To predict 72-h and 9-day emergency department (ED) return by using gradient boosting on an expansive set of clinical variables from the electronic health record.This retrospective study included all adult discharges from a level 1 trauma center ED and a community hospital ED covering the period of March 2013 to July 2017. A total of 1500 variables were extracted for each visit, and samples split randomly into training, validation, and test sets . Gradient boosting models were fit on 3 selections of the data: administrative data , data available at triage, and the full set of data available at discharge. A logistic regression (LR) model built on administrative data was used for baseline comparison. Finally, the top 20 most informative variables identified from the full gradient boosting models were used to build a reduced model for each outcome.A total of 330 631 discharges were available for analysis, with 29 058 discharges (8.8%) resulting in 72-h return and 52 748 discharges (16.0%) resulting in 9-day return to either ED. LR models using administrative data yielded test AUCs of 0.69  and 0.71(95% CI 0.70–0.72), while gradient boosting models using administrative data yielded test AUCs of 0.73 (95% CI 0.72–0.74) and 0.74 (95% CI 0.73–0.74) for 72-h and 9-day return, respectively. Gradient boosting models using variables available at triage yielded test AUCs of 0.75 (95% CI 0.74–0.76) and 0.75 (95% CI 0.74–0.75), while those using the full set of variables yielded test AUCs of 0.76 (95% CI 0.75–0.77) and 0.75 (95% CI 0.75–0.76). Reduced models using the top 20 variables yielded test AUCs of 0.73 (95% CI 0.71–0.74) and 0.73 (95% CI 0.72–0.74).Gradient boosting models leveraging clinical data are superior to LR models built on administrative data at predicting 72-h and 9-day returns. To test the impact of additional input features, we build our models on 3 subsets of variables: administrative data , data available at the time of triage, and data available at the time of discharge. We use gradient boosting (XGBoost), a powerful classification algorithm suited for EHR data due to its ease with missing values, to model nonlinear relationships.Retrospective data on all adult visits was obtained from 2 EDs covering the period of March 2013 to July 2017 to ensure a 1-year historical time frame from the study start period of March 2014 as well as a 9-day prospective time frame for all visits. The represented EDs include a level 1 trauma center with an annual census of approximately 85 000 patients and a community hospital-based department with an annual census of approximately 75 000 patients. Both EDs are located within the same city and are part of a single hospital system utilizing the Epic EHR  and the Emergency Severity Index for triage. This study adhered to the Transparent Reporting of a multivariable prediction model for individual prognosis or diagnosis statement.Two binary outcomes were defined for every ED visit ending in discharge: return within 72 h and return within 9 days. Given the overlap in patient population and frequent transfers between the 2 EDs included in the study, a return visit at either ED was considered valid regardless of the location of the initial visit.The dataset used for this study derives from our prior study on predicting hospital admission at triage, which made use of patient demographics, time and location of presentation, triage vitals, chief complaint, hospital usage statistics, past medical history, outpatient medications, historical vitals, historical labs, and historical imaging and EKG counts.Samples were randomly split into a training set of 264 631 (80%), a validation set of 33 000 (10%), and a held-out test set of 33 000 (10%). All categorical variables were converted to numeric variables using one-hot encoding.LR models were trained on all samples excluding the test set using the RMSprop optimizer in the Keras interface.P < .001).From the 346 656 ED discharges in the study period, a total of 330 631 visits were available for analysis after filtering for inclusion criteria . Of these, 29 058 discharges (8.8%) resulted in a return visit within 72 h while 52 748 discharges (16.0%) resulted in a return visit within 9 days. Major characteristics of each outcome group are shown in LR models using administrative data yielded test AUCs of 0.69 (95% CI 0.68–0.70) and 0.71 (95% CI 0.70–0.72), while XGBoost models using administrative data yielded test AUCs of 0.73 (95% CI 0.72–0.74) and 0.74 (95% CI 0.73–0.74) for 72-h and 9-day return, respectively. XGBoost models using variables available at triage