and no virological failures were observed. Overall, five patients experienced a virological relapse . No lossp = 0.18). Among those with TE results ≥20 KPa (64.5%), 62 of 96 patients who underwent upper endoscopy had endoscopic evidence of portal hypertension and only one patient had a relapse (p = 1.0).Upon analysis by TE results ≥20 KPa, SVR12 was 99.1% (95% CI 95.7–99.8) compared to 95.7% (95% CI 89.5–98.3) registered in patients with lower KPa results ( relapse . By evidp = 1.0).Overall, 64 patients had ultrasonographic evidence of portal hypertension, SVR12 in this subgroup was 98.4% (95% CI 91.7–99.7). This response rate was not different from the 97.0% (95% CI 92.6–98.8) SVR12 attained in patients without ultrasonographic signs of portal hypertension (p = 0.53).Having identified the threshold of the PLT count of 150,000 µL as highly suggestive of portal hypertension, patients were analyzed accordingly. SVR12 was 99.2% (95% CI 95.7–99.8) for 133 patients with lower PLT counts and 94.4% (95% CI 86.5–97.8) for 72 patients with higher PLT count results (p = 1.0). Of the 22 patients who had both low albumin levels and a low PLT count, 100% achieved SVR12.Albumin levels lower than 3.5 g/dL were registered in 30 patients. All of them achieved SVR12, in contrast with 170 of 175 with higher albumin levels (SVR12 97%) (95% CI 93.4–98.7) (p = 0.28).Finally, patients were also analyzed by CTP score A5 versus higher scores. In 162 patients with CTP A5, SVR12 was 98.1% (95% CI 94.7–99.3) as compared to 95.3% (95% CI 84.5–98.7) in 43 patients with higher CTP scores (p = 0.23). In 10 patients with HCC, SVR12 was 100% as compared to 96.2% (95% CI 93.4–98.5) achieved in patients without HCC (p = 1.0).In patients with ascites, SVR12 was 88.9% (95% CI 56.5–98.0) as compared to 95.3% (95% CI 84.5–98.7) achieved in patients without ascites . Headaches were registered in 15%, insomnia in 10% and diarrhea and abdominal pain in 3% of patients during treatment.SAEs were rare; three patients had urinary tract infections, two had tracheobronchitis and one had bronchopneumonitis. No SAEs events were considered to be related to the antiviral treatment. Three patients died after follow-up completion, one of lung cancer, one of worsening liver disease and ascites complication and another of HCC.Laboratory abnormalities were infrequent. Few patients experienced anemia (2%) and 1.4% experienced an increase in bilirubin levels.In this multicenter prospective real-world cohort, GT3-infected patients with cirrhosis, half of whom exhibited the presence of portal hypertension by non-invasive evaluation, achieved an SVR12 rate of 97.6%. SVR12 rates of 99.1% were observed in the subgroup of patients considered at high risk of portal hypertension.3, suggestive of portal hypertension [Randomized controlled trials demonstrated that SVR12 rates were associated with SOF/VEL range from 91% in the ASTRAL-3 study to 93% in a combined analysis of the 212 GT3 patients with compensated cirrhosis included in the ASTRAL program, and to 96% observed in the POLARIS-3 trial . No detartension , or endo3 were treated without ribavirin. SVR12 rates of 91.0% and 94.7% were registered in these subgroups, respectively. These rates are in keeping with the 99.1% and 96.2% registered in the corresponding subgroups from our cohort. Moreover, in our study SVR12 rates were confirmed to be high in patients with evidence of portal hypertension, either endoscopic (98.2%) or ultrasonographic (97.1%).The effectiveness observed in our cohort is comparable to that reported in a multinational cohort on over 400 GT3 patients with compensated cirrhosis from around the globe. That study compared SOF/VEL to SOF/VEL plus ribavirin . Indeed,The majority of our patients had cirrhosis of CTP class A, therefore they cannot be compared to those with decompensated cirrhosis included in the ASTRAL-4 study focusing on CTP B patients and supporting the addition of ribavirin for 12 weeks of treatment [Our data give reason to implement the strategy to treat all compensated cirrhotic patients without ribavirin, including those with F1–F2 varices or hypertensive gastropathy, and avoid unnecessary pre-treatment genotyping based on the high SVR12 achieved in this GT3 cohort of patients with advanced cirrhosis. Very recent results have shown that, while the decompensated cirrhosis outcome was not significantly ameliorated by DAA treatment, patients with compensated cirrhosis who achieved sustained virologic response after DAA treatment experienced similarly decreased mortality and cancer risk decline as compared to patients with chronic hepatitis . Of noteIn the era of DAA, patients with GT3 and cirrhosis are also considered difficult to treat with Glecaprevir and Pibrentasvir (G/P). In the SURVEYOR-2 study, 12 weeks of treatment led to an SVR12 rate of 98% in treatment naïve patients, while 16 weeks were required to achieve an SVR12 of 96% in treatment experienced patients . In a reDespite its small sample size, this study included very well-characterized patients either in terms of liver disease or in terms of co-morbidities from alcohol abuse. Given the SVR12 of 99.1%, the lack of a control group of patients receiving ribavirin does not appear to be a limiting factor. On the other hand, the number of patients with cirrhosis is currently declining after 6 years of availability of oral DAA; moreover, test and treat strategies using an STR as SOF/VEL can now be easily and safely adopted regardless of genotype, fibrosis and even portal hypertension.A limitation of our study might be the lack of data on NS5A-associated RASs at baseline. However, according to previous evidence generated by our group showing that baseline NS5A associated RASs are quite rare in our geographical area , no furtSimplified treatment is currently recommended by the WHO in order to reach HCV elimination by 2030 . GT3 patIn conclusion, this study shows the effectiveness of STR based on SOF/VEL without the addition of ribavirin in cirrhotic patients with 10% 5-year risk of mortality, as patients with evidence of portal hypertension. The high SV12 rate and the absence of any treatment-related SAEs suggest that this regimen can be adopted without risk in the perspective of a test and treat strategy required to achieve WHO 2030 goals.


