Due to differences in the circulation of influenza viruses, distribution and antigenic drift of A subtypes and B lineages, and susceptibility to infection in the population, the incidence of symptomatic influenza infection can vary widely between seasons and age-groups. Our goal was to estimate the symptomatic infection incidence in the Netherlands for the six seasons 2011/2012 through 2016/2017, using Bayesian evidence synthesis methodology to combine season-specific sentinel surveillance data on influenza-like illness (ILI), virus detections in sampled ILI cases and data on healthcare-seeking behaviour. Estimated age-aggregated incidence was 6.5 per 1000 persons : 4.7–9.0) for season 2011/2012, 36.7 (95% UI: 31.2–42.8) for 2012/2013, 9.1 (95% UI: 6.3–12.9) for 2013/2014, 41.1 (95% UI: 35.0–47.7) for 2014/2015, 39.4 (95% UI: 33.4–46.1) for 2015/2016 and 27.8 (95% UI: 22.7–33.7) for season 2016/2017. Incidence varied substantially between age-groups . Integration of all relevant data sources within an evidence synthesis framework has allowed the estimation – with appropriately quantified uncertainty – of the incidence of symptomatic influenza virus infection. These estimates provide valuable insight into the variation in influenza epidemics across seasons, by virus subtype and lineage, and between age-groups. Characteristics of the seasonal influenza epidemic that annually occurs in the northern hemisphere winter vary greatly between seasons . Although an epidemic can be expected to occur every winter, the attack rate, timing, affected age groups and therefore the disease burden of the epidemic can vary widely. Of the drivers of variation in transmission intensity between seasons , the domAt the national level, a frequently applied – although indirect – approach for monitoring the seasonal influenza epidemic and measuring variation in intensity across seasons is through the incidence of influenza-like illness (ILI) from sentinel general practitioner (GP) networks, and/or the incidence of severe acute respiratory infection (SARI) from hospital surveillance . Becauseinfluenza-associated ILI or SARI incidence is the recommended indicator, which takes into account the proportion of ILI/SARI cases that are influenza virus-positive [Furthermore, for the calculation of population-level influenza burden   in the Netherlands, stratified by season and by age-group and to compare SI incidence across seasons according to the predominantly circulating influenza virus A subtype and B lineage. These stratified SI incidence estimates are vital for the calculation of disease burden, economic burden  and for investigating the effects of preventive measures at the national level [In the current study, we aim to estimate the incidence of al level . AlthougIn a previous study, we had estimated SI incidence for a ‘typical’ influenza season within the Bayesian multi-parameter evidence synthesis (MPES) framework, by aggregating the relevant data over the period 2005–2007 . Here, wIn the main analysis – and as an improvement over previous work  – we resThe number of patients that consulted their GP with ILI were obtained from the Nivel Primary Care Database sentinel surveillance . Since thist from Ref. [Internet-based monitoring of ILI in the general population  proA subset of patients who presented with ILI at a Nivel sentinel GP were sampled by taking a nose swab and throat swab; these were combined into a single tube with virus transport medium for virological diagnostic testing by real-time reverse-transcription polymerase chain reaction (PCR) (RT-PCR) at RIVM . A patiea priori knowledge regarding model parameters underlying SI incidence, via Bayesian MPES [We implemented a previously published approach for estimating SI incidence by combining all relevant data with ian MPES . MPES isian MPES , 20, butTo investigate between-season variation in SI incidence according to influenza virus A subtype and B lineage, we also specified a model in which data were stratified by subtype (A(H1N1)pdm09 and A(H3N2)) and lineage . We report SI incidence by dominant subtype/lineage, and with respect to the presence of a co-circulating strain(s) (in which co-circulation was defined as two (sub)types that each represented 40–60% of all influenza virus detections). Due to low counts, it was not feasible to stratify by subtype/lineage and age-group concurrently.NSI, stratified by season and age-group, can be estimated using the ‘multiplier method’, as:NILI is the total number of ILI patients from sentinel surveillance, δGP|ILI refers to the proportion of persons reporting ILI who consult their GP (from Influenzanet), ρInfl is the influenza virus positivity rate from virological testing and SeInfl is the estimated clinical sensitivity of this test. The above equation represents a ‘user-friendly’ version of the directed acyclic graph (DAG) stipulating the relationships (stochastic and deterministic) between the parameters and data sources  that comprise the MPES model.The incidence of SI, NSI. Data sources and other required parameters are described in The main task of the MPES model is to estimate i and season i − 1 is multifactorial; for instance, genetic drift of the virus, subtype and lineage dominance, waning immunity rate [i and season i − 1. Thus, we defined the priors for the ILI rates to allow for potential autocorrelation, by specifying an autoregressive process of order 1, AR(1).Our model was developed under the assumption that ILI incidence rates over the different seasons should not be considered as independent data. Considering ILI incidence rates over a long (21-seasons) analysis period, there is a small positive lag-1 correlation in this parameter apparent for all age-groups . We reasoned that for identical parameters estimated across multiple seasons, one should allow for possible non-independence of these parameters across time. The origin of a positive temporal dependence in ILI incidence rates between season ity rate , and relThe age-aggregated incidence rates of SI with influenza per 1000 persons were 6.5 (95% UI: 4.7–9.0) for season 2011/2012, 36.7 (95% UI: 31.2–42.8) for 2012/2013, 9.1 (95% UI: 6.3–12.9) for 2013/2014, 41.1 (95% UI: 35.0–47.7) for 2014/2015, 39.4 (95% UI: 33.4–46.1) for 2015/2016 and 27.8 (95% UI: 22.7–33.7) for season 2016/2017; see Supplementary Material, Table S1. In the total Dutch population, these rates correspond to 109 thousand, 616 thousand, 153 thousand, 694 thousand, 670 thousand and 475 thousand SI cases in seasons 2011/2012 through 2016/2017, respectively.The incidence of SI was highest for the age-group <5 years in four out of six seasons , mainly due to the low proportion of influenza positives among swabbed ILI patients in this stratum. In contrast to SI incidence, ILI incidence was always underestimated by medically attended ILI , because underascertainment factors were always positive.The age-group pattern differed if influenza virus positivity and underascertainment were excluded from the MPES; considering only those ILI patients who consult their GP, the highest and second highest ILI incidence rates were observed for the <5 and 65+ years age-groups, respectively . Furthermore, reliance on medically attended ILI rates alone would either under- or over-estimate SI incidence . For instance, in the 2016/17 season ILI underestimated SI incidence in all age-groups under 65+ years, yielding multiplication factors of 1.4–2.9, but in season 2011/12 among persons aged 65+ years SI incidence was substantially In five of the six seasons assessed, an influenza virus A subtype was dominant in terms of the highest SI incidence among all four subtypes/lineages . For seaTotal SI incidence tended to be highest in those seasons in which both an A subtype and a B lineage were dominant contributors. Aggregating over all six seasons, A(H3N2) resulted in more symptomatic infections than any of the other subtypes/lineages ; the next highest was A(H1N1)pdm09 .We estimated the incidence of symptomatic infection with influenza virus in the Netherlands, stratified by season and by age-group and compared SI incidence across seasons according to virus A subtype or B lineage . This moAccounting for underascertainment of ILI represents an improvement over influenza intensity indicators that are restricted to medically attended ILI cases; measurement of GP-attended ILI only yields divergent age-group patterns compared with SI incidence  and thus would invite a different interpretation. SI incidence estimates are also important for estimating the subtype/lineage- and age-stratified morbidity burden of influenza, and they may serve as inputs for cost-effectiveness studies that assess national influenza prevention strategies such as vaccination.Interestingly, in the 2015/2016 season, in which influenza virus subtype A(H1N1)pdm09 was dominant, estimated SI incidence was lowest for the 65+ years age-group, which is consistent with the observation that people born before 1957 have better pre-existing immunity to influenza virus subtype A(H1N1) that was dominantly circulating from 1918 until 1957 , compareThe well-established influenza surveillance in Europe is primarily based upon medically attended ILI incidence, to monitor the characteristics – timing and intensity – of an influenza season. In the Netherlands, the long-standing sentinel GP surveillance system has provided extremely valuable information on ILI since 1970 . HoweverδGP|ILI is narrower than in reality. Note that this also applies to other observed data, for which no information on measurement error was available. Third, the precision of influenza virus positivity rates was limited by the relatively few patients swabbed per GP practice per week. However, restriction of analyses to the winter period (week 40 through week 20) ensured that weeks with extremely sparse data did not contribute to seasonal totals. Additionally, it was assumed that the swabbed ILI patient population is an unbiased representation of all ILI patients who consult the GP, but sampling of ILI patients conducted randomly.Strengths of our study include the use of all available, relevant data sources for the estimation of SI incidence and the extension of our previous approach  to proviFinally, GP sentinel surveillance only delivered data for community dwelling elderly persons. The elderly living in nursing homes were excluded, as they would normally not consult physicians outside the facility. This may have affected the estimated SI incidence for the 65+ years age-group. However, in 2015 approximately 117 000 persons lived in nursing or elderly homes in the Netherlands ; this fiIn conclusion, integrating all relevant data sources – season-specific ILI surveillance, virological testing and data on healthcare-seeking behaviour – has allowed estimation of SI incidence. Compared with the ‘direct method’, the evidence synthesis framework adds value in terms of flexibility , and appropriately quantified uncertainty. These estimates provide valuable insight into the variation in influenza epidemic intensity across seasons, by influenza virus A subtype and B lineage and between age groups.