yielded test AUCs of 0.75 (95% CI 0.74–0.76) and 0.75 (95% CI 0.74–0.75), while those using the full set of variables yielded test AUCs of 0.76 (95% CI 0.75–0.77) and 0.75 (95% CI 0.75–0.76) for 72-h and 9-day return, respectively . The traVariables with high information gain for the full XGBoost models included markers of ED usage such as number of prior ED visits, number of prior admissions, ECG counts, urinalysis counts, as well as historical vital signs and a comorbidity of alcohol- or substance-related disorders . VariablStatistical measures for XGBoost models built on data available at triage, at 3 different cutoffs , are shown in In this study, we predict 72-h and 9-day ED return using gradient boosting on an expansive set of 330 631 ED discharges and 1500 variables. We are able to predict 72-h and 9-day return with similar accuracy, with small but significant improvement from including comprehensive elements of clinical data. Our best-performing models for predicting 72-h and 9-day ED return yielded test AUCs of 0.75 and 0.76, respectively, while our reduced models built on the top 20 informative variables yielded a test AUC of 0.73 for both outcomes. We expand on a prior model that predicted 72-h return in a Veteran’s Affairs population, enhancing performance by using a nonlinear algorithm on a large number of clinical variables.,,We confirm that visits that result in early ED return differ in several ways from those that do not result in return. Previously identified risk factors such as arrival mode, insurance status, number of previous ED visits, number of hospital admissions, and types of diagnoses were found to be significant.,,,The AUCs of our best models do not exceed 0.8 despite the inclusion of numerous clinical variables. The challenge of predicting early ED return may in part be due to the multifactorial nature of ED returns. Although the EHR can accurately encode quantitative variables such as presence of comorbidities and frequency of hospital utilization, a patient’s decision to return to the ED may also be contingent on emotional factors like fear and uncertainty that are not well-captured in the EHR.,,,We anticipate multiple clinical applications of ED return risk prediction models. It may be valuable to predict ED return visits early in the ED course in order to trigger intervention. We show that most informative variables are available by the time of triage, suggesting that care coordinators or social workers may begin interventions as early as triage. This suggestion is further supported by prior studies that have shown that patient disposition  can be robustly predicted at triage.,Another potential clinical application may be to use the model to retrospectively screen return cases for those associated with lapses in care.This study has several limitations. The study is a retrospective study that lacks a separate validation cohort. The study did not take account into scheduled versus unscheduled ED return, given that there is no formal definition for scheduled visits and that scheduling a return visit does not necessarily result in return. The study also restricts patient data to those collected from prior ED visits and does not include data from outpatient clinic or inpatient wards, which may provide important information regarding a patient’s healthcare usage pattern. Although our study included 2 neighboring EDs within the same city, it did not take account of return visits to EDs outside the city or to EDs in a different hospital system and likely under-represents ED return.Machine learning is able to predict 72-h and 9-day return with modest accuracy, with small but significant improvement from the use of clinical variables. The highest impact features are available by the completion of triage, supporting earlier intervention for patients with high risk of return. Further studies are needed for clinical implementation of such a model.Journal of the American Medical Informatics Association online.WSH, ADH, and RAT conceptualized the study. WSH and RAT curated the data. WSH preprocessed the data, trained the models, and created the figures. WSH and ADH developed the methodology and wrote the original draft. All authors revised the manuscript and approved its final version. RAT supervised all steps of the work.Woo Suk Hong was supported by the Yale School of Medicine Medical Student Research Fellowship. Adrian Daniel Haimovich and Richard Andrew Taylor received no specific funding for this study.Conflict of interest statement. None declared.ooz019_Supplementary_DataClick here for additional data file.