Predictors for glucose intolerance postpartum were evaluated in women with gestational diabetes mellitus (GDM) based on the 2013 World Health Organization (WHO) criteria. 1841 women were tested for GDM in a prospective cohort study. A postpartum 75g oral glucose tolerance test (OGTT) was performed in women with GDM at 14 ± 4.1 weeks. Of all 231 mothers with GDM, 83.1% (192) had a postpartum OGTT of which 18.2% (35) had glucose intolerance. Women with glucose intolerance were more often of Asian origin , had more often a recurrent history of GDM , higher fasting glycaemia (FPG) , higher HbA1c , and higher triglycerides . Sensitivity of glucose challenge test (GCT) ≥7.2 mmol/l for glucose intolerance postpartum was 80% (63.1%–91.6%). The area under the curve to predict glucose intolerance was 0.76 (0.65–0.87) for FPG, 0.54 (0.43–0.65) for HbA1c and 0.75 (0.64–0.86) for both combined. In conclusion, nearly one-fifth of women with GDM have glucose intolerance postpartum. A GCT ≥7.2 mmol/L identifies a high risk population for glucose intolerance postpartum. Gestational diabetes mellitus (GDM) is defined as diabetes diagnosed in the second or third trimester of pregnancy provided that overt diabetes early in pregnancy has been excluded . TreatmeThe ‘International Association of Diabetes and Pregnancy Study Groups’ (IADPSG) and the World Health Organization (WHO) recommend a universal one-step approach with the 75 g oral glucose tolerance test (OGTT) for screening of GDM with the use of more stringent diagnostic criteria for GDM ,9. The IClinicalTrials.gov (NCT02036619). The study protocol was approved by the Institutional Review Boards of all participating centers (B322201420693). Participants provided informed consent before inclusion in the study. The study was registered in The Belgian Diabetes in Pregnancy study (BEDIP-N) was a multi-centric prospective cohort study that has previously been described in detail ,14,15. W2 and obesity as a BMI ≥30 Kg/m2. Excessive weight gain was defined according to the 2009 Institute of Medicine (IOM) guidelines  and β-cell function , were measured, as previously described ,20,21,22The analyses of the FPG at 6–14 weeks and the glucose measurements of the OGTT were performed locally at each center. The analyzes of the GCT’s, insulin, lipids and HbA1c levels were performed centrally at the lab of UZ Leuven  and these results were not communicated to participants and health care providers during the study. Plasma glucose was measured by an automated colorimetric-enzymatic method on a Hitachi/Roche-Modular P analyzer . Insulin was measured by the immunometric ECLIA (Roche Modular E170). HbA1c was measured by Tosoh Automated Glycohemoglobin Analyzer HLC-723G8. Lipid levels were measured by the immunoassay analyzer Cobas 8000 . Coefficients of variance are 1% for glucose, 6% for insulin, about 2% for lipids and 2% for HbA1c in the Lab of UZ Leuven.p-value <0.05 (two-tailed) was considered significant. Analyses were performed by A. Laenen using SAS software (version 9.4).Descriptive statistics were given for the two groups where continuous variables are presented as means with standard deviation if normally distributed, or as medians with interquartile range otherwise; categorical variables are presented as frequencies with percentages. Logistic regression models were used for data analysis with postpartum glucose intolerance as a binary response variable and subject characteristics as explanatory variables. Results were reported as odds ratios (OR) with 95% confidence intervals. For variables during pregnancy that were significantly different between women with and without postpartum glucose intolerance, adjusted OR for age and BMI in early pregnancy were analyzed. The discriminative power of continuous variables for postpartum glucose intolerance is presented by receiver operating curve (ROC) curves and estimated as the area under the curve (AUC) with 95% confidence interval. The AUC ranges between 0.5 (discrimination no better than chance) and 1 (perfect discrimination). Diagnostic accuracy of binary or dichotomized variables is estimated as sensitivity, specificity, positive and negative likelihood ratio, and post-test probability, with 95% confidence intervals. A Prospective multicentric cohort study with 1841 women tested for GDM with a 75 g OGTT. GDM was diagnosed in 12.5% (231) of all participants. Of all women with GDM, 83.1% (192) attended the postpartum 75 g OGTT. The OGTT was performed at 14.4 ± 4.1 weeks after delivery. Of all women with an OGTT postpartum, none had T2DM and 18.2% (35) had glucose intolerance postpartum of which 37.1% (13) had IFG, 54.3% (19) had IGT, and 8.6% (3) had IFG and IGT combined.p = 0.008; HOMA-IR 16.8 (8.9–24.8) vs. 10.2 (7.3–15.6), OR 1.07 (1.02–1.11), p = 0.002], had a lower β-cell function , lower HDL-cholesterol , higher fasting triglycerides  and breastfed less often  . Compared to NGT women, women with glucose intolerance postpartum were more insulin resistant  . There w= 0.009] . Women wp = 0.021], were more often multiparous , had more often a recurrent history of GDM , a higher fasting glycaemia (FPG) , a higher HbA1c , and higher fasting triglycerides  at the time of the OGTT during pregnancy , regnancy . After aregnancy . The AUCregnancy . Evaluatregnancy . The AUC for FPG alone in early postpartum to predict glucose intolerance was 0.76 (95% CI 0.65–0.87), the AUC for HbA1c alone was 0.54 (95% CI 0.43–0.65) and the AUC for FPG and Hba1c combined was 0.75 (95% CI 0.64–0.86) .By diagnosing GDM, a group of women at high risk to develop T2DM and cardiovascular disease later in life was identified ,5. LifesData on the risk to develop T2DM in women with GDM diagnosed by the one-step approach and 2013 WHO criteria are limited. Before the introduction of the 2013 WHO screening strategy, studies have shown that 30%–50% of women with GDM develop T2DM within the first 10 years after the index pregnancy . The useFinding the risk factors for glucose intolerance postpartum after a recent history of GDM is important to identify a subgroup of women at a particularly high risk to progress to T2DM on the long-term. A more intensive follow-up and treatment of this high risk group might be cost-effective to prevent T2DM . The mosAs can be expected, we show that women with GDM who developed glucose intolerance in early postpartum were more insulin resistant and had an impaired beta-cell function compared to NGT women after delivery. During pregnancy insulin sensitivity and beta-cell dysfunction were not significantly different between both groups. However, women who develop GDM, often already have a subclinical metabolic dysfunction prior to conception compared with NGT women . We specIn our study, women with glucose intolerance postpartum breastfed less often than NGT women but without a difference in the duration of breastfeeding or in the exclusivity of breastfeeding between both groups. However, the timing of the postpartum OGTT varied between participants and we have no prospective data to evaluate the association between the duration of breastfeeding and risk for glucose intolerance postpartum. In addition, we have no data on breastfeeding beyond three months after delivery. There is now strong evidence that lactation duration (>3–6 months) is independently associated with a graded reduction in the incidence of T2DM ,38. ThisFinally, we show that a GCT can also have a role in predicting postpartum glucose intolerance. We have previously shown that a GCT threshold of 7.2 mmol/L has the best trade-off between sensitivity and specificity to screen for GDM when using the 2013 WHO criteria . Here weThe strengths of our study are the large prospective multiethnic cohort with the availability of many clinical and biochemical variables in early pregnancy. In addition, we provide data on the predictive value of HbA1c and FPG in early postpartum to detect glucose intolerance. Moreover, we present novel data on the predictive value of a GCT when used in a two-step screening strategy for GDM with the 2013 WHO criteria to predict glucose intolerance postpartum. The limitations are the lack of a control group postpartum and the lack of long-term data postpartum. In conclusion, we show that nearly one-fifth of women with GDM based on the 2013 WHO criteria have glucose intolerance in early postpartum and these women have a more adverse metabolic profile. An FPG and HbA1c in early postpartum are not accurate enough to replace a 75 g OGTT to detect glucose intolerance. In addition, a GCT threshold of 7.2 mmol/L has the best sensitivity to predict glucose intolerance in early postpartum. Combining CGT and risk factors identified in the present study could identify a group of women who are at the highest risk for postpartum glucose intolerance and where recall incentives may be most needed.