The aims of this study were to determine the recurrence rate and the prognostic factors for recurrence-free survival (RFS) in esophageal squamous cell carcinoma (ESCC) patients without lymph node metastasis (LNM).Between January 2011 and June 2017, 101 patients with ESCC were treated and pathologically confirmed to be lymph node negative. The clinicopathological parameters were evaluated to identify the prognostic factors for RFS using Cox proportional hazards models.n = 11; 57.9%), followed by distant recurrence ; one patient developed local and distant recurrence simultaneously. The results of multivariate analysis showed that the independent prognostic factors for decreased RFS in node-negative patients were a tumor located in the upper chest , 1.523–14.916, P = 0.007), the presence of lymphovascular invasion , and a preoperative serum carcinoembryonic antigen level ≥ 5 μg/ml .Nineteen out of 101 patients (18.8%) developed recurrence, and the median RFS was 41 months. The most common pattern of relapse was local recurrence (The aforementioned parameters were the prognostic factors in node-negative ESCC patients, and they associated with a higher probability of recurrence after surgery. These patients should be followed closely, and adjuvant therapy should be considered. Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor occurring in Chinese individuals . At presIn the present study, we evaluated the recurrence rate and analyzed the prognostic factors for recurrence-free survival (RFS) in node-negative patients at a single institution. Our findings are likely to provide a reference point for clinicians to better assess the degree of tumor malignancy and to influence the use of adjuvant treatment in node-negative patients.This study was approved by the Ethics Committee of the Peking University First Hospital. We retrospectively reviewed data from 301 patients with esophageal cancer (EC) who underwent esophagectomy for curative intent between January 2011 and June 2017 in the Thoracic Department. The following were excluded: (1) patients with a pathological type of nonsquamous cell carcinoma; (2) patients with pathologically confirmed LNM; (3) patients with tumors that had a positive margin (R1 or R2); (4) patients who received neoadjuvant or adjuvant chemotherapy or chemoradiotherapy; (5) patients who died of non-neoplastic causes; and (6) patients who were lost during follow-up.Preoperative examinations, which included a thorough physical examination, a thoracic computed tomography (CT) scan, a bone scan, an endoscopic examination and a biopsy, a cervical ultrasound, an abdominal CT scan or an ultrasound, a cardiopulmonary function test, and a hematological profile, were performed to rule out distant metastasis and to evaluate the feasibility of surgery. Positron emission tomography–computed tomography (PET-CT) scans were not routinely performed in our department. They were only used for the preoperative examination of patients with highly suspected metastases, such as lymphadenopathy (> 1 cm in diameter on a CT scan).All operations were performed under general anesthesia and double lumen tracheal intubation by qualified surgeons. The surgery consisted of transthoracic esophagectomy and at least two field en-bloc lymphadenectomies . Cervical lymph node dissections were only reserved for patients with suspected supraclavicular LNM before surgery. Gastric conduit was used for esophageal reconstruction in all patients, except one. The tumor was resected with a negative margin distance > 5 cm. For the resection of the upper ESCC, however, the distance of the proximal margin from the tumor might be < 5 cm, such that the proximal clearance was > 3 cm at least. There were two surgical approaches: (1) the right-sided transthoracic approach, where esophagectomy and mediastinal lymphadenectomy were performed through the right thoracic cavity. Stomach mobilization and abdominal lymph node dissection were performed at the abdominal stage. An anastomosis was made at the top right thoracic cavity or the left neck. The right-sided approach was used for cases of thoracic ESCC, regardless of the tumor location, except for those tumors located at the esophagogastric junction (EGJ). In the left-sided transthoracic approach, esophagectomy and thoracic and abdominal lymph node dissection were performed through the left thoracic cavity. An esophagogastric anastomosis was made underneath the aortic arch or at the left neck. The left-sided approach was used for cases of middle and lower ESCC and for tumors located at the EGJ. The extent of lymph node (LNs) dissection was as follows. During the left-sided procedure, the LNs at the paraoesophageal region , the mediastinum , and the upper abdominal region  were removed. During the right-sided procedure, in addition to the LNs that were dissected in the left-sided approach, the LNs along the bilateral recurrent nerves were also removed. After surgery, the specimens were examined by an experienced pathologist to determine whether LNM was present or absent and whether the surgical margin was free of the tumor (R0 resection). When the distance of the proximal margin from the tumor was less than 1 cm, the pathologist reported the precise distance between the surgical margin and the tumor.General information: age, sex, smoking history, body mass index (BMI), preoperative serum carcinoembryonic antigen (S-CEA) level, and preoperative serum squamous cell carcinoma antigen (S-SCC) level.Surgery-related information: surgical approach, operation time, blood loss during the surgery, and postoperative complications .pT stage), total number of dissected lymph nodes (TLN), extent of lymphovascular invasion (VI), extent of perineural invasion (NI), tumor grade (well-differentiated (G1) /moderately differentiated (G2)/poorly differentiated (G3)), and TNM stage. The dissected lymph nodes were evaluated by well-trained pathologists. Each patient was assigned a pathological staging, according to the AJCC TNM Classification of Carcinoma of the Esophagus and Esophagogastric Junction (8th Edition) [Postoperative pathology: tumor size, depth of tumor invasion  was calculated from the operation to the last follow-up or death. The recurrence-free survival (RFS) was calculated from the date of surgery to the first recurrence. Based on the location of the relapse, recurrence was divided into locoregional recurrence or distant metastasis. Locoregional recurrence was defined as a recurrence restricted to the anastomotic site, the area of the original tumor bed or regional lymph nodes . Distant recurrence was defined as any recurrence with features beyond those of a locoregional recurrence.P < 0.1 in univariate analysis were included in multivariate Cox regression analysis. All statistical tests were 2-sided, and a P-value < 0.05 was considered statistically significant.SPSS 22.0 software  was used for statistical analysis. Continuous variables were expressed as the mean ± standard deviation (SD) or median (range). Categorical variables were expressed as a percentage. All variables, including the demographic data, operative information and tumor characteristics, were analyzed. Kaplan–Meier methods were used to construct the RFS curves. The survival difference of each variable on RFS was analyzed by the log-rank test. The number of patients at risk was calculated for the beginning of each time period. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. Variables that had a significance level of n = 8); (2) patients with pathologically confirmed LNM (n = 147); (3) patients with tumors that had a positive margin (R1 or R2) (n = 1); (4) patients who received neoadjuvant or adjuvant chemotherapy or chemoradiotherapy (n = 34); (5) patients who died of non-neoplastic diseases (n = 8); and (6) patients who were lost during follow-up (n = 2). As a result, 101 patients were enrolled in this study.There were 301 patients with EC who were treated with surgery during this period. The following were excluded: (1) patients with a pathological type of nonsquamous cell carcinoma (P < 0.001) , with a median age of 64 years (range: 47–85 years). The mean S-CEA and S-SCC levels were 2.6 ± 1.5 μg/ml (range: 0.6–8.7 μg/L) and 2.5 ± 7.0 μg/ml (range: 0.3–70.0 μg/ml), respectively. The tumor was located in the upper third of the esophagus in 11 patients (10.4%), the middle segment of the esophagus in 58 patients (56.5%), and the lower third of the esophagus and the gastroesophageal junction in 32 patients (33.0%). Surgery was performed using left and right approaches in 44.3 and 55.7% of patients, respectively. Complications developed in 27.7% of patients after surgery. The TLN was 23 (8–56) for the entire study population. The median number of TLNs stratified by surgical approach was 23 (9–55) for the left-sided approach and 22.5 (8–56) for the right-sided approach, respectively. Among the 101 patients, 9 (8.9%) underwent cervical LN dissection or sampling, and the median number of removed LNs was 3 (1–9). All patients underwent thoracic and abdominal LN dissection, and the median numbers of removed LNs were 13 (1–41) and 9 (1–25), respectively. VI and NI were present in 5.9 and 14.9% of patients, respectively. The tumor grade was well differentiated (G1) in 16 patients (15.8%), moderately differentiated (G2) in 70 patients (69.3%), and poorly differentiated (G3) in 15 patients (14.9%). There were 51 patients (50.1%) with stage I disease and 50 patients (49.9%) with stage II disease. Among the 101 patients, 19 (18.8%) patients experienced recurrence. Compared with patients without recurrence, the prognosis of patients with recurrence was significantly worse , followed by distant recurrence ; one patient developed local and distant recurrence simultaneously. The details of the recurrences are presented in Table Follow-up was performed until October 2018 or recurrence. The median follow-up time for the 101 patients was 41 months (range: 4–92 months). For the 19 patients who developed recurrence, recurrence  usually occurred within the first three years after surgery. Recurrence peaked within the first year  and then dropped rapidly after the third year after surgery (1 patient per year). The most common pattern of relapse was local recurrence (P = 0.011), presence of VI (P < 0.001), CEA level (P = 0.003), and tumor grade (P = 0.011) were significantly associated with the RFS of patients. The results of multivariate analysis using the Cox proportional hazards regression model with stepwise selection also demonstrated that the independent prognostic factors for decreased RFS in patients with node-negative disease were a tumor located in the upper chest , CEA ≥5 μg/ml  and the presence of VI . The details are summarized in Tables The results of univariate analysis indicated that tumor location      Fig. a, althouRecurrence was common for ESCC patients after esophagectomy. Previous studies have reported that the recurrence rate following curative radical resection by open thoracotomy ranged from 42 to 52% , 13. TheThe present study evaluated the risk factors that influence the development of recurrence in node-negative patients after radical esophagectomy and showed that the presence of VI, a primary tumor located in the upper chest, and a higher S-CEA level were independently associated with decreased RFS after esophagectomy. The presence of VI and NI has been increasingly reported as an adverse prognostic marker in various malignancies –16. A siP = 0.006). We put forward two explanations for this: Firstly, almost all tumors could be completely resected, regardless of location, with the strict selection of surgical patients and the improvement of surgical techniques. However, for tumors located in the upper chest, it was more difficult to achieve a wide resection than for those located in the middle and lower chest. Secondly, several studies have reported that patients with ESCC tumors located in the upper esophagus have a higher rate of cervical LNM [In regard to tumor location, previous studies have reported conflicting results. Eloubeidi and colleagues  reportedical LNM –23. Howeical LNM , 23, andical LNM , 23. TheTumor markers have been reported to associate with the prognosis of ESCC. Zhang and colleagues  studied P = 0.007). The degree of tumor differentiation has been reported to be associated with the prognosis in ESCC patients. Situ and colleagues5 studied 317 ESCC patients with stage T2N0M0 and found that tumor grade was an independent prognostic factor (P = 0.011). In a study of 292 patients, it was reported that histologic differentiation was an independent prognostic factor for the survival of patients with ESCC (P < 0.001) [Although the degree of tumor differentiation was not an independent prognostic factor in multivariate analysis, it was found to be a prognostic factor in the log-rank test (< 0.001) . The res< 0.001) .This study had several limitations. First, our study was a retrospective study with potential bias. Second, the number of patients included in our study was relatively small. Third, the follow-up time was relatively short, and long-term follow-up information was lacking.In conclusion, the presence of VI, a tumor located in the upper chest, and a preoperative S-CEA level ≥ 5 μg/ml were the prognostic factors in node-negative ESCC patients, and they associated with a higher probability of recurrence after surgery. These patients should be followed closely, and adjuvant therapy should be considered.