Anemia is a common finding in patients with diabetes, even in the absence of kidney disease and is a risk factor for adverse outcomes, including all-cause and cardiovascular mortality. Despite this, relatively little is known about the burden of anemia among adults with diabetes in sub-Saharan Africa. The aim of this study was to determine the prevalence of anemia and its association with renal disease among diabetic adult outpatients attending a hospital in Northeast Ethiopia.A cross-sectional study was conducted among 412 diabetic adults at the diabetes clinic of Dessie Referral hospital in Northeast Ethiopia, from January to April 2018. Each patient provided a blood sample for hemoglobin and serum creatinine levels and urine for albuminuria. Anemia was defined by World Health Organization criteria (<13 g/dl for men and <12 g/dl for women). Glomerular filtration rate (GFR) was estimated using the 4-variable Modification of Diet in Renal Disease (MDRD) equation. Chronic kidney disease (CKD) was classified into 5 stages based on the eGFR and albuminuria.P < 0.001). The prevalence of anemia increased with stage of CKD, from 22.6% at stage 1 to 100% at stage 4. Fifteen percent of the patients had anemia below the treatment threshold of 11 g ⁄dl. In multivariate analysis, older age ; type 2 diabetes ; presence of hypertension ; high systolic BP ; serum creatinine  and low GFR  were independently associated with greater odds for the presence of anemiaAnemia was present in 26.7% of the participants, and CKD in 43.0%. Anemia was more prevalent in patients with CKD (39.5%) than those without CKD (17.0%; Anemia is commonly present among diabetic adults attending our diabetes outpatient clinic in Northeast Ethiopia, including those without kidney disease. Our findings highlight the need for incorporating anemia screening into routine diabetes care to enable early detection and treatment of anemia and hence improve the overall care of patients with diabetes. Anemia is a common and often unrecognized complication of diabetes associated with all-cause and cardiovascular disease mortality ,2. Anemi12), autoimmune diseases, drugs and advanced glycation [The risk of anemia in patients with diabetes is estimated to be two- to three fold higher than that of patients without diabetes ,10. Evidlycation ,18.Despite the high prevalence of anemia in patients with diabetes, even in the absence of renal disease and its significant adverse effect on disease outcomes ,19, veryWe conducted a cross-sectional study at the out-patient diabetic clinic of Dessie Referral Hospital (DRH) in Northeast Ethiopia, from January to April 2018. DRH is found in Dessie town of Amhara regional state, which is located 401 km northeast of the capital Addis Ababa, Ethiopia, and it serves as a referral center for the Wollo and surrounding zones. The hospital registers and treats all diagnosed diabetic patients and provides tertiary diabetes patient care in the region. Patients with diabetes receive chronic care at the out-patient section of the diabetes clinic of the hospital. Patients with diabetes receiving care in the unit undergo an annual evaluation that includes amongst others: a clinical evaluation, an assessment of diabetes control (fasting blood glucose), chronic complications and cardiovascular risk factors assessment. Adult patients (≥ 18 years) attending the outpatient diabetes clinic of the hospital for routine follow up during the study period were included. All patients with a known hematologic disease, any acute condition within the last 2 weeks, or those who received a blood transfusion in the preceding 3 months were excluded. Patients were also excluded if they were pregnant (women) or hospitalized.The required sample size of the study was determined using single population proportion formula by considering: 50% prevalence rate of anemia, Z = 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 non-respondents, a total of 422 diabetic patients were included in the study. A systematic random sampling technique  was employed to select the study participants. Ten patients with missing clinical data were excluded, leaving 412 patients for the final analysis.2). Blood pressure (BP) was taken using a manual