Treatment of patients with knee osteoarthritis is challenging. Unloader braces have been developed with various success. Unloader One® Knee Brace is light, easily-fitted and shown to be effective by the unloading of the affected compartment. The aim of the study was to assess the clinical outcome of the brace vs. a placebo on patients with knee osteoarthritis.Initially 150 patients were randomized to receive either the Unloader brace or a control placebo group look-alike brace where the active strips had been removed. The patients were followed up at 6,12,26 and 52 weeks with Knee Society Score (KSS) and Knee injury and Osteoarthritis Outcome Score (KOOS). The reason for dropout was recorded.p = 0.009). The study group improved in KSS function from 67.0 to 78.6 (p < 0.001) and KOOS for knee related symptoms increased/improved from 64.3 to 72.4 (p < 0.001). Activity of daily living increased/improved from 65.3 to 75.2 and Sports/Recreation from 24.6 to 40.2 (p > 0.001) whereas the control group did not show significant improvements in any of the scores. The dropout was higher in the control group, 40 vs. 25.A total of 149 patients were included, 74 in the study and 75 in the control group. The mean age was 59.6 vs. 60.2, BMI was 27.5 vs. 26.9, 37% vs. 44% were women in the study vs. control group. Both groups showed improvement in KSS over 52 weeks, with the study group showing higher improvement in mean scores. KSS increased from 64.3 to 84.0 for the study group and from 64.0 to 74.6 for the control group (The brace seems to be more effective and better tolerated than the placebo.NCT03454776) on March 6th 2018.The trial was retrospectively registered with ClinicalTrials.gov ( Osteoarthritis (OA) is a progressive degenerative joint disease that involves damage to the joint cartilage and changes in the subchondral bone and connective tissue of the joints . Risk faThis study was a randomized placebo controlled study in patients with mild to moderate knee OA. All patients included were initially treated in primary care settings, with patient education, physical therapy and analgesic use. Patients visiting our outpatient clinic who met the inclusion criteria were asked to join the study. Some patients were recruited after responding to advertisements in local newspapers and social media. The first patient was included in April 2012 and the last in August 2014. The follow up time was 12 months. An orthopedic surgeon evaluated symptoms and radiographic evidence before inclusion in the study. All patients between 30 to 70 years of age, with knee pain for more than three months, with arthroscopic or radiographic evidence of knee OA (Allbäck or Kellgren-Lawrence grade 1–2) [150 patients were randomized to either a study group or a control group and followed for one year. The study group received an Unloader One® knee brace   and answThe data was analyzed and 95% confidence interval was calculated using a mixed model repeated measures analysis of variance. This method uses all available information and includes justifications for baseline imbalance with stratification of randomized confounding factors. An independent statistician analyzed all data presented. Statistical analysis was performed with STATA  Power and sample size calculation was done with the PS: Power and Sample Size Calculation program by William D. Dupont and Walton D. Plummer, Jr. version 3,0. 2009 [The results of data collected from KSS and KOOS are presented as estimated marginal mean difference at 6, 12, 24 and 52 weeks and 95% confidence interval.In total 149 patients answered questionnaires at baseline, 74 in the study group and 75 in the control group. All patients had unilateral medial OA, 80 in the left knee and 68 in the right knee. After 52 weeks 85 patients were still participating in the study, 50 in the study group and 35 in the control group. The reasons for dropout are shown in the flowchart below  to 84.0 (95% CI 79.5–88.5) compared to 64.0 (95% CI 60.3–67.6) to 74.6 (95% CI 69.3–80.0). The difference at twelve months between the two groups is 9.4 (95% CI 2.4–16.4).  to 78.6 (95% CI 74.7–82.5) at 52 weeks, with a difference of 10.6 (95% CI 4.1–17.1). The control group showed slight improvement from 67.1 (95% CI 64.0–70.3) to 70.8 (95% CI 66.2–75.3) with the difference between the groups 7.8 (1.9–13.8) . This difference is not considered clinically important.On the pain subscale of KOOS, the study group improved from 61.2 (95% CI 58.7–63.7) to 68.9 (95% 66.0–71.8) at 52 weeks, with a difference of 7.7 where a difference of 10 is considered a clinically detectable difference. The control group showed less improvement with a difference between the groups of 5.2 (The study group improved from 64.5 (95% CI 61.8–67.2) to 72.4 (95% CI 74.7–82.5) whereas the control group shows hardly any improvement from baseline. The difference at 52 weeks from baseline for the study group is 7.9 which is slightly less than 10, the clinically detectable cut off mark traditionally used in KOOS scores.p < 0.001) while the control group shows no statistically significant difference from baseline.The sub score for ADL shows an increase in the study group from 65.3 (95% CI 62.9–67.7) to 75.2 (95% CI 72.4–78.0) and a difference of 9.8 (p < 0.001). The control group did not show much improvement during this time and the difference between the groups at 52 weeks was 12.5 (p < 0.001), which is both a clinical and statistical significant difference.The sub score for sport and recreation increased for the study group during the whole follow-up. The scores for the study group increased from 24.6 (95% CI 21.6–27.6) to 40.2 (95% CI 36.7–43.8) with a difference of 15.7 (p = 0.02) which is not a clinically detectable difference.The sub score for quality of life did not show much change for either group, the study group increasing from 52.2 (95% CI 49.4–55.0) to 55.7 (95%52.5–59.0) and the control group actually decreasing from 52.3 (95% CI 49.5–55.1) to 49.5 (95% CI 45.4–53.6). The difference between the groups at 52 weeks was 6.1 (This exploratory study indicates that pain and function (activity of daily living and sports and recreation) can be improved in comparison to placebo during one-year use of the Unloader brace. The study showed some improvements in primary endpoints except in the KOOS subgroup measuring quality of life at one year follow up. The differences between the groups at 6, 12 and 24 weeks are hard to interpret due to poor return at these intervals. Most patients showed up at some of the follow up appointments i.e. either at six weeks or three months but not both, and some showed up at all appointments. Some subjects stated that they did not have time for all the follow-up visits. Some patients choose not to come to the appointment but sent in the self-administered questionnaires, and others showed up at the appointments but neglected to send in the self-reported questionnaires.There were more dropouts from the control group than the study group, mostly due to mechanical issues, such as the brace sliding of the leg or hurting the patient.It is interesting to note that improvements in KSS score are not evident at the six week follow up and the difference between groups does not become apparent until after six months suggesting that long term follow up is needed to see the difference. Many studies are limited by short follow up times. This also suggests that it may take time for the patients to adjust to the brace in the clinical setting and full results should not be expected immediately.The high dropout rate in the control group compared to the study group shows in our opinion that the design of the brace matters and supports the theory that unloading the affected compartment can relief pain and improve function. With this said, it should be kept in mind that the placebo braces might still have clinical effect as shown in biomechanical studies even though the dynamic tension strap has been removed. This effect might be due to diminished