With prolonged survival and aging of persons with HIV on combination antiretroviral therapy (ART), hypertension has emerged as a significant cause of morbidity and mortality globally. However, little is known about the burden of this comorbid condition among adults living with HIV in sub-Saharan Africa. In this study, we aimed to determine the prevalence and factors associated with hypertension among HIV-infected patients receiving ART in Northeast Ethiopia.  A cross-sectional study was conducted at the ART clinic of Dessie Referral Hospital, Northeast Ethiopia, between January and May 2018. HIV-infected patients who were on ART for at least 12 months were included in the study. Demographic, clinical, and laboratory data were collected from each participant. Hypertension was defined as a systolic blood pressure (BP) of ≥140 mmHg and/or diastolic BP of ≥90 mmHg or a reported use of antihypertensive medication. Univariable and multivariate analyses were performed to identify factors associated with hypertension.  A total of 408 patients were studied with a mean (±SD) age of 37 ± 10.3 years, and 66.9% were female. The prevalence of hypertension was 29.7% . Nearly 75% of the patients with hypertension were previously undiagnosed. In a univariate analysis, older age, male gender, a family history of hypertension, duration of HIV infection, duration on ART, high body mass index, low CD4 count, diabetes, and renal impairment were associated with hypertension. Multivariate analysis revealed older age , male gender , longer duration on ART , high body mass index , and diabetes  as independent risk factors of hypertension.  Hypertension is highly prevalent among HIV-infected patients on ART attending our clinic in Northeast Ethiopia but is mostly undiagnosed. These findings highlight the need for integrating hypertension management into routine HIV care to prevent adverse outcomes and improve health of people living with HIV on ART. The widespread introduction of combined active antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection has dramatically reduced AIDS-related mortality and increased life expectancy among people living with HIV in both high- and low-income countries –3. With People living with HIV on combination ART are at an increased risk for hypertension than HIV-uninfected individuals , 11. DatWith the new “test and treat” policy of the WHO , it is oDetailed methods for the study have been published previously . In brieZ = the level of statistical significance with a 95% confidence interval (CI) of 1.96, and a precision level of 0.05, then the minimum sample size obtained was 384. After adding 10% to account for nonrespondents, a total of 422 HIV-infected individuals on combination ART were consecutively included in the study. Fourteen patients with missing clinical data were excluded, leaving 408 patients for the final analysis.The sample size was determined by considering 50% prevalence rate of hypertension. Using single population proportion formula, with the following parameters: 2). Blood pressure was measured in the right arm three times at an interval of at least 5 min using a manual sphygmomanometer, with the patient remaining in a sitting position for at least 15 min before the first measurement. The average of the three measurements was recorded.Eligible patients were approached and invited to participate while they were attending the ART clinic for their regular follow-up. For consenting patients, structured questionnaires were used to collect data on the sociodemographic, medical and family history, and behavioral  factors. Anthropometric data such as weight (in kilograms), height (in meters), and blood pressure (in mm Hg) were also measured. Body weight and height were measured in light clothes without footwear, and the body mass index (BMI) was calculated as weight divided by height squared , duration on ART, and types of ART regimens, were abstracted from patients' database. Blood samples were collected in the early morning after an overnight fasting. Fasting plasma glucose was measured by enzymatic GOD-PAP method as mg/dl using A25 Biosystems clinical chemistry analyzer . Serum creatinine was measured using Jaffe kinetic method (as mg/dl) for estimating glomerular filtration rate (eGFR) according to the Modification of Diet in Renal Disease (MDRD) study equation . CD4 cel2) and overweight/obese (BMI ≥25 kg/m2).Hypertension was defined as a self-reported use of antihypertensive medication and/or systolic blood pressure of ≥140 mmHg and/or diastolic blood pressure of ≥90 mmHg on at least two occasions. Diabetes mellitus was defined as fasting plasma glucose level of ≥126 mg/dl on two consecutive study visits  or a preχ2) test was used to compare categorical variables, whereas the t-test was used to compare continuous variables. Multivariate stepwise logistic regression analysis with backward selection of variables with univariate P values of <0.25 was conducted to identify factors independently associated with hypertension in the study population. Odds ratios (OR) and 95% confidence intervals (95% CI) were also obtained. A P value of <0.05 was used to indicate statistical significance.Data were entered into “EpiData version 3.1” and exported to SPSS version 20.0 statistical software for analysis. Data were reported using mean ± standard deviation (SD) for continuous variables and proportions for categorical variables. The chi-square (P=0.002). All patients were on combination ART, and overall 150 (36.8%) were on AZT regimen. Sixteen participants (3.9%) were current smokers, and 62 (15.2%) alcohol users, with significant sex differences (both P < 0.01). Men compared with women were more likely to have a family history of hypertension (P=0.048). Mean (SD) body mass index was 20.4 (2.1) kg/m2, with no difference between men and women (P=0.233). The mean (SD) systolic and diastolic blood pressures did not differ between men and women (both P > 0.05). Overall, the proportion of participants with diabetes mellitus was 8.8% and 9.8%, respectively, and had moderate to severe renal impairment (eGFR < 60 ml/min/1.73 m2), with significant difference between men and women .A total of 408 patients were included in the analysis . Demographic and clinical characteristics are shown in P < 0.001). Higher prevalence of hypertension was also observed in men than in women and in those with longer duration of HIV infection, longer duration of ART exposure, a family history of hypertension, high BMI, low CD4 count, diabetes mellitus, or impaired renal function (low eGFR). No significant difference was found in hypertension prevalence for the other characteristics recorded . Of the 121 patients with hypertension, 31 (25.6%) were already on pharmacotherapy and 90 (74.4%) were newly diagnosed. The prevalence of hypertension was significantly higher in older patients (older than 45 years: 53.8%) than in their younger counterparts , male sex , longer duration on ART , high BMI , and diabetes mellitus  were independently associated with an increased risk of hypertension , and it was previously undiagnosed in about three quarters (74.4%). The overall prevalence of hypertension in this study was comparable to 34% prevalence reported from Southwest Ethiopia  but high.7%, and The increased risk of hypertension observed among older HIV-infected patients on ART in our study is consistent with previous studies , 31, 34.The multivariate analysis of the present study however found an independent association between duration of ART exposure and the presence of hypertension, and this is consistent with other related studies , 23, 34.Higher BMI was found to be associated with an increased risk of prevalent hypertension in our patients. This finding was in agreement with the study from the country  and otheLimitations of the present study are mainly related to the observational design and cross-sectional nature of the current analyses. The results reported herein are only associations from which no conclusions regarding causality can be drawn. Given the drugs were studied in combinations, we were unable to assess the association between exposure to each of the individual ARV drugs and the risk of hypertension. On the other hand, information on other environmental factors, such as physical activity or diet and salt intake, was not collected. The stratification of renal function has been made based on the MDRD-4 formula, the validation of which is lacking among Ethiopian adults. Finally, our study reports data from large, public-sector government ART clinic, so our results may not be generalizable to the overall population.In conclusion, hypertension was highly prevalent among HIV-infected adults on ART attending our clinic in Northeast Ethiopia, but it was mostly undiagnosed. Older age, male gender, ART duration, high BMI, and comorbid diabetes were independently associated with hypertension. These findings highlight the need for integrating hypertension screening, diagnosis, and treatment into routine HIV care to prevent disease-related adverse outcomes and improve health of people living with HIV on ART.