sphygmomanometer in a sitting position, after 5 minutes of rest and three measurements were averaged to be recorded. Hypertension was defined as systolic BP ≥140 mmHg and/or diastolic BP ≥90 mmHg or a self-reported presence of hypertension/use of antihypertensive medication.Eligible patients were approached and invited to participate while they were attending the clinic for their regular follow-up. Consenting patients were interviewed by the study investigators to collect information on demographic , medical history (presence of hypertension/use of antihypertensive medication) and behavioral factors  using a structured questionnaire. Anthropometric data such as weight (kg), height (m), and blood pressure (mm Hg) also were collected by the attending nurse or doctor. The body mass index (BMI) was calculated as weight divided by the square of height  values were determined using the hematology analyzer Cell-Dyn 1800 . Five ml of venous blood was obtained by using the serum separation tube (SST) and centrifuged within 1 hour for serum separation for creatinine measurement. Serum creatinine was measured using Jaffe kinetic method as mg/dl. Urine samples were also collected from the participating patients to detect albuminuria using dipsticks .2 with albuminuria; and stage 2, eGFR = 60–89.9 ml/min/1.73 m2 with albuminuria; and stages 3, 4 and 5 as an eGFR of 30–59.9, 15–29.9 and < 15 ml/min/1.73 m2, respectively. CKD was defined as K/DOQI CKD stages 1–5  [Anemia was defined according to the World Health Organization (WHO) criteria: Hgb concentration <13 g/dl for males and < 12 g/dl for females . A seconminuria) .2) test was used for comparison of categorical variables while the Student t-test  was used to compare continuous variables. Because only 32 patients had both eGFR < 60 ml/min/1.73 m2 and albuminuria, we examined the association between eGFR and albuminuria with anemia prevalence using chi-square test. For this, patients with eGFR< 60 ml/min/1.73 m2 were combined and compared with those eGFR ≥ 60 ml/min/1.73 m2. We also evaluated the associations of anemia with level of renal function (eGFR) using logistic regression. We repeated the analyses using mildly reduced eGFR (eGFR 60–89.9 ml/min/1.73 m2) in place of eGFR ≥ 90 ml/min/1.73 m2, as the reference group. Multivariate logistic regression analysis (backwards stepwise) was conducted and the corresponding adjusted odds ratios (AOR) and 95% confidence intervals (CI) were used to identify factors independently associated with anemia. P < 0.05 was used to indicate statistical significance.The data was entered in to “EpiData version 3.1” and was exported to SPSS version 20.0 statistical software for analysis. Normally distributed and continuous variables were expressed as mean (± SD), and non-normally distributed variables were presented as medians (quartiles 25 and 75%). Chi squared . Mean BMI was 22.23 ± 3.2 Kg/m2. Mean systolic and diastolic BP was 120 ± 19 and 79 ± 32 mmHg, respectively and 117 (28.4%) patients were hypertensive. The mean eGFR was 100.1 ± 39.8 ml/min/1.73 m2, and 59 patients (14.3%) had eGFR < 60 ml/min/1.73 m2. One hundred and fifty patients (36.4%) had albuminuria. On the other hand, about 79% of patients with albuminuria had eGFR ≥60 ml/min/1.73 m2. CKD was present in 43.0% of patients; of these, 35.0% had stage 1, 31.6% had stage 2, 32.2% had stage 3 and only one patient (0.6%) had stage 4 CKD  and anemia prevalence progressively increased with age: 18.3% for age 18–30 years, 14.6% for 31–45 years, 31.4% for 46–60 years and 52.4% for >60 years (P < 0.001). Interestingly, males were more likely to be anemic than females (P < 0.001), but this may be related to the higher threshold of hemoglobin for males in the definition used. Patients with anemia were more likely to have type 2 diabetes, longer duration of diabetes, alcohol consumers, hypertension, high current systolic BP, fasting blood glucose, serum creatinine and low eGFR . There was no significant difference in the educational status, smoking, BMI and diastolic BP of patients with and without anemia.Of the 412 study patients, 110 (26.7%) had anemia according to the WHO criteria. Characteristics of patients with or without anemia are summarized in P < 0.001). The