muscular co contractions rather than compartmental unloading as suggested by Ramsey et al. .The KSS questions for pain and function ask patients to remember their experience the previous four weeks and the KOOS questionnaire the previous week. It is a thought for mind if these kinds of outcome scores are appropriate for measuring outcomes for a device that is only used during activity rather than continuous pain relieving effect such as medication or operations. This might explain larger differences between groups in functional scores rather than pain and symptom scores.There is a clinically and statistically significant improvement in KSS and KSS function for both groups at one year compared with baseline. These results are compatible with previously published studies , 18. TheIn the study protocol we had set the economic aspects of brace use as the secondary endpoint. This proved to be more complex than we anticipated and beyond the scope of this article and therefore the secondary endpoint was limited to the dropout rate.The results of this study reflect our experience in clinical practice, there are many who find braces useful and there are those who don’t think that they add anything to their treatment. Further research should be focused on identifying the responders and non-responders of this treatment applying OARSI criteria  for resp


The aim of this study was to assess the biocompatibility of positively charged imidazolium-based ionic liquid-protected nanosilver solution (AgNPs) root canal irrigant. -8 M/L (group 5) were randomly injected in 5 sites of dorsal skin of each rat. Tissue inflammatory reaction were evaluated histopathologically after 2 h, 48 h and 14 days. Statistical analysis was done with SPSS version 21 and the Kruskal-Wallis H and Dunn tests were used to find statistically significant differences. The level of significance was set at 0.05.  Eighteen male 4- to 5-month old Sprague-Dawley rats, weighing 200-300 gr were selected and randomly divided into 5 groups: Normal saline 0.9% (group 1), 5.25% NaOCl (group 2), 2.5% NaOCl (group 3), 2.0% chlorhexidine solution (group 4) and AgNPs at 5.7×10P<0.05). Group 2 displayed higher inflammatory response in comparison with group 5 (P<0.05). Tissue reaction to group 5 was not more severe than the reaction to group 3 or 4. It also would irritate less inflammatory response compared to group 2 (P<0.05). All solutions irritated the highest tissue response after 48 h. Group 1 showed lower inflammatory response compared to groups 2 and 4 (Comparing with NaOCl and CHX, it is possible to label AgNPs as a tissue compatible agent.  One of the purposes of root canal therapy is to eradicate debris, tissue remnants and bacteria from the root canal system , 2. Due Recent studies have focused on nanosilver (NS) products as possible alternative for endodontic irrigants , 10. Thevia non-specific oxidative destruction and silver inducing cytotoxicity [et al. [et al. [Enterococcus faecalis. It is revealed that AgNP has lower cytotoxic effect on L929 in comparison to NaOCl and CHX [Biocompatibility of NS is still a controversial topic. Some evidence warn about the adverse effects of NS on human health toxicity , 15. Fro [et al.  displaye [et al.  proved t and CHX . Since p◦C along with a 40% of relative humidity. The rats were allowed ad-libitum access to a normal standard chow diet and water. Animals received human care and all the experiments were performed in conformity with the guidance for care and use of experimental animals approved by a local ethic committee in Shiraz University of Medical Sciences, Shiraz, Iran (#95-01-03-12208).Eighteen male 4- to 5-month old Sprague-Dawley rats, weighing 200-300 gr were selected for the present study. Animals were housed in plastic cages over hardwood bedding. The temperature was set at 24-8 M/L which was prepared according to the method suggested by Abbaszadegan et al. [Animal tissue reaction was evaluated against irrigants as follows; group 1, normal saline 0.9% (control group); group 2, 5.25% NaOCl ; group 3, 2.5% NaOCl ; group 4, 2.0% chlorhexidine solution  and group 5, AgNPs at 5.7×10via a light microscope  under ×400 magnification. An average of the inflammatory cells for each group was obtained from 5 separate areas in histologic sections. Tissue reaction of each irrigant was assessed via sum of inflammatory cells existing in its corresponding histopathologic sections [The animals received anesthesia with xylazine (10 mg/kg) and ketamine (25 mg/kg) and the dorsal skin was shaved, cleaned and disinfected with 10% iodine solution. By a transplant template, 5 circles were marked on dorsal of each rat, leaving 3 cm between the circles. Then 0.1 mL of each irrigant was injected subcutaneously into the circles. Tissue reaction was evaluated in three period times after the injections as following; 2 h, 48 h and 14 days. Rats were sacrificed at each period by overdose of xylazine/ketamine solution. The dorsal skin tissue with the thickness of 2-3 mm was excised with a surgical blade and fixed in 10% buffered formalin. The tissue processing and histopathological slide preparation were performed according to the procedure introduced by Gomes-Filho’s . The hissections . P-value less than 0.05 was considered as statistically significant.Data were expressed as mean and standard deviation (SD) and analyzed using Statistical Package for the Social Sciences software . The Kruskal-Wallis H and Dunn’s tests were used to find statistical significant differences between different irrigants. A Tissue reaction to irrigants after each period of time-8 M/L AgNPs solution was mild to moderate (According to data presented in moderate  to 4.P<0.05). In addition, group 2 displayed higher inflammatory response compared to group 5 (P<0.05).Considering all groups in mentioned time periods, statistically significant differences in tissue inflammatory response were found that are reported in Considering lack of studies on the biocompatibility of AgNP solution as an intracanal endodontic irrigant, this study designed to evaluate the compatibility of AgNP with vital tissue comparing with 2.5% NaOCl, 5.25% NaOCl and 2% CHX. An optimal endodontic irrigant should have the ability to dissolve tissue, eliminate resistant bacteria and does not induce inflammatory reaction in case of inadvertent extrusion , 20, 21.in vitro study has demonstrated that 5.7×10-8 M/L AgNPs was effective against bacteria, especially E. faecalis whilst it had only a mild cytotoxicity to L929 fibroblasts [et al. [An roblasts  and alsoroblasts , 27, so In the present study, 0.9% sterile saline as control group created only a mild inflammation in first 48 h. This finding is in agreement with other studies , 20, 21 It was found that AgNPs had no statistically significant difference in comparison with 2.5% NaOCl and 2% CHX in promoting tissue reaction at all periods of time. In addition, AgNPs was significantly more tolerated by the tissue than 5.25% NaOCl at 2 h, 48 h and 14 days et al. [et al. [et al. [AgNPs showed mild inflammatory response at 2 h and increased to moderate reaction until 14 days. Gomes-filho  [et al.  after im [et al. , 21, 30. [et al. . NaOCl w [et al. . Besides [et al.  which co [et al. . Consequet al. [The 2% CHX and 2.5% NaOCl presented mild inflammatory reaction at 2 h. The inflammation increased to moderate after 48 h. After 14 days, a decrease in the number of inflammatory cells was found which could be the result of connective tissue repair process. This Also accords with the studies evaluating 0.12% CHX as an endodontic irrigant which reported that the mean number of inflammatory cells dropped after 14 days , 21. Alset al.  showed tet al. [The tested materials injected subcutaneously at the dorsum of rats, using the method described by Gomes-filho et al. . In thisThis study concluded that, AgNPs solution was biocompatible in comparison to 2.5% NaOCl and 2% CHX, but more future studies are needed to confirm the observed results.