Odds Ratios (ORs) for the risk of metabolic diseases increased from the lowest to highest TBF/CI quartile . Young adults with increased TBF/CI had higher risk of hyperhomocysteinemia (Hhcy) . There was a 1.407 increase in the odds of obesity, a 1.112 increase in the odds of hyperlipidemia (HLP) and a 1.094 increase in the odds of diabetes mellitus (DM) per standard deviation (SD) increase in TBF/CI . TBF/CI showed higher predictive values for obesity, HLP, DM and Hhcy than weight adjusted total body fat . Young adults with increased TBF/CI had higher ratios of metabolic diseases, which suggested that TBF/CI can be a good indicator and had a close relationship with metabolic diseases.The aim of the study was to evaluate the usefulness of conicity index (CI) adjusted total body fat (TBF), which was defined as TBF/CI, in various metabolic diseases in young adults. A cross-sectional study was carried out in Chinese PLA General Hospital and a total of 1365 young adults (age 20–40 years) who underwent a health check-up examination were finally included in the analysis from February 2016 to 2017. Linear Regression and logistic regression were used to further examine relationship between the index and metabolic diseases. The average age was 34.5 years.  As market aged with a booming economy and increasing material riches, metabolic diseases are becoming more prevalent among younger group. Scientists have paid more and more attention to the situation, but the evaluation markers were still outdated to assess the risk of metabolic diseases effectively.1. The technique has drawn more attention over the past years. Weight adjusted total body fat, measured by BIA, was closely associated with many diseases, including cardiovascular diseases, diabetes mellitus (DM) and osteoporosis et al.2.Body Composition Analysis, namely bioelectric impedance (BIA), is largely based on the perspective that the electric resistance varies in different human body tissues, thus low-level and safe current which pass through tissues can be detected specifically, quickly and painlessly1. However, the value of it remains controversial. Several reports suggested that the trend of it and BMI differed in adults, thus there was no agreement about cut-off points for it to diagnose obesity3. Furthermore, the application of it is less convenient than BMI. Nevertheless, as the understanding of BIA gradually deepen, appendicular skeletal muscle mass index (ASMI), measured by BIA and then calculated by different formulas, have been used as indicators for the diagnosis of sarcopenia in the elderly people4. But the fomulas of weight adjusted body fat and ASMI could not consider the adipose distribution of participants well, even adjusted by BMI.Previous studies indicated that weight adjusted total body fat may be a better predictor of high blood pressure (HBP), hyperglycemia and hyperlipidemia (HLP), which exemplified cardiovascular risk factors, than body mass index (BMI)7. Even though CI adjusted indices have potential value in clinical application, little is known about the role of CI adjusted total body fat (TBF), namely TBF/CI, in metabolic diseases assessment. We firstly calculated the TBF/CI to determine whether it can be a good indicator and had a close relationship with metabolic diseases in young adults.Conicity index (CI), which was a good tool to assess fat mass distribution and figure in young adults as previous studies suggestedIn total, 1365 ambulatory adult subjects aged between 20 and 40 years who underwent a routine health examination including BIA in Chinese PLA General Hospital from February 2016 to 2017 were enrolled. The inclusion criteria were young adults (age 20–40 years) who accepted BIA test. Participants were excluded for any of the following reasons: acute phase of chronic diseases, thyroid disorders, immune diseases, chronic renal failure, anorexia, malignant tumors, gastrointestinal surgery history and incomplete data. All participants were given oral informed consent to take part in the study and their data to be used. The study protocol was approved by the Institutional Review Board of Chinese PLA General Hospital. All methods were performed in accordance with the relevant guidelines and regulations.8. The 10 h fasting forearm vein blood was obtained in the early morning, and an Architect Ci8200-intergrated system  was used to detect the level of blood glucose, whole-blood glycohemoglobin A1c (HbA1c), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total plasma homocystein (tHcy). Postprandial glucose was determined at 2 h after the administration of 75 g glucose.Past history, personal history and family history were collected by trained healthcare providers. All subjects wore light clothes and stood in the upright position without shoes during BIA measurement using a body composition analyzer . Body mass, height, waist circumference (WC) and hip circumference (HC) were measured to an accuracy of 0.1(kg or cm) according to the standard protocolBMI, waist-hip ratio (WHR), CI, percentage body fat (PBF) were calculated from the following equations:9. Obesity was defined as BMI ≥ 28 kg/m2 and/or WC in male ≥ 90 cm while in female ≥ 85 cm10. HLP was defined as one of following: TC ≥ 5.2 mmol/L, LDL-C ≥ 3.4 mmol/L, HDL-C < 1.0 mmol/L, TG ≥ 1.70 mmol/L or previously diagnosed HLP11. DM was defined as fasting blood glucose ≥ 7.0 mmol/L, or blood glucose ≥ 11.1 mmol/L at 2 h after loading glucose, or HbA1C ≥ 6.5%, or previously diagnosed DM12. Hyperhomocysteinemia (Hhcy) was defined as tHcy ≥ 10 μmol/L or currently supplementing folate14.In the present study, age, BMI, LDL-C, HbA1c and tHcy were incorporated into continuous metabolic risk variables. Smoking history was defined as daily smoking more than 1 and accumulating 100 or more cigarettes, cigars or pipes for at least 6 monthsodds ratios (ORs) of metabolic diseases varied with increasing quartile of TBF/CI. Moreover, linear regressions without adjustment were constructed to assess association of continuous risk variables with TBF/CI. Logistic Regressions were used to examine ORs of metabolic diseases identified by TBF/CI. Receiver Operator Curves (ROCs) were created to determine the predictive value of different anthropometric indices for various metabolic diseases. A two-sided P value < 0.05 was considered statistically significant.All the data were analyzed using the Statistical Package for the Social Sciences, version 22.0 . All continuous variables were approximately under normal distribution, and were expressed as mean (standard deviation (SD)). Categorical data were expressed as count (percentage). Linear trend tests were used to evaluate trends of TBF/CI changed in continuous risk variables  and chi-quare trend tests were used in assessing The mean age was 34.5(5.0) years. TBF/CI in all the population, male and female were 14.99(5.25), 16.07(5.25), 13.22(4.76), respectively. 45.1%, 55.4%, 6.4% and 43.4% of young adults had obesity, HLP, diabetes and Hhcy, separately . Besides, the increasing trends were also found in women and men respectively  except age quartile in men (P trend = 0.115) and tHcy quartile in both men and women (P trend = 0.565 in men and 0.207 in women)  . Young adults with increased TBF/CI had higher risk of Hhcy . There was a 1.407 increase in the odds of obesity, a 1.112 increase in the odds of HLP and a 1.094 increase in the odds of DM per SD increase in TBF/CI after adjusted for age, sex and smoking history .In whole, multivariate-adjusted ORs for the risk of Obesity, HLP, DM were also found in women and men respectively . Men with increased TBF/CI had higher risk of Hhcy . There was a 1.528 increase in the odds of obesity in men, a 1.264 increase in women per SD increase in TBF/CI after adjustment . A 1.159 increase in the odds of HLP in men (P < 0.001) and a 1.055 increase in women (P = 0.008) were found as one TBF/CI unit increasing. The odds of DM increased 1.098 in men (P < 0.001) and 1.090 in women (P = 0.046) with TBF/CI changed per SD  = 0.845) showed a higher predictive value for obesity than WHR (AUC = 0.841) and PBF (AUC = 0.771), but lower than BMI (AUC = 0.921) . In HLP, DM and Hhcy, TBF/CI  also displayed higher predictive values than PBF , but lower than both BMI  and WHR  , TBF/CI can better evaluate the risk of Hhcy.Our study reported for the first time that the usefulness of TBF/CI in various metabolic diseases in young adults. CI is an useful and accuracy tool to evaluate the distribution of fat mass. As BMI cannot distinguish fat mass and lean mass clearly and its prediction is prone to be disturbed by the difference of samples in adipose distribution and body build, use of the BMI as a predictor for body fatness has been criticized17 demonstrated that intramuscular fat was associated with HLP risk. As mentioned above, we observed that the risk of HLP increased with added TBF/CI as well.A cross-sectional study carried out in American middle age populationet al.18, which pinpointed that volatile fatty acid instead of whole-body skeletal muscle mass measured by magnetic resonance imaging was a significant predictor of abnormal glucose metabolism. Nonetheless, Son JW et al.19 did a prospective cohort study (follow-up 2 years) in South Korean community residents aged 40–69, which exhibited that a hazard ratio of 1.35 increase for the risk of developing type 2 diabetes per SD decrease in ASMI using multi-frequency BIA that strongly consistent with dual-emergency x-ray absorptiometry (DXA). In our present work, there were a 1.094 increase in the odds of DM per SD increase in TBF/CI. The results suggested that the diabetes risk increased with augmented TBF/CI in young adults and BIA maybe a viable, portable and accessible instrument for community and clinic-based assessment recommended by the Asian Working Group of Sarcopenia (AWGS)20. Disparate measurements in heterogeneous ethnic groups may account for the argumentative results21.Even before-mentioned studies have pointed out that there were close ties between body composition and metabolic risk factors, controversy has emerged till now regarding the relevance between body fat and abnormal glucose metabolism. Canadian aged 20–69 and consisted of