prevalence of anemia increased with stage of CKD, from 20.6% at stage 1 to 100.0% at stage 4 (P < 0.001). The prevalence of anemia at a Hgb level <11 g/dl increased significantly with worsening of CKD stages as expected. stage 4 . Sixty-t2 had WHO-defined anemia compared with 21.2% of those with eGFR ≥ 60 ml/min/1.73 m2 (P < 0.001). In addition, logistic regression revealed that patients with moderate renal impairment (eGFR = 30–59.9 ml/min/1.73 m2) were 6.3 times  as likely to have anemia as patients with normal renal function (eGFR ≥ 90 ml/min/1.73 m2). Patients with moderate renal impairment were also 4.2 times  as likely to have anemia as patients with mild renal impairment (eGFR = 60–89.9 ml/min/1.73 m2) (data not shown). When stratified by albuminuria status, 38.7% of those with albuminuria had anemia, about 2.5 times higher than the 19.8% in those without albuminuria (P < 0.001).More than half (59.3%) of the patients with eGFR < 60 ml/min/1.73 mP = 0.026); type 2 diabetes ; presence of hypertension ; current high systolic BP ; high serum creatinine  and low eGFR  were independently associated with greater odds for the presence of anemia . This prevalence of anemia is comparable to 14.3% prevalence reported from sub-Saharan African study in Cameroon . Our preP < 0.001). This finding is significant given that anemia has previously been shown to be associated with cardiovascular disease and all-cause mortality and higher cost of managing diabetic patients with CKD [Our findings of increased rates of anemia among diabetic patients with CKD compared to those without CKD is consistent with findings from previous studies evaluating the relationship of diabetes to anemia ,14,32. Iwith CKD ,17. The with CKD –35, whicwith CKD .2 had anemia compared with 21.2% of those with eGFR ≥ 60 ml/min/1.73 m2. Renal function as measured by GFR equations was the strongest risk factor for prevalent anemia and may help to identify diabetic patients at greater risk [2). This is in accordance with previous findings [Similar to previous reports, anemia in our study was more common among diabetic patients with impaired renal function (low eGFR) ,24,25,37ter risk . It has ter risk . Howeverfindings ,19,25,37The multivariate analysis of this study showed that older age is independently associated with greater odds for the presence of anemia and this is consistent with other related studies ,34,37. TThis study also showed a significant association between anemia and the presence of hypertension and current high systolic blood pressure. This association is of concern considering that hypertension in diabetic patients’ increases the risk of renal impairment and thus increasing the subsequent development of anemia. In the above study by Thomas et al, a weak association between systolic blood pressure and hemoglobin level was detected in diabetic patients with renal disease after adjusting for differences in renal function . HoweverThe present study has some limitations: the study lacks control groups and did not assess the causes of anemia in diabetic patients. Renal disease was assessed based on a one-time GFR and proteinuria measurements, possibly leading to overestimation of the actual prevalence of CKD. The cross-sectional nature of the study, where better relationship between anemia and different potential factors affecting it progressively cannot be well established, so a longitudinal study is needed to assess the relationship over time. However, the study has provided some data to inform decision-makers to improve current care and management of diabetic persons.In conclusion, anemia is commonly present among diabetic adults attending our outpatient diabetes clinic in Northeast Ethiopia, including those without kidney disease. More than a quarter (26.7%) of our diabetic outpatients had anemia and 15.3% had anemia below the recommended treatment threshold of 11 g ⁄ dl. Our findings highlight the need for incorporating anemia screening into the routine assessment of diabetic complications particularly for those with significant risk factors to enable early detection and treatment of anemia and hence improve the overall care of patients with diabetes.S1 File(XLS)Click here for additional data file.