Determine in a cohort of patients with normal hearing and chronic tinnitus if self-reported history for temporomandibular joint (TMJ) dysfunction and a positive modulation of tinnitus in the TMJ region could be suggestive of an underlying TMJ disorder.The study included 226 patients presenting to the Head and Neck Service of our University Hospital. Following audiological and somatic tinnitus evaluation, patients were divided into two groups. The study group (n = 134) included subjects that met both the following criteria: A) a self-reported history for TMJ dysfunction and B) a positive modulation of tinnitus following somatic maneuvers in the TMJ region. The control group (n = 92) included patients with similar demographic and tinnitus characteristics that did not meet the proposed criteria for somatic tinnitus. Afterwards, patients underwent clinical TMJ evaluation in the Service of Clinical Gnathology of our University.One hundred thirty-one patients (57.9%) received a clinical diagnosis of TMJ disorder according to DC/TMD Axis I; 79.1% in the study group and 27.2% in the control group. Ninety-five (42.1%) patients were negative for TMJ disorders; 20.9% in the study group and 72.8% in the control group. A significantly higher number of TMJ disorders was found in patients in the study group compared to the control group (p<0.0001). Most patients had joint disorders (67.2%), followed by other (29.8%) and pain disorders (29%). Logistic regression analysis in the study group showed that female gender was more prevalent in patients with TMJ disorders.Our findings in patients with chronic tinnitus and normal hearing suggest that self-reported history for somatic dysfunction and modulation of tinnitus, when occurring simultaneously in the TMJ region, can be useful to preliminarily identify patients with TMJ disorders. Tinnitus is defined as the perception of sound without an accompanying external auditory stimulus. Tinnitus can follow hearing loss, ototoxicity, and psychiatric comorbidity –9. In a Patients with somatic tinnitus can benefit from specific treatments of the associated somatic disorder –32; howeTinnitus modulation by movements of the head and neck, limbs and eyes following interactions between the auditory and the somatosensory systems has been proposed as a possible indicator for somatic tinnitus , 35, 36.In a recent study from our group, we proposed that the correlation between a positive self-reported history for a somatic dysfunction and positive tinnitus modulation in the same region could be suggestive of a somatic disorder underlying tinnitus . The aimThis study was conducted in 226 patients with normal hearing and chronic tinnitus recruited among those presenting to the Head and Neck Service of our University Hospital  between January 2016 and June 2017 with tinnitus as their chief compliant.Clinically normal hearing was defined as an individual hearing threshold ≤25 dB HL at frequencies from 250 to 4,000 Hz at the octave scale in both ears according to the American Academy of Otolaryngology and American Council of Otolaryngology . ChronicExclusion criteria were pulsatile tinnitus, history of acoustic trauma, middle or inner-ear disease , significant interaural hearing asymmetry, retrocochlear disease, previous ear surgery, concurrent medical treatment for tinnitus except for antioxidant drugs.All patients underwent audiological and somatic tinnitus evaluation in the Tinnitus Unit of the Department of Sense Organs. Following audiological and somatic evaluation, patients were divided into two groups. The study group (n = 134) included subjects that met both the following criteria: A) a self-reported history for TMJ dysfunction and B) a positive modulation of tinnitus following somatic maneuvers in the TMJ region. The control group (n = 92) included patients with similar demographic  and tinnitus  characteristics that did not meet the proposed criteria for somatic tinnitus. Afterwards, patients underwent clinical TMJ evaluation in the Service of Clinical Gnathology of the Department of Oral and Maxillofacial Sciences.Patients signed a written informed consent; the procedures performed were in accordance with the ethical standards of the ethics committee on human experimentation of the Department of Sense Organs of the Sapienza University of Rome, that specifically approved this study, and with the Helsinki Declaration.Patients underwent anamnestic evaluation, a full otolaryngology examination and audiological test battery including pure tone audiometry (PTA) and acoustic immittance test. PTA was measured at frequencies of 0.125, 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, and 8 kHz; hearing was considered symmetrical if thresholds for each ear occurred within 10dB of each other. Tinnitus side, pitch, and loudness were tracked for each patient; characteristics included tinnitus side  and tinnitus description from a predefined set of possibilities including “buzzing”, “whistle”, “high-pitched”, “low-pitched” and “other”. All subjects were asked to complete the Italian versions of the Tinnitus Handicap Inventory (THI) , HearingSomatic tinnitus evaluation included anamnestic investigation of self-reported history for TMJ dysfunction and assessment of tinnitus modulation.Self-reported history for TMJ dysfunction was considered positive if the patient reported one or more of the following events occurring before the onset of tinnitus: 1) head trauma involving TMJ region; 2) intensive manipulation of teeth or jaw; 3) recurrent pain episodes in the TMJ region; 4) increase of both TMJ pain and tinnitus at the same time; and 5) intense periods of bruxism during day or night .Tinnitus modulation assessment was performed as previously described , 15. FivTMJ was evaluated by a specialized dentist according to Diagnostic Criteria for Temporomandibular Disorders Axis I (DC/TMD), the most commonly used diagnostic criteria for TMJ disorder evaluation characterized by simple, reliable, and valid operational definitions for the history, examination, and imaging procedures needed to render physical diagnoses in both clinical and research settings . PatientStatistical analyses were performed using Prism GraphPad v7. Descriptive statistics, mean, and standard deviation were calculated for numeric variables; frequency and percentages were calculated for categorical variables. Unpaired t test was used to evaluate differences between patients in the two groups for numeric variables. Chi-square test of association was performed to assess association between two categorical variables. Multiple logistic regression was performed to identify the variables associated with TMJ disorders. The p-value for assessing statistical significance was an alpha of 0.05.Two hundred twenty-six patients were enrolled in the study; 134 were included in the study group and 92 in the control group. One hundred twenty-eight patients were males (56.6%) and 98 were females (43.4%). Study workflow is shown in In the study group, 78 (58.2%) were males, and 56 (41.8%) were females. Mean age was 48.5 years . Average PTA thresholds were 16.8 dB HL (0.125–2 kHz), 20.4 dB HL (2–4 kHz), and 26.6 (4–8 kHz) with no significant interaural asymmetries. The average duration of tinnitus at the time of the first admission was 4.9 years (SD = 6.6). Tinnitus was bilateral in 75 patients (55.9%) and unilateral in 59 (44.1%). Tinnitus was described by patients as “high-pitch” in 43 patients (32.1%), “low-pitch” in 35 (26.1%), “whistle” in 32 (23.9%), “buzzing” in 14 (10.4%), and “other” in 10 (7.5%). Mean THI score was 46.9 (SD = 20.8), mean HHI was 15.1 (SD = 17.1), mean HQ was 14.2 (SD = 7.8) and mean GUF score was 10.2 (SD = 7.2).In the control group, 50 (54.3%) were males, and 42 (45.7%) were females. Mean age was 45.9 years . Average PTA thresholds were 14.9 dB HL (0.125–2 kHz), 19.8 dB HL (2–4 kHz), and 27.1 (4–8 kHz) with no significant interaural asymmetries. The average duration of tinnitus at the time of the first admission was 4.5 years (SD = 5.6). Tinnitus was bilateral in 51.1% of cases and unilateral in 48.9%. Tinnitus was described as “high-pitch” in 10.9%, “low-pitch” in 16.3%, “whistle” in 36.9%, “buzzing” in 26.1%, and “other” in 9.8% of cases. Mean THI score was 27.5 (SD = 20.1), mean HHI was 10.1 (SD = 7.3), mean HQ was 13.1 (SD = 10.6) and mean GUF score was 8.6 (SD = 6.7).Table 2.No significant differences between groups were found for age (p = 0.1639), gender (p = 0.5670), tinnitus length (p = 0.6225), laterality (p = 0.4715), HQ (p = 0.4017) and GUF (p = 0.0992). A significant difference was found for the THI (p<0.0001) and HHI (p = 0.0084) questionnaire scores. Demographic and tinnitus characteristics and questionnaire results are presented in One hundred thirty-one patients (57.9%) received a clinical diagnosis of TMJ disorder according to DC/TMD Axis I; 106/131 (79.1%) were in the study group and 25/131 (27.2%) in the control group. Ninety-five (42.1%) patients were negative for TMJ disorders; 28/95 (20.9%) in the study group and 67/95 (72.8%) in the control group. The difference between the study and control groups was statistically significant (p<0.0001) .Among patients with a TMJ disorder diagnosis, 69 (52.7%) were males and 62 (47.3%) were females. Mean age was 47.4 years . The average duration of tinnitus was 5.2 years (SD = 6.9); tinnitus was bilateral in 53.4% of cases and unilateral in 46.6%. Mean THI score was 43.9 (SD = 22.1), HHI was 14.3 (SD = 15.3), HQ was 14.3 (SD = 8.4) and mean GUF score was 10.2 (SD = 7.1).Among patients without TMJ disorders, 59 (62.1%) were males and 36 (37.9%) were females. Mean age was 47.5 years . The average duration of tinnitus was 4.3 years (SD = 5.4); tinnitus was bilateral in 54.7% of cases and unilateral in 45.3%. Mean THI score was 32.3 (SD = 21.6), HHI was 11.4 (SD = 12.3), HQ was 12.9 (SD = 9.8) and GUF was 8.5 (SD = 6.7).No significant differences were found between patients with and without a diagnosis of TMJ disorder for age (p = 0.9432), gender (p = 0.0929), tinnitus length (p = 0.2524), laterality (p = 0.6443), HHI (p = 0.1295), HQ (p = 0.2431) and GUF (p = 0.0625). A significant difference was found only for the THI (p = 0.0001) questionnaire.When comparing patients with and without TMJ disorders within the study and control groups, a higher prevalence of the female gender was found in patients with a diagnosis of TMJ disorders in the study group (48.1% vs 17.8%) (p = 0.0037), while no significant differences for age (p = 0.4360), tinnitus length (p = 0.9629), and laterality (p = 0.8893) were found. Similarly, self-administered questionnaire scores did not differ. No significant differences for demographic and tinnitus characteristics and questionnaire scores were found between patients with and without TMJ disorders in the control group. Detailed results are presented in Relative proportions for gender and tinnitus laterality within groups are shown in Logistic regression analysis was performed in the study group to investigate demographic characteristics, tinnitus length, and questionnaire variables associated with a positive diagnosis of TMJ disorder; analysis indicated that male gender was 0.21 times less common in patients with TMJ disorders than in patients without . No differences were seen for age, questionnaire score and tinnitus length .TMJ disorders were classified according to DC/TMD Axis I classification. Results are shown in Among patients with TMJ disorders, 88 patients (67.2%) had a diagnosis of joint disorder , 38 (29%) of pain disorder  and 39 (29.8%) were classified as “other” . One hundred seven patients (81.6%) had only one clinical examination item (joint OR pain OR other), 13 (9.9%) had two concomitant items  and 11 (8.5%) had all three items. No significant differences were found between patients in the study and control groups for TMJ disorder type and number of concomitant clinical examination items.The present study evaluated the presence of TMJ disorders in tinnitus patients with normal hearing matching the criteria proposed by the authors for somatic tinnitus compared to a sample of patients with similar demographic and tinnitus characteristics that did not match such criteria. TMJ disorders were diagnosed according to standardized diagnostic criteria. A significantly larger number of patients in the study group received a clinical diagnosis of TMJ disorder compared to the control group. These results suggest that history and modulation of tinnitus, when occurring simultaneously in the TMJ region, may have a role for the preliminary selection of tinnitus patients that are more likely to have a TMJ somatic disorder, although an association between somatic disorders and pure somatic origin of tinnitus in these patients cannot be confirmed by the present data. Our findings are also supported by the prevalent theories in the current literature , 27, 37.About 70% of the patients with a clinical diagnosis of TMJ disorder had joint disorders, followed by pain disorders and other disorders of the TMJ. This is consistent with Buergers, who reported that most patients in their sample had joint disorders . Most ofLogistic regression analysis indicated that female gender was more prevalent in patients with TMJ disorders than in patients without TMJ disorders in the study group; this is consistent with results previously reported by other authors that indicated a higher prevalence of female gender in patients with TMJ disorders , 26, 48.No significant differences were found for tinnitus characteristics, such as pitch and duration, between patients with and without a diagnosis of TMJ disorder. This is consistent with previous findings from Wright et al and Vernon et al , 49.The association of tinnitus with somatic disorders has been reported by many authors , 50–53; The present study shows that a larger number of patients in the study group compared to the control group received a clinical diagnosis of TMJ disorder; however, a direct correlation between the TMJ disorder and tinnitus can be only speculative. Future studies on the effects of specific TMJ therapy in patients selected according to these criteria are necessary to confirm the direct relationship between tinnitus and the underlying TMJ disorder in these patients.This study has some limits. The effects of TMJ disorder treatment on tinnitus have not been investigated, as the present study was limited to the diagnosis of TMJ somatic disorders in patients matching the proposed criteria. Although tinnitus improvements in patients with somatic tinnitus have been extensively described after treatment of TMJ disorders by many authors , 25–32, Hidden and high-frequency hearing loss were not studied in our patients; audiological evaluation investigated frequencies up to 8 kHz and was not extended to higher frequencies. Given the spread of hidden hearing loss among the general population, and especially among tinnitus sufferers , 55, theThe significantly higher number of clinically diagnosed TMJ disorders in patients with chronic tinnitus and normal hearing matching the proposed criteria compared to subjects in the control group suggests that self-reported history for somatic dysfunction and modulation of tinnitus, when occurring simultaneously in the TMJ region, can be useful to preliminarily identify patients with TMJ disorders. Given the elevate correlation between somatic disorders and tinnitus reported in the literature, such criteria should always be investigated when approaching tinnitus patients, as they could be relevant for specific tinnitus patient subtyping.