overweight or obesity were enrolled in a cross-sectional study by Kuk JL et al.22 elucidated that tHcy was positively related to fat-free mass and lean body mass in middle-aged Italian by a cross-sectional study. Whereas another study targeting Chinese hypertension population aged 45–75 years23 expatiated that nearly 20% decrease in OR for Hhcy in subjects with high physical activities. A significant positive correlation between tHcy and TBF/CI were displayed in our study. There maybe two possible explanations. For one thing, attention has been paid more to animal protein intake accompanied with fat intake which may lead to the ignorance of vegetable consumption in young’s diet24. As is known to us, vegetable was a vital and natural source of folate and vitamin B complex, and the lack of which could cause Hhcy. For another, the young was fit in a stage of good absorption and vigorous synthetic metabolism. Age, various health status, diet structure and surroundings may contribute to the disputable results25.Accordingly, another association between body fat and tHcy has remained controversial. Battezzati A 15. Secondly, seen from our study, CI adjusted TBF showed higher predictive values for metabolic diseases than traditional definition, namely weight adjusted total body fat. Even the AUC of TBF/CI was less than the obesity gold standard-BMI, TBF/CI showed a higher predictive value for obesity than WHR. The function of TBF/CI is worth of further exploration in the future, maybe it could replace WHR sometimes especially in some patients who could not measure hip circumference for some special causes, like hip bedsore26. Thirdly, the AUCs of TBF/CI were nearly between 0.7 and 0.9, which manifested median value in assessing metabolic diseases.Generally speaking, TBF/CI has its irreplaceable advantages. Firstly, compared with biochemical test, TBF/CI was non-invasive. It was convenient and costless for early metabolic syndrome assessmentHowever, as a cross-sectional study, the work had inevitable limitations. Firstly, biases were unavoidable since it was a single center research specific to check-up population. Secondly, we did not evaluate hormone level and nutritional status that could be confounding factors that affected TBF/CI. Last but not least, we only discussed the relationship between TBF/CI and metabolic diseases. So cause-and-effect relationship could not be demonstrated in the study. Therefore, a large and longitudinal cohort is warranted to further analyze the association between TBF/CI and metabolic diseases.In conclusion, Young adults with increased TBF/CI had higher ratios of metabolic diseases, which suggested that TBF/CI can be a good indicator and exhibited a close relationship with metabolic diseases.


To assess toxicity and quality-of-life (QOL) after carbon ion radiotherapy (CIRT) at the Shanghai Proton and Heavy Ion Center (SPHIC) and identify clinical factors that correlate with urinary, bowel and sexual function.Sixty-four patients with localized prostate cancer admitted from July 2015 to January 2018 underwent CIRT. At baseline and 5 time-points after radiotherapy, we assessed patients’ QOL using the 26-item edition of the Expanded Prostate Cancer Index-Composite (EPIC-26) Chinese version. Logistic regression was performed to identify clinical factors associated with acute genitourinary (GU) toxicity and relative QOL.p < .001). For urinary incontinence, bowel and sexual QOL, the scores remained stable at 2-year follow-up. The occurrences of acute Grade 1 and 2 GU toxicity were 20.3 and 10.9%, respectively, and of late Grade 1 and 2 GU toxicity were 3.1 and 1.6%, respectively. No acute or late gastrointestinal (GI) toxicity occurred. Transurethral resection of the prostate (TURP) was a risk factor that predicted a decline in urinary related QOL, and age made a difference to bowel-related QOL. For sexual QOL, castration status was a remarkable risk factor. An international prostate symptom score (IPSS) ≥8 increased the risk of Grade 1–2 acute GU toxicity 5.3-fold.By the end of CIRT, urinary irritation/obstruction temporarily declined (− 7.92 ± 1.76, Patients with prostate cancer had favorable QOL after CIRT. IPSS ≥8 was a risk factor to acute GU toxicity, and TURP predicted a decline in urinary QOL. Age was related to bowel QOL, and castration status was associated with sexual QOL.NCT02739659. Registered April 15, 2016.Carbon Ion Radiotherapy for the Treatment of Localized Prostate Cancer,  Prostate cancer is the most common urologic cancer among Chinese men with largest increase in incidence of all cancers in 2015 . FurtherSome clinical trials have reported excellent disease control and favorable toxicity of CIRT for prostate cancer –8. HowevA total of 102 patients with prostate cancer were consecutively admitted from July 2015 to January 2018. The inclusion criteria were men with pathologically confirmed localized prostate cancer who received CIRT as the radical treatment. Patients with visceral or bone metastases , postopeBefore CT simulation and every irradiation, patients were required to empty the rectum and fill the bladder. Patients are instructed to drink 330 ml of water 30–60 min prior to simulation and daily treatment. The organ at risk, including the rectum and bladder, was contoured on the basis of the Male Radiation Therapy Oncology Group (RTOG) Normal Pelvis Atlas . The cliHormonal therapy was not administrated to low-risk patients, while intermediate-risk patients recommended to receive at least 6 months of hormonal therapy. And high-risk and very high-risk patients recommended for at least 2 years hormonal therapy.Before CIRT initiation, demographic characteristics, international prostate symptom score (IPSS) and past medical history including transurethral resection of prostate (TURP) and hemorrhoids was recorded. Given the IPSS symptom questions were responsive to lower urinary tract symptoms , we diviThe urinary-related adverse events, such as obstruction and cystitis, were defined as genitourinary (GU) toxicity, whereas the bowel-related adverse events were defined as gastrointestinal (GI) toxicity. Acute toxicity was evaluated using the Common Toxicity Criteria Adverse Event 4.03. Late toxicity was assessed by the RTOG/European Organisation for Research and Treatment of Cancer (EORTC) Late Radiation Morbidity scoring scheme .The Expanded Prostate Cancer Index-Composite (EPIC) is a well-established patient-reported outcome questionnaire to monitor QOL among prostate cancer survivors. The 26-item version of EPIC, also known as the EPIC-26, contains symptom-based domains, such as urinary incontinence, urinary irritation/obstruction, sexual and bowel. The EPIC-26 domains, scoring from 0 (poorest) to 100 (best), can be tracked over time to understand symptom burden, functional outcomes and the impact of adverse effects  was usedThe QOL assessment was performed at the following 6 times: before CIRT (baseline), immediately after treatment finished, and at 3 months, 6 months, 1 year and 2 years after CIRT. The assessment was conducted in various ways, including face-to-face interviews, letters and online questionnaires; however, every patient had at least one face-to-face interview QOL assessment by an interviewer with a psychology background. The castration conditions were asked at each follow-up.P < .10) were then analyzed in a multivariate regression model [P values less than.05 were considered significant. Age was considered as a confounding factor [SPSS version 23.0 software was used. Nonparametric test and chi-square test were performed to assess patient characteristics and QOL scores between two prescribed dose groups. First, generalized estimating equations  were useon model , 21. P vg factor .Table Until January 2018, the median follow-up was 19 months . 31 (48.4%) patients were not castrated at last follow-up and the reasons were low risk, completed hormonal treatment and intolerable drug toxicity.p < .001) but then recovered or was even superior to baseline in subsequent follow-up. At the end of treatment, when urinary function was at its poorest, patients reported “a moderate or big problem” mostly in urinary frequency (41.4%) and a weak stream (20%); however, these issues subsequently greatly resolved (Table p = .057). Some 32 (50%) patients had poorer bowel function at last follow-up. Nevertheless, after 6 months of follow-up, the number of patients who reported moderate or large problems with bowel function was zero. When it came to sexual QOL, the scores remained persistently stable after CIRT, but the baseline score was low (21.7 ± 18.5), with over 70% of patients complaining of multiple poor sexual functions  and 7 (10.9%), respectively, and the most common GU toxicity was urinary frequency . There were also 5 cases of Grade 1 hematuria and 3 cases of Grade 1 dysuria. None of the patients developed grade 3 or higher acute GU toxicity. There were no acute GI toxicity occurring. With regard to late toxicity, there were 2 (3.1%) cases of Grade 1 and 1 (1.6%) case of Grade 2 cystitis for GU toxicity, with no late GI toxicity observed.Table The history of TURP did not make a difference to the acute GU toxicity during the CIRT, though it was a remarkable risk factor of declined urinary QOL in the relative long term. Hormonal therapy made no difference, either. However, IPSS ≥8 made a significant difference and it predicted a 5.3-fold higher probability of acute GU toxicity during CIRT Table .p = .035). Castration status was a potential risk factor for declined bowel QOL with an odds ratio (OR) of 2.8, but it did not reach significance after multifactorial adjustment (p = .079). Neither aspirin therapy nor hemorrhoid history were significantly related to impaired bowel QOL.When focusing on bowel QOL, the baseline score was a significant influence factor, with a higher baseline score tending to indicate a decrease in subsequent QOL. Though the discrepancy was not significant in the univariate analysis, it was significant in the multivariate analysis  GyE/20 fx CIRT at the National Institute of Radiological Sciences, using the Functional Assessment of Cancer Therapy (FACT) Prostate scale. Prostate cancer-related QOL scores (FACT-P) decreased