At a multivariate analysis, only mitotic count  and N2 resection  were found to be predictive factors of recurrence. The number of metastatic lymph nodes and mitotic count is the most significant predictive factors of recurrence after pancreatic surgery for nonmetastatic nonfunctioning pNENs.Nodal involvement  is an important prognostic factor after surgery for pancreatic neuroendocrine neoplasms (pNENs). We aim to evaluate the predictive role of the number of nodal metastases after pancreatic resection for pNENs. We analyzed from a prospectively maintained database all pancreatic resections for nonmetastatic nonfunctioning pNENs performed in our institution from 2011 to 2016. According to the number of nodal metastases, enhancing the actual categorization, we distinguished the following: N0, no nodal metastases; N1, 1-3 metastatic lymph nodes; and N2, metastases in 4 or more regional lymph nodes. Recurrence and disease-free survival (DFS) were evaluated. The predictive value in terms of recurrence for each clinicopathological data, including the number of metastatic lymph nodes, was calculated. Univariate and multivariate analyses were conducted. 77 patients underwent pancreatic surgery for pNENs. N0, N1, and N2 resections were found in 52 (67.5%), 16 (20.8%), and 9 (11.7%) cases, respectively. Mean follow-up of the entire cohort was 48 (±25) months. The recurrence rate was 11.8%, and the mean time of recurrence was 12 (±14) months. DFS was 83.7 months (76.0 - 91.5). At a univariate analysis, factors associated with recurrence were mitotic count (OR 1.19,  Pancreatic neuroendocrine neoplasms (pNENs) are rare neoplastic diseases, with an estimated incidence of 1/100000 people and account for about 1-2% of all pancreatic tumors .The presence of nodal metastases is currently considered as one of the most powerful prognostic factors , and an The aim of the current study was to evaluate the prediction role of nodal involvement and of the number of metastatic lymph nodes in our series of standard pancreatic resections for sporadic nonmetastatic pNENs.All consecutive standard pancreatic resections performed with curative intent for nonmetastatic nonfunctioning pNENs between 2011 and 2016 at our center were retrospectively analyzed from a prospective collected database. Functioning metastatic pNENs and those associated with genetic syndrome were excluded.All pancreatic resections were performed by expert pancreatic surgeons in a high-volume pancreatic center. Standard resections included pancreaticoduodenectomy (PD), distal pancreatectomy (DP), and total pancreatectomy (TP). The different resections were performed according to the preoperative work-up and to the decision of multidisciplinary board. All PD and TP were performed with an open approach, while a laparoscopic approach was adopted for selected cases of DP. Standard lymphadenectomy was performed in all resections . In all Database was queried for demographic data, perioperative, intraoperative, and postoperative detail findings. Postoperative complications were collected and defined according to Clavien-Dindo's classification  with graPathological reports were collected and evaluated. T and N stages were classified according to the current ENETS classification . The numAccording to that, pNEN G1 has a mitotic count < 2/10 high power fields (HPFs) and Ki-67 index < 3%, pNEN G2 has a mitotic count 2–20/10 HPFs and/or Ki-67 index 3–20%, pNEN G3 has a mitotic count > 20/10 HPFs and/or a Ki-67 index > 20% and is classified as well differentiated by pathologist, and pNEC has a mitotic count > 20/10 HPFs and/or a Ki-67 index > 20% but is classified as poorly differentiated.Mitotic counts was assessed in at least 50 HPFs in most mitotically active areas, and Ki-67 labeling index was measured in 500-2000 cells in the most proliferative “hot spot” areas.Taking in consideration the discrepancies between mitotic count and Ki-67, the final grade was based on which ever index places the tumor in the highest grade category.R1 resection was defined as the presence of neoplastic involvement of the evaluated margin (0 mm rule).All cases were discussed at a multidisciplinary board for the decision of eventual treatments and of timing of follow-up. Generally, a 6-month follow-up including at least a high-quality imaging procedure was proposed to all the patients for the first 2 years and yearly thereafter as a minimum for at least 5 years from surgery. Ga68-PET was usually repeated at one year from surgery in neoplasm positive to the preoperative evaluation.Information regarding general health status, disease recurrence, or progression was collected during scheduled outpatient visits. Disease-free survival (DFS) was defined as the time from diagnosis to first evidence of disease recurrence at imaging and was censored at the last follow-up date if no events had occurred. Overall survival (OS) was defined as the time from diagnosis to the last