The evidence on diagnostic test accuracy (DTA) of diabetic retinopathy (DR) screening utilising photographic studies by non-ophthalmologist personnel in low and middle-income country (LMIC) settings is scarce. We aimed to assess DTA of DR screening using a nonmydriatic hand-held digital camera by trained general physicians in a non-ophthalmic setting.This study is a validation of a screening intervention. We selected 700 people with diabetes (PwDM) > 18 years of age, not previously screened or treated for DR, presenting at a tertiary medical clinic in Sri Lanka. Two-field retinal imaging was used to capture fundus images before and after pupil dilatation, using a hand-held non-mydriatic (Visuscout 100®-Germany) digital retinal camera. The images were captured and graded by two trained, masked independent physician graders. The DTA of different levels of DR was assessed comparing physician’s grading with a retinologist’s clinical examination by mydriatic bio-microscopy, according to a locally adopted guideline.Seven hundred eligible PwDM were screened by physician graders. The mean age of participants was 60.8 years (SD ±10.08) and mean duration of DM was 9.9 years (SD ±8.09). Ungradable image proportion in non-mydriatic imaging was 43.4% . This decreased to 12.8%  following pupil dilatation. In comparison to detection of any level of DR, a referable level DR (moderate non-proliferative DR and levels above) showed a higher level of DTA. The sensitivity of the defined referable DR was 88.7% (95% CI 81.7–93.8%) for grader 1  and 92.5% (95% CI 86.4–96.5%) for grader 2 (PPV 68%), using mydriatic imaging, after including ungradable images as screen positives. The specificity was 94.9% (95% CI 93.6–96.0%) for grader 1  and 96.4% (95% CI 95.3–97.3%) for grader 2 (NPV 99.4%).The Physicians grading of images from a digital hand-held non-mydriatic camera at a medical clinic, with dilatation of pupil of those who have ungradable images, provides a valid modality to identify referable level of DR. This could be a feasible alternative modality to the existing opportunistic screening to improve the access and coverage.ISRCTN47559703. Date of Registration 18th March 2019, Retrospectively registered.Current Controlled Trials The online version of this article (10.1186/s12886-019-1092-3) contains supplementary material, which is available to authorized users. Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM), leading to sight loss if not detected and treated in time . The IntThe most common method of detecting DR in LMICs is direct ophthalmoscopy and slit-lamp bio-microscopy. The direct ophthalmoscopy has a low sensitivity and specificity even at the hands of experienced eye care specialists . The mydDifferent models of DRS have been implemented in many parts of the world. In resource poor LMICs development of a DRS model is complex . The lacRetinal fundus photography is the most common DRS method used globally  and digin = 25 participants, 49 eyes) [Hand held retinal cameras use for DRS in various settings and outcomes mainly depend on the image quality. Yogesan et al., 2000) reported that images captured by a hand-held camera were not suitable for tele-screening due to poor quality . However in this study sample size was very low (49 eyes) . A study00 report49 eyes) . A revie49 eyes) . TherefoTo the authors’ knowledge there is no evidence on DRS using digital retinal imaging from Sri Lanka. A situational analysis conducted in the Western province showed a large gap in DRS services delivery compared to the estimated need . This stEthics approval was obtained from both ethics review committees of the London School of Hygiene & Tropical Medicine-United Kingdom and the National Eye Hospital-Sri Lanka. This study adhered to the tenets of the ‘Declaration of Helsinki’ and written informed consent was obtained from all participants. A prospective screening intervention validation study was conducted between May 2017 and May 2018 at a tertiary level, public sector out-patient medical clinic in the Western province of Sri Lanka. The main outcome measure was detection of signs of DR (any DR or a referable level) by physician graders using captured digital images, according to a locally adopted guideline. The protocol of this validation study has been published in Journal of Medical Internet Research (JMIR-doi:10.2196/10900) and a summary is outlined below .0 field of view angle. The camera comprised of 9 fixation targets and resolution of the camera is 800 × 480 (5 megapixels). Guidelines were used to standardize reporting of image quality, and ungradable images were classified based on the proportion of the retina visible for grading , identification of signs of DR using images and DR grading according to an adopted classification system based on the United Kingdom - National Screening System  , with an expected sensitivity of 70% and prevalence of moderate NPDR among PwDM of 20%. This included an additional 25% to take account of ungradable images . Interim analysis was undertaken to ascertain the level of ungradable images and, to take account of a higher than expected proportion of ungradable images, the sample size was increased to 700 PwDM. A consecutive sample (n = 700) of diagnosed PwDM (> 18 years) without previous DRS at an eye clinic who were included in the study following written informed consent. Participants were identified at a tertiary level medical clinic, in the Western province of Sri Lanka.A sample size of In the index test imaging, two-field   were graded by the retinologist to calculate inter-grader agreement.For quality assurance, 15% of each non-mydriatic and mydriatic image sets were evaluated by the retinologist for technique, ability to image the required field and gradability. Fifteen percent of each hundred image sets were given back to the physician graders for double grading to assess the repeatability and intra-grader agreement in the 1st and 2nd attempts of grading images. A sample of the same image sets (Data were entered in to an MS Excel-16.0 worksheet and transferred to SPSS-Version-20.0 (Armonk-NY-IBM Corp-2011) and STATA/IC-Version-14.2  for analysis. DTA variables of sensitivity, specificity and predictive values and agreement analyses (kappa statistics) were calculated with 95% confidence intervals, for each grader and each pupil status compared to the reference standard using individual eyes as the unit of analysis, considering each gradable eye as a separate case. Two approaches were used in the calculations to examine the impact of ungradable images on the outcomes. i.e., by excluding the ungradable images and by including ungradable as test positive in the analysis. As ungradable images indicate a requirement for referral to an eye clinic, we analysed ungradable images as screen positives to examine the sensitivity and specificity of detecting a need for referral. This also allows comparisons with previous studies, which have used both methods.Subgroup analysis conducted for identification of presence/absence of DR (any DR), moderate NPDR and above with / without macular signs, to make recommendations for a referable criterion for the local context. We used different referable criteria i.e., by pupil status, level of DR, level of visual acuity and presence of macular signs in the analysis to understand the variation in DTA to assess the most suitable and accurate cut off level of DR without overloading the eye clinic and also facilitating safe practice at a non-ophthalmic setting.Of the 826 eligible PwDM identified from medical clinical records, response rate was 84.7% (700/826). Mean age of the participants was 60.8 years SD ±10.08) and majority were women . Only 27.9% (195/700) of the participants were employed and 79.1% (554/700) lived in the capital city of Colombo and hailed from low income families . Of these, 98.4% (689/700) had type 2 DM and 1.6% (11/689) were diagnosed with DM at age < 30 years and were on insulin. The mean age at diagnosis of DM was 50.9 years (SD ±11.03) and mean duration of diabetes was 9.9 years (SD ±8.09). Mean fasting plasma glucose with in the last 3 months was 140.4 mg/dl (SD ±55.43). Additional co-morbidities included; hypertension (70%), hyperlipidaemia (57.3%), ischaemic heart disease (31.9%), nephropathy (9%) and neuropathy (35%). The Table 0.08 and n = 69-no consent and n = 57-did not attend for imaging)  we left with 1342 image sets (by eyes) in DTA analysis. Overall there were 1041 DR positive eyes and 301 DR negative eyes as identified at the reference test. The technical failure rates by the area of visibility of the retinal fields for each image set in the index test by pupil status and grader level (by eyes) are described in the Table Seven hundred PwDM were included in the study and 126  were excluded  in grader 1 and 78.3% (95% CI 73.7–82.5%) in grader 2. However, since they were referring a higher proportion of ungradable, probably those who did not have the disease, specificity values dropped to 70.4% (95% CI 67.6–73.1%) in grader 1 and 76.2% (95% CI 73.6–78.7%) in grader 2 in non-mydriatic imaging. In mydriatic imaging when we included the ungradable images in the analysis sensitivity was 79.3% (74.7–84.8%) in grader 1 and 78.0% (95% CI 73.4–82.2%) in grader 2. The specificity value of grader 1 was 89.2% (95% CI 87.2–90.9%) and grader 2 was 91.5% (95% CI 89.7–93.1%). The sensitivity, specificity, NPV, PPV