immediately after CIRT, but returned to near baseline at 12 months. The global QOL scores (FACT-G) continued to decline slowly. At 5 years of follow-up, the FACT-P and FACT-G scores were significantly lower than the baseline level, but the deviation was only about 2 points. The main reason for the decline in scores was the decline in Social/Family well-being scores.Ishikawa et al.  used theHabl et al.  used EORTo summarize the QOL after CIRT of prostate cancer: 1) At the end of CIRT, there was a significant decline in QOL, but it soon recovered. In general, CIRT had little impact on the QOL. 2) All 3 studies found that hormonal therapy was associated with a late-stage decline in QOL scores. The 2 Japanese studies used QOL scales based on global QOL without prostate cancer-related symptoms. The German study found that urinary symptoms were the most affected, but the sample size was small and the follow-up time was short. Above all, differences in QOL scales make it difficult to compare their results. Although our study used the EPIC-26 scale, which is different from the above studies, the results were highly consistent with that of the Heidelberg Ion-Beam Therapy Center, which had indicated that CIRT had little impact on the QOL of patients with prostate cancer. The results of our study are based on the symptoms associated with prostate cancer treatment and will help clinicians better explain the management of symptoms after CIRT for prostate cancer.In addition to QOL changes, we showed results for acute and late radiation toxicity. The short-term acute toxicity was similar to the results reported in Japan and Germany, where Grade 1–2 acute toxicity was well tolerated and Grade 3+ acute toxicity was not observed , 25, 26.Several studies have proposed that TURP was a predictor of decline in urinary QOL and GU morbidity in photon external radiotherapy –29. ThisAdvanced age was a significant protective factor against poorer QOL. A previous review by Blank and Bellizzi observed that older cancer survivors when diagnosed had a higher QOL, given that earlier cancer detection can lead to increased stress and an overall negative psychological perception . NeverthWith respect to the sexual domain, castration status was a remarkable adverse factor, increasing the risk of impaired sexual QOL by approximately 13-fold. The adverse sexual effects of hormonal therapy have been well investigated, including decreased sexual desire and inability to achieve orgasm , 36. HowIshikawa, H. et al. reported that acute GU toxicity was a risk factor for the occurrence of persistent toxicity after CIRT . TherefoOur study has several limitations. First, patients were not followed up long enough to observe enough clinical endpoint events; thus, the late toxicity of CIRT needs to be further updated in subsequent reports from our center. Furthermore, the number of the patients in this study was not as large as that using photon radiotherapy, possibly resulting in selection bias. However, as far as 2016, the number of all carcinoma patients receiving CIRT per year was 938, and only 5 of all 11 heavy ion centers admitted over 1000 patients, with 4 facilities located in Japan and 1 in Germany .Patients with prostate cancer had favorable QOL after CIRT. IPSS ≥8 was a risk factor for acute GU toxicity, and TURP predicted a decline in urinary QOL. Age was related to bowel QOL and castration status was associated with sexual QOL.


Pseudomonas aeruginosa. Ethyl gallate (EG), PG, and butyl gallate (BG) inhibited biofilm formation and virulence factors including elastase, pyocyanin, and rhamnolipid, in P. aeruginosa without affecting cell viability by antagonizing the QS receptors LasR and RhlR. PG exhibited the most potent activity. Interestingly, hexyl gallate (HG) inhibited the production of rhamnolipid and pyocyanin but did not affect elastase production or biofilm formation. Notably, OG inhibited the production of rhamnolipid and pyocyanin but stimulated elastase production and biofilm formation. Analysis of QS signaling molecule production and QS gene expression suggested that HG inhibited RhlR, while OG activated LasR but inhibited PqsR. This mechanism was confirmed using QS mutants. Additionally, PG prevented the virulence of P. aeruginosa in Caenorhabditis elegans and a mouse model. This is the first report of the differential effects of alkyl gallates on QS systems and PG has great potential as an inhibitor of the virulence and biofilm formation of P. aeruginosa.Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Among alkyl gallates, propyl gallate (PG) and octyl gallate (OG) are used as food preservatives. Here we found that alkyl gallates differentially affect virulence, biofilm formation, and quorum sensing (QS) in  Many pathogenic bacteria utilize QS for the production of virulence factors and biofilms. Thus, the inhibition of QS is considered a new promising target for the control of bacterial infections. Traditional strategies for the prevention and treatment of bacterial infections are based on the use of antibacterial compounds that kill bacteria or inhibit the growth of bacteria. These strategies have resulted in substantial stress on target bacteria, causing the rapid growth of resistant populations4. Virulence factors and biofilm are not essential for bacterial survival. Thus, interference with bacterial virulence factors and biofilms is a new therapeutic strategy because this method produces less selection pressure for the development of resistance than traditional strategies5.Quorum sensing (QS) is a bacterial communication system that uses small diffusible signal molecules known as autoinducers. Bacterial population behaviors regulated by QS include bioluminescence, sporulation, biofilm formation, virulence factor production, and antibiotic resistancePseudomonas aeruginosa is a Gram-negative bacteria that is highly resistant to existing antibiotics and cause many opportunistic and nosocomial infections. In particular, chronic lung infections with P. aeruginosa are the major causes of mortality in cystic fibrosis (CF) patients6. P. aeruginosa has been shown to form biofilms in the CF lung, which increase bacterial resistance to antibiotics7, and also produce several virulence factors including elastase, rhamnolipid, and pyocyanin. Thus, the inhibition of virulence factor production and biofilm formation may be highly attractive for the prevention and treatment of P. aeruginosa infections8.P. aeruginosa is tightly regulated by three main QS systems organized in a hierarchical manner3. LasR-LasI and RhlR-RhlI use acyl homoserine lactones (AHLs) as signaling molecules, while PqsR-PqsABCD uses 2-alkyl-4-quinolones. LasI and RhlI, the AHL synthases, synthesize N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL) and N-butanoyl homoserine lactone (BHL), respectively, whereas PqsABCD, the quinolone synthase, produces 2-heptyl-3-hydroxy-4(1 H) quinolone (PQS). When activated by OdDHL, the LasR-OdDHL complex activates the transcription of lasI and lasR, which leads to autoinduction. The LasR-OdDHL complex also initiates the expression of the rhl and pqs QS systems and directs the gene expression of biofilms and virulence factors such as elastase9. The RhlR-BHL complex, in turn, activates the expression of rhlI and activates many QS-dependent virulence factors, such as rhamnolipid and pyocyanin. The PqsR-PQS complex also activates the gene cascades associated with the PQS system and virulence factors such as pyocyanin and rhamonolipids10. Recently, IQS (2-(2-hydroxyphenyl) thiazole-4-carbaldehyde), was discovered as a fourth QS signal molecule. The IQS system is tightly controlled by las under normal conditions, but take over the functions of the central las system under phosphate depletion stress conditions11.QS in Terminalia bellerica12. Notably, PG and OG have been approved for use as antioxidant additives in the food and pharmaceutical industries for over 60 years because these compounds exhibit significantly low toxicity both in vitro and in vivo15. PG and OG are used at concentrations of up to 0.015% (0.7 mM) and 0.4% (1.4 mM), respectively, in foods16. To date, in addition to their antioxidant activity, alkyl gallates have been reported to exhibit various other biological activities and the chain lengths of alkyl gallates differentially affects the pharmacological activity of these compounds depending on the specific pharmacological activity18. Alkyl gallates were reported to have antimicrobial activity against Gram-positive bacteria and some fungi, but almost no antibacterial activity against Gram-negative bacteria21. Very recently, phenolic compounds, including methyl gallate (MG), have been reported to exhibit antibiofilm activity against Streptococcus mutans and anti-QS activity against P. aeruginosa, but the antivirulence activity of alkyl gallates, especially PG and OG, has not been studied to date24.Gallic acid (GA) and alkyl gallates are found in several natural and industrial products. For example, GA and propyl gallate (PG) are abundant in green tea and octyl gallate (OG) has been found in the medicinal plant P. aeruginosa virulent factors from a microbial metabolite library, we identified gallate-like compounds. Here, we report the differential effects of alkyl gallates on virulence factors and biofilms, the in vivo antivirulence activity of PG against P. aeruginosa, and the differential effects of alkyl gallates on QS systems.While screening for an inhibitor of P. aeruginosa PAO1 cells were evaluated. Six alkyl gallates , PG, butyl gallate (BG), hexyl gallate (HG), and OG) and GA were tested. The antibacterial activity of the alkyl gallates was also examined against each virulence factor using an optical-density-based assay. MG, EG, and PG inhibited elastase production in PAO1 cells in a dose-dependent manner without affecting cell viability  and key QS-controlled genes  in PAO1 strains was investigated  with almost no effect on the other genes. In contrast, OG dramatically activated the transcription of las genes, while repressing the transcription of the pqs, rhlB, and phzE1 genes, with almost no effect on the transcription of the rhlI and rhlR genes . The transcription of