follow-up.T-student, chi-square). DFS was assessed using the Cox regression model and Kaplan-Meier curves with log-rank comparison. Odds ratio (OR) was reported with the 95% confidence interval (CI). Univariate and multivariate analyses were conducted. Statistical significance was stated with a p value < 0.05. No analysis of OS was carried out due to a single disease-specific event during the follow-up.Data were analyzed using the IBM SPSS software version 13 and are expressed as percentage or mean ± standard deviation. Descriptive and inferential statistics were carried out with the parametrical analytical models adequate for the type of variable studied  and were included in the analysis: 22 PD (28.6%), 50 DP (64.9%), and 5 TP (6.5%).p < 0.001). The overall rate of nodal metastases was 32.5%. According to the number of metastatic lymph nodes, resections were classified as N1 and N2 in 16 (20.8%) and 9 (11.7%) cases, respectively. Mean LNR was 0.09 ± 0.2, without a significant difference between the surgical procedures.Demographic and clinicopathological features are listed in p < 0.001)). The mean postoperative length of stay was 10 days ± 5, and the readmission rate was 7.8%.Only in one case 90-day postoperative mortality was recorded (1.3%): the patient died in postoperative day 7 after TP for a septic shock. Major postoperative complications occurred in 15 cases (19.5%); the POPF rate was 22.2% . Three other patients died during the follow-up period due to other causes not related to disease. The recurrence rate was 11.7% (8 cases), and the mean time of recurrence was 12 ± 14 months. DFS was 83.7 months (76.0 - 91.5).p < 0.05). When we considered the number of nodal metastases, at a univariate analysis, N2 resection was a significant prognostic factor (OR 30.12 (2/44-244.94), p < 0.002). No significant correlation was found with the patients' characteristics or type of procedure.The univariate analysis of DFS predictive factors was described in The better ability to catch variation in DFS of the proposed nodal involvement categorization is also clear evaluating the Kaplan-Meier for DFS of this different grouping variables Figures  and 2, aIn a model accounting for the mitotic count, the Ki67 value, and the presence of lymph node metastases, at a multivariate analysis, only mitotic count remains an independent prognostic factor (OR 1.51 (1.0-2.29)), while the presence of metastatic lymph nodes loses its significance (OR 26.71 (0.60-1180.48)). Performing the multivariate analysis adopting LNR instead of the presence of nodal metastases, LNR seems a more robust variable but still not significant (OR 11.60 (0.91-148.04)). On the other hand, adopting the proposed categorization , at the same multivariate analysis, N2 resection was found to be an independent prognostic factor (OR 134.74 (2.19-8261.17). In all these three evaluated models, at a multivariate analysis, mitotic count remains an independent prognostic factor.The correct prognostic evaluation of a neoplastic patient is crucial to determine his specific cure pathway. This aspect is even more relevant in patients affected by pNENs: in fact, these patients could have a very long survival , but thep < 0.001). Similar results were found by Zhang et al. [p < 0.001). At a multivariate analysis, mitotic count  and number of nodal metastases  were found to be the only independent prognostic factors. Mitotic count is theoretically a less reproducible variable with respect to Ki67 proliferation index, despite that, it perform better in the multivariate model. Some aspect of collinearity and the low recurrence rate should be taken into account when evaluating this result.Many different classifications for pNENs have been proposed, with the aim to frame in the best way the patients' prognosis . Nodal ig et al.  evaluatiInterestingly, in our series N0 and N1 patients showed similar mean DFS (81.80 (75.74-87.86) and 80.0 (61.42-98.58) months, respectively). This result demonstrated that N1 patients have a biological behaviour more similar to N0 patients, if compared with N2 ones. Evaluating together N1 and N2 patients, we could risk not only an overall prognostic ability but also to neglect a clinical relevant subgroup of patients that could benefit of a different management.This study has several limitations. The first one is its retrospective nature. Another weak points are the low number of recurrence in our series that could weaken the statistical power of the study. Unfortunately, national database usually lacks the specific data of number of metastatic lymph nodes to answer this query. As shown in the national retrospective analysis performed by Zhang et al. , more thThe number of metastatic lymph nodes in nonfunctioning resected pNENs seem to be a better prognostic factor, if compared with the simple presence of nodal metastases. This finding should be taken into account for the development of the actual staging classifications.