and kappa agreement at different levels of DR after including the ungradable images are described in Table The DTA estimates were calculated after excluding ungradable images (< 50% of the field visible) in the next step as an accuracy measure of the modality. In the comparison physician’s grading using 2-field imaging against the clinical reference standard, in detection of any level of DR, there was no significant difference in DTA by pupil status, in each grader. Similar results were observed in detection of macular signs. Table As a pragmatic approach for a resource poor non-ophthalmic setting, we reported the DTA of DRS using non-mydriatic imaging and dilatation of the pupils of only those who have ungradable images (two-step process). In this sub-analysis, the eye which was ungradable even following mydriasis were considered as screen positives. We derived a sensitivity of referable level of DR 81.1% (95% CI 72.9–87.9%) for grader 1 and 82.1% 95% CI 74.0–88.6%) for grader 2. The specificity values were 95.4% (95% CI 94.2–96.5%) for grader 1 and 97.1% (95% CI 96.1–97.9%) for grader 2 in this approach. We observed an improved level of PPV (59.7–70.2%) and NPV (98.4–98.5%) in this strategy. The details are described in Additional file .0–88.6% We combined the DTA of the detection of referable level DR (moderate NPDR and above) with positive macular signs, using non-mydriatic imaging, where the sensitivity was 79.0% for grader 1 and 70.8% for the grader 2. These estimates improved to 84.5 and 85.8% respectively for grader 1 and 2 after dilatation. For the same referable level specificity values were 96.6 and 98.0% for grader 1 and 2 respectively and there was no significant change with the pupil dilatation .We also incorporated visual acuity threshold for referrals  and found a sensitivity of grader 1 was 98.3% (95% CI 94.9–99.7%) and grader 2, 97.4% (95% CI 93.5–99.3%). However, in the same referable level specificity values showed an overall reduction , probably due to high number of PwDM referred to the next level without ≥ moderate NPDR. These approaches will be useful in making recommendations for a referable level for a non-ophthalmic setting.The percentage of image gradability agreement, between index graders and retinologist (inter-grader agreement), in non-mydriatic imaging; grader 1 was 85.2%  and grader 2, 78.5% . In mydriatic imaging, inter-grader gradability agreement of grader 1 was 76.2%  and grader 2, 72.7% .We proposed grading the same images by the retinologist would provide a fair comparison for the physician graders in agreement analysis. However, here the concerns were limitations in the degree of view of a hand-held retinal camera and skills of capturing images by the physicians. In this analysis, in the grading of DR and macular signs, we found that inter-grader agreement was mostly > 0.8 except for the grader 2 non-mydriatic images . We observed a satisfactory level of agreement of the physician graders findings using a 2-field modality. The overall concordance of the results is described in Table The index graders re-graded the coded image sets in a masked fashion independently without having access to the first attempt data. In this, first attempt vs second attempt weighted kappa agreement was calculated to assess the repeatability of DR grading at level of retinopathy. The kappa value of grader 1 was 0.69 (95% CI 0.60–0.78) and grader 2, 0.66 (95% CI 0.58–0.73). In comparison of grader 1 vs grader 2, first attempt kappa was 0.82 (95% CI 0.76–0.89) and in second attempt it was 0.74 (95% CI 0.66–0.83%)  had lens opacity. Among these 29.8% (85/285) eyes had significant level of lens opacity which required cataract assessment. Following reference test 37 eyes were identified as having lens opacities that required urgent cataract surgery. Overall, 75.6% of the participants had no DR, 16.7% mild DR (R1), 3.6% moderate NPDR (R2), 0.4% severe NPDR (R3) and only 1% had PDR (R4). Among the ungradable images in non-mydriatic imaging, 66.5% (274/412) had no retinopathy (R0), 19.9%-mild NPDR (R1), 1.7%-moderate NPDR (R2), 0.7%-severe NPDR (R3) and 1.5%-proliferative DR (PDR-R4)  . Six hundred and ninety-two PwDM completed the reference test examination and 98% (684/692) of them underwent retinologists examination < 4 weeks period.We demonstrated that DRS by general physicians using a mydriatic two field technique was a feasible modality to detect a defined level of referable DR  in a non-ophthalmic setting, considering the level of DTA achieved. This may be suitable for LMIC settings where it would be difficult to implement full population based DRSP due to resource and information constraints. Compared to a locally accepted clinical reference standard, DRS using mydriatic 2-field strategy by general physicians showed an accepted level of DTA which most of the HIC screening programs follow (sensitivity of > 80% and specificity of > 95%) . We assuDigital retinal imaging showed promising results in DRS . The digThe optimum number of retinal fields in a DRS strategy is a key factor that affects accuracy. The ETDRS 7-field strategy is considered to be the gold standard for DR detection but is not practical in a screening program . PreviouIn some settings, several non-ophthalmological personnel had been employed in DRS. In our study we proposed DRS by trained general physician at medical clinic following assessment of barriers. The estimates from previous studies are comparable with the finding of our study . In a stIn our study 75.6% of the participants had no DR, 16.7% mild DR (R1), 3.6% moderate NPDR (R2), 0.4% severe NPDR (R3) and only 1% had PDR (R4). A study conducted among the slum populations  in India, using a hand-held nonmydriatic camera reported 8.1% severe NPDR and 6.8% PDR which are higher prevalence than our study . One reaNon-mydriatic imaging has lower resolution and lower image quality leading to poorer detection of DR , 49. HowWe excluded PwDM with previous eye screening or treatment, which reduced the proportion of people with DR, which may have introduced spectrum bias. However, the resulting sample included a wide range of pathologies, albeit with fewer people with more advanced disease. When considering any DR as referable level, there were 301 DR positive and 1041 DR negative eyes in the analysis. There were only 69 DR positive eyes when considering moderate NPDR and above as the referable level. However, PwDM who already visited the eye clinic would not usually participate in a screening programme, therefore the sample examined in this study reflects the PwDM who would be eligible for DRS. Another limitation was high proportion of ungradable images from non-mydriatic imaging compared to other studies. However, the populations in LMICs have a higher prevalence of untreated cataracts, which would prevent adequate retinal view and would require referral. In a potential DRSP, these participants will be referred to the next level of eye care and the patient would benefit from the imaging even if the DR status remains unknown.The most common gold standard for a reference test would be the ETDRS 7-field image grading by an expert grader. However, it was not possible in this setting for a large sample due to resource and time limitations. In addition, any misclassifications in the clinical reference test could have been mitigated, with a second reference grader. In order to have a higher precision of the DTA, the sample size should be adjusted according to the reported low prevalence of higher grades of DR such as severe NPDR and PDR.Our proposed DRS modality of using a hand-held non-mydriatic retinal camera at a medical clinic may be more appropriate for a resource poor LMIC setting, with the rising prevalence of DM. However, the caution is quality of the images. Our findings may not applicable to a HIC setting where there are more resources and avenues for development of a population-based DRS program using table-top digital imaging systems. On the other hand, this modality can be piggy back in a population-based program in any setting, to improve the access.In this study we demonstrated that the diagnostic test accuracy of the physician graders was closer to the standard practice of national level screening programs in other settings. We conclude that 2-field retinal imaging using a hand-held digital camera at a medical clinic, by physician graders, with dilatation of pupil of those who have ungradable images, provides a valid modality to identify referable level of diabetic retinopathy. This strategy is an accurate screening method of detection of a referable level in a health care facility-based people with diabetes who are at risk of developing sight threatening diabetic retinopathy.Additional file 1:Figure S1. Evaluation of image quality. Figure S2. Two retinal images captured. Table S1. Diabetic retinopathy classification system (DOCX 989 kb)Image quality evaluation and diabetic retinopathy grading classification system. Additional file 2:Detailed flow chart of the number of participants and image sets used in the analysis (DOCX 57 kb)Additional file 3:Prevalence of lens opacity and other condition that would affect image gradability and reference test examination (DOCX 16 kb)Additional file 4:Diagnostic test accuracy for two step grading process (DOCX 14 kb)Additional file 5:Intra-grader agreement analysis of double grading (DOCX 13 kb)