QS-regulatory genes . As a control experiment, in low phosphate Pseudomonas medium supplemented with 4 mM K2HPO4 in which IQS is regulated by las11, the transcription level of ambB was significantly reduced in the LasI and LasR-deficient strains compared with the levels in the wild-type strain (PA14) and the alkyl gallates inhibited the transcription levels of ambB in the three strains as expected  of PG and OG used in all experiments were at least two-times lower than those  used in food.EG, PG, and BG inhibited virulence factor production and biofilm formation in the P. aeruginosa are organized hierarchically, with LasR at the top of the signaling cascade9, and are also interconnected34. When activated by OdDHL, the LasR-OdDHL complex triggers the onset of both the rhl and pqs systems by activating the transcription of rhlI, rhlR, pqsR, and pqsH. PqsR  is the transcriptional regulator of the PQS biosynthesis operon pqsABCD, and PqsH completes the final step of PQS synthesis. The PqsR-PQS complex enhances the transcription of rhlI, thus influencing the overall expression of the rhl QS system36. However, the RhlR-BHL complex represses PQS signal production by inhibiting the expression of pqsR and pqsABCD38. Thus, it has been suggested that the ratio of OdDHL to BHL determines the activation of PQS synthesis38. LasR-OdDHL induces the production of virulence factors, such as the LasA and LasB elastases and alkaline protease, and biofilm formation10. RhlR-BHL and PqsR-PQS direct gene expression of several virulence factors, such as rhamnolipid, pyocyanin, and hydrogen cyanide35. In this study, similar to FC, PG inhibited virulence factor production  and biofilm formation by antagonizing the QS receptors LasR and RhlR. Additionally, as shown by using the rhlR mutant 22 and MG44 were reported to have antibacterial activity against Salmonella strains but not against other Gram-negative bacteria including P. aeruginosa, E. coli etc.Alkyl gallates have been reported to exhibit various biological activities, including cardiovascular protection45. Because each assay for biofilm formation and virulence factor production used different media and incubation conditions, the antibacterial activity of the alkyl gallates in each assay was measured in parallel to determine whether the inhibitory activity of these compounds on biofilm formation and virulence factor production is due to growth inhibition activity. Indeed, the alkyl gallates tested exhibited differences in antibacterial activity among the various assays performed in this study. Interestingly, PG had no antibacterial activity in all the assays, even at 2000 μM, but alkyl gallates with shorter or longer alkyl chains than PG exhibited antibacterial activity against P. aeruginosa in this study  at 600 nm, the cultures were centrifuged at 4000 rpm for 10 min and the elastase activity of the culture supernatants was measured.Elastase activity in the supernatant of 47 and rhamnolipid48 production and biofilm formation49 in P. aeruginosa PAO1 and PA14 were measured as previously described (Supporting Information). After incubation for 24 h, cell viability was assayed by measuring the OD at 600 nm, and pyocyanin and rhamnolipid were then extracted and determined at 520 and 421 nm, respectively. Biofilm cells were measured using crystal violet staining.PyocyaninA. tumefaciens NT1 and C. violaceum CV02627. Overnight cultures of each strain were grown in LB medium at 30 °C. One mL of the 20-fold-diluted overnight culture was transferred into a 15-mL conical tube and 5 μL of X-gal (20 g/L) were added to A. tumefaciens NT1. Ten microliters of 100 μM OdDHL (Sigma) and 50 mM BHL  were then added to A. tumefaciens NT1 and C. violaceum CV026, respectively. Finally, 10 μL of each test compound dissolved in DMSO were treated to the cultures, followed by incubation at 30 °C for 48 h. After cell viability was assayed by measuring the OD at 600 nm using a microtiter ELISA reader, color changes in C. violaceum CV026 and A. tumefaciens NT1 were measured at 590 nm and 545 nm, respectively.An AHL-based QS competition assay was carried out using two reporter strains, namely, 50 and QS gene expression51 were determined as previously described. The culture method for P. aeruginosa PAO1 and PA14 was the same as that used for rhamnolipid and pyocyanin assays. For the measurement of three QS signaling molecules, 3 mL of the culture supernatants were extracted with 3 mL of ethyl acetate acidified with 0.1% acetic acid, and the organic layer was then evaporated and measured by LC-MS/MS in MRM mode (Supporting Information). For the determination of QS gene expression, total RNA was extracted using TRIzol reagent (Invitrogen), followed by synthesis of cDNA for RT-qPCR detection of the expression of target genes and measurement by RT-qPCR using a Bio-Rad CFX-96 real− time PCR system  with the primers listed in Table rpoD gene.The effects of alkyl gallates on QS signaling molecule productionP. aeruginosa-C. elegans infection assay was conducted as previously described52. C. elegans nematodes were grown on NGM plates containing an ample lawn of E. coli OP50 as a food source at 20 °C for 48 h. Killing and control plates were prepared by spreading 5 μL of overnight cultures of PAO1 and OP50 cells, respectively, on a 35-mm petri plate with 4 mL of PGS agar. Test compounds dissolved in DMSO were treated to the killing plates. The plates were then incubated at 37 °C for 24 h to make a bacterial lawn and then shifted to 23 °C for 24 h. Thirty L4-stage worms were placed on each plates and incubated at room temperature. Nematodes were counted for survival every 5 h for 30 h.A 7 PAO1 cells. Bacteria was precultured for 20 h and diluted 5-fold in fresh MHA medium or in MHA supplemented with 10 or 100 μM PG. After shaking incubation for 7 h, the PAO1 cultures were centrifuged, and the pellets were washed with saline, and then resuspended to 5 × 108 cfu/mL. Anesthesia was induced prior to infection by intraperitoneal injection with Zoletil 50 (50 g/L) + Rompun (23.32 g/L) at 0.006 mL/10 g + 0.004 mL/10 g. Mice were infected with a dose of 2.5 × 107 cfu/mouse in 50 μL PBS via the intranasal inhalation (n = 15 for each group). Infected mice were observed for survival for 72 h.Twenty 5-week-old BALB/C inbred female mice  were infected with 2.5 × 10P-values for testing statistical differences between measurements were estimated by the unpaired Student’s t-test . A p-value < 0.05 was considered statistically significant.Data are presented as mean ± standard deviation (SD) values. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Handong Global University (South Korea) (protocol #HGU-20170921-006). All experiments were performed in accordance with the approved guidelines of the Institutional Ethical Committee, adhered to Guide for the Care and Use of Laboratory Animals of National Research Council (USA).Supplementary Information


Diffusion weighted imaging (DWI) has a good diagnostic value for malignant thyroid nodules, but the published protocols suffer from flaws and focus on the apparent diffusion coefficient (ADC). This study investigated the diagnostic performance of multiple MRI parameters in differentiating malignant from benign thyroid nodules.This was a retrospective study of 181 consecutive patients . The patients underwent conventional MRI, DWI, and dynamic contrast-enhanced MRI before surgery. The chi-square test and the Student t test were used to compare the conventional features and ADC value between malignant and benign groups. Multivariate logistic regression was used to identify the independent predictors and to construct a model. Receiver operator characteristic (ROC) curve analysis was used to assess the diagnostic performance of the independent variables and model.P < 0.05). The multivariable analysis revealed that ADC value , irregular shape , ring sign in the delayed phase , and cystic degeneration  were independent predictors. Among them, ADC performed the best in discriminating benign from malignant nodules, with an area under the curve (AUC) of 0.95, 0.90 sensitivity, and 0.91 specificity. When the independent factors were combined, the diagnostic performance was improved with an AUC of 0.99, 0.97 sensitivity, and 0.95 specificity.Tumor diameter, ADC value, cystic degeneration, pseudocapsule sign, high signal cystic area on T1-weighted imaging, ring sign in the delayed phase, and irregular shape showed significant differences between two groups (all ADC value could discriminate between benign and malignant thyroid nodules with a good performance. Subjective features such as the ring sign, irregular shape, and cystic degeneration associated with malignant thyroid nodules could provide complementary information for differentiation. About, 19–67% of the healthy, asymptomatic individuals are diagnosed with nodules of the thyroid , 3. WithAlthough ultrasound (US) is widely used to detect thyroid nodules and determine their malignant potential, the US features and index of each grade of the Thyroid Imaging Reporting and Data System (TI-RADS) remain controversial , 7. US-g− 3/m2) [DWI reflects the random Brownian motion of water molecules within a voxel of tissue. The movement of the tissue water molecules between the two opposing gradients will result in dephasing, which is seen as signal loss. ADC can be quantified calculated with repeating the sequence in different magnetic field strengths . The ADC− 3/m2) , 14. DWI− 3/m2) , but DWI− 3/m2) , 18.Therefore, in the present study, we sought to improve the image quality with a special coil on the neck surface and a breath-hold technique, and to investigate the value of multiple MRI parameters for the diagnosis of thyroid carcinoma. Quantitative and qualitative parameters were examined.This was a retrospective diagnostic study. Between January 2013 and December 2016, 479 consecutive patients underwent thyroidectomy at our hospital. Among them, 254 patients had undergone MRI examination within one week before surgery at the Department of Radiology. This study was approved by the institutional review board of our hospital. The requirement for individual written informed consent was waived.n = 39); 2) poor image quality deemed to be non-diagnostic after review (n = 15); or 3) lesions with complete cystic changes (n = 19).The inclusion criteria were: 1) thyroid nodule that underwent surgery; and 2) available MRI data obtained within one week of surgery. The exclusion criteria were: 1) lesion size < 3 mm by US  equipped with an 8-channel special neck surface coil . All patients were examined using the same machine, coil, and scan series.2. Imaging parameters for DWI were: TR/TE, 6550 ms/minimum; FOV, 14 cm; NEX, 4; matrix, 128 × 128; slice thickness, 4 mm; and gap = 0.5 mm. Contrast enhancement studies were implemented using axial T1WI obtained with a fast-spoiled gradient recalled echo . Gadolinium  was intravenously injected at 0.2 ml/kg body weight and 3 ml/s, followed by a 20 ml saline flush. In each patient, a phase was performed prior to the injection of contrast medium. Six phases were obtained after injection of the contrast agent at 30, 60, 120, 180, 240, and 300 s. In the contrast-enhanced protocol, breath-hold was performed during each phase. Spatial saturation bands were also used to remove signal from overlying fat and adjacent tissues. All patients received training on the breath-hold technique before the MRI examination.The MR imaging protocol included T1-weighted image(T1WI), T2-weighted image(T2WI), DWI, and contrast enhanced T1WI of thyroid. Coronal fat-suppressed T2WI (repetition time (TR)/ echo time (TE), 1280 ms/85 ms; slice thickness; 4 mm; gap, 1 mm; matrix, 288 × 192; number of excitations (NEX), 4; field of view (FOV), 18 cm); axial T1WI ; axial fat-suppressed T2WI . DWI was performed using a single-shot echo planar imaging sequence with the diffusion gradient b factor = 800 s/mmTwo radiologists (13 and 18 years of MRI experience) reviewed all images using an AW4.5 workstation . Each radiologist was blinded to histopathology results. All images were reviewed independently by the two radiologists. Discrepancies were solved by discussion.2. The ADC values were measured twice and then the average value was taken.Lesions were evaluated for location, size (the largest linear dimension of nodules), shape (regular or irregular), margin (clear or vague), signal intensity heterogeneity (homogeneous or heterogeneous on T2WI), cystic degeneration, high signal cystic area on T1WI, ADC value of solid tumor area, and degree and pattern of enhancement. ROI (region of interest) of solid tumor area were manually drawn in the largest slice of lesions on ADC maps. The solid tumor areas were defined as a bright signal intensity identified on DWI images and dark signal intensity on ADC maps. Meanwhile, obvious areas of cystic changes, hemorrhage, calcification, and lesion margins need to avoid based on combined T2WI, contrast enhanced T1WI and DWI. The size of ROIs was determined to correspond with the darkest portion of lesions on ADC maps ranged 10-50 mmIn this study, blood vessels, thyroid tissue, and muscles were used to assess subjectively the degree of early enhancement of the nodule, which was categorized as: mild (the enhancement was similar to that of adjacent muscle tissues); moderate (the enhancement was higher than that of adjacent muscle tissues but lower than that of blood vessels); and marked (the enhancement approached that of blood vessels). The pattern of enhancement reflected the dynamic contrast enhancement of thyroid nodules after the injection of the contrast agent in the delayed phase with ‘wash in’ or ‘wash out’. The ring sign was defined as the central area of the nodule showing ‘wash out’, whereas the peripheral area showed persistent enhancement. The pseudocapsule sign was defined as the tumor area showing a clear capsule after contrast agent administration in the delayed phase.P-values < 0.05 were considered statistically significant. Binary logistic regression was used to identify the features that were independently predictive of malignant thyroid tumor. Variables demonstrating a significant association with malignant thyroid lesions were entered into the model in a forward stepwise method. The final model was selected based on variables with P < 0.05. The odds ratios (OR) and 95% confidence intervals (95%CI) were used as a measure of the relative magnitude of an association between predictor variables and malignant tumor. ROC curves were used to determine the cutoff value to differentiate the parameters of malignant from benign tumors.Categorical data were presented as percentage and analyzed using the chi-square test. Continuous data were tested using the Kolmogorov-Smirnov test. Normally distributed continuous data were presented as means ± standard deviation and analyzed using the Student t test. Non-normally distributed data were presented as medians (range). All statistical analyses were performed with SPSS 23 . Two-sided P = 0.700) or gender (P = 0.419) were found between the benign and malignant nodule groups . The ADC of the benign group  was significantly higher than that of the malignant group  (P < 0.001). Cystic degeneration, the pseudocapsule sign, and high signal cystic area on T1WI were more common in benign thyroid nodules . The ring sign in the delayed phase and irregular shape after contrast agent was significantly more common in the malignant group (both P < 0.001) .Table 1) Figs. . There wP < 0.001), irregular shape , ring sign in the delayed phase , and cystic degeneration  were independently associated with malignant thyroid nodules, with 0.961 accuracy.Table The ROC curve analysis revealed that the best cut-off of ADC values achieved AUC of 0.95, 0.90 sensitivity and 0.91 specificity. Cystic degeneration, ring sign, and irregular shape had AUC of 0.76, 0.82, and 0.91, respectively. When the independent factors were combined, the diagnostic performance was improved to an AUC of 0.99, 0.97 sensitivity, and 0.95 specificity  was used, which could better reflect the actual diffusion characteristics in this study. In addition, we used special techniques to improve image quality. A relatively small FOV (14 × 14 cm) was used to reduce susceptibility artifacts. Shim blocks were used to optimize magnetic homogeneity in the thyroid region. All patients received respiratory training to improve movement-related problems. We used a breath-hold technique on dynamic contrast-enhanced MRI phase to reduce breathing motion artifacts and added saturated zone to reduce carotid artery pulsatile artifacts. Therefore, 239 of the 254 patients showed excellent image quality in this study.The incidence of thyroid cancer is rapidly increasing, with a 3% estimated annual increase in the United States . SimilarSome studies have shown that DWI can differentiate benign from malignant thyroid nodules , 14, 26.Dynamic contrast enhancement can play a complementary role in the diagnosis of thyroid carcinoma. During the delayed phase, the ring sign  was seen in a large number of malignant thyroid nodules, which was not reported in previous studies. The central tumor area with washout indicates active growth of tumor cells, whereas the peripheral area is mainly composed of loose connective tissue with abundant intercellular matrix. Peripherally enhanced areas in malignant thyroid tumors during the delayed phase may also be related to the fibrous stroma of the tumor and the presence of vascular fibrotic stroma. Malignant thyroid nodules in the present study showed irregular shape after contrast agent. The histopathological characteristics of thyroid carcinoma indicate the invasive and heterogeneous growing pattern. One recently published study  also shoIn the present study, cystic degeneration, high signal cystic area on T1WI, and the pseudocapsule sign were significantly more frequent in benign thyroid nodules than in malignant nodules. Nodular goiter was the main pathological type of benign thyroid nodules in this study. Due to the relative abundance of colloid follicles and hemorrhage, nodular goiter showed cystic changes and high signal intensity in cystic areas. Shi et al.  showed sIn this study, the enhancement degree between the two groups was significantly different, but there was major overlap between the two groups. Nodular goiter and adenoma showed moderate or marked enhancement with abundant hyperplasia of thyroid follicles. Follicular thyroid carcinoma showed marked enhancement because of abundant hyperplasia of thyroid follicles and neovascularity. Papillary thyroid carcinomas demonstrated moderate or marked enhancement with increased cell density, severe desmoplastic response, and cell proliferation, which were consistent with previous studies , 29.Taken together, the present study strongly suggests that multiple MRI parameters should be considered when evaluating thyroid nodules. While irregular shape, ring sign, and cystic degeneration can be subjective and dependent upon the radiologist’s experience, ADC can provide quantitative information to differentiate thyroid carcinoma from benign thyroid nodules. On the other hand, whether the parameters observed in the present study are better than other modalities such as US, computed tomography, and scintigraphy  require This study has several limitations. Firstly, this study was retrospective in design, leading to selection bias and therefore undermining the validity of the results. Prospective studies with larger sample size would increase the credibility of the results. Secondly, thyroid nodules measuring < 3 mm were not included. Improvements in MRI software and using smaller slice gaps may facilitate the detection of smaller lesions in future studies. Importantly, it has been shown that small thyroid lesions are at higher risk of malignancy than larger ones . TherefoMultiple MRI parameters could be helpful to differentiate malignant thyroid nodules from benign nodules. The logistic regression showed that ADC value could discriminate between benign and malignant thyroid nodules with a good performance. Subjective signs such as the ring sign, irregular shape, and cystic degeneration associated with malignant thyroid nodules could provide complementary information for differentiation. Combining subjective MRI features to a quantitative measurement could improve the diagnostic performance of MRI for malignant thyroid nodules.