Objectives: In this study, we aimed to analyse the genetic diversity Kelch 13 (K13) propeller allele of the Plasmodium falciparum isolates mainly imported from Southeast Asia and Africa in southern China, including the provinces of Yunnan and Guangxi.Methods: At enrolment, we collected blood samples from patients with confirmed cases of malaria infection between January 2012 and December 2017, for analysis. Individual patient information was obtained via a malaria surveillance system. The malaria infections and P. falciparum K13 mutations were diagnosed by using a nested polymerase chain reaction (PCR) method.Results: The K13 mutations were identified in 283 P. falciparum isolates from 18 counties in Yunnan and 22 counties in Guangxi. Of Forty-six isolates  that harbored K13 mutant alleles were detected: 26.8% in Yunnan (33/123) and 8.1% in Guangxi (13/160). A total of 18 different K13 mutations were detected. Only the F446I mutation was detected in Yunnan isolates, and F446I was more frequent  than other alleles. Further, the temporal distribution of the F446I mutation ratio from 2012 to 2015 exhibited no significant difference in Yunnan Province . A578S allele was the main K13 mutation  from Africa. The K13 mutants were present in 33.3% of indigenous isolates, 27.4% of isolates from Southeast Asia, and 7.9% of isolates from Africa. The analysis of 10 neutral microsatellite loci of 60 isolates showed that at the TAA109 locus, the expected heterozygosity of F446I (He = 0.112 ± 0.007) was much lower than that of wild type and other mutation types in Myanmar isolates. With respect to geographic distribution, TAA109 also exhibited a significant difference between isolates from Southeast Asia (He = 0.139 ± 0.012) and those from Africa (He = 0.603 ± 0.044).Conclusions: The present findings on the geographic diversity of K13 mutant alleles in P. falciparum may provide a basis for routine molecular surveillance and risk assessment, to monitor artemisinin resistance (ART) in China. Our results will be helpful for enriching the artemisinin resistance database in China during the elimination and post-elimination phases. Plasmodium falciparum malaria infection in endemic regions by the World Health Organization (WHO). ACTs have contributed to a sharp reduction of the malaria burden in many countries  have been recommended as first-line drugs for the treatment of ountries . Howeverountries , 3. Therountries . ART andountries . For exan to ART . It wouln to ART , 8. Suchrovinces . Besidesast Asia .PfKelch 13 propeller (K13) has been identified as a molecular marker for ART-resistant isolates. More than 5% of surveyed patients carrying K13 resistance-confirmed mutations, and all of whom have been found to have either persistent parasitaemia by microscopy on day 3 or a half-life of the parasite clearance slope ≥ 5 h after treatment are considered as confirmed endemic artemsinin resistance  , or rapiAll individual cases included in this study from the web-based reporting system (China Information System for Diseases Control and Prevention) were carefully reviewed and analyzed.PF3D7_1343700) was amplified using a nested PCR method as described previously (http://blast.ncbi.nlm.nih.gov/). Multiple nucleotide sequence alignment and analysis were performed using the DNAMAN sequence alignment editor (https://www.lynnon.com/pc/framepc.html).Parasite genomic DNA from all blood-spot samples collected into microcentrifuge tubes and one filter spot (diameter 5 mm) was extracted using a QIAamp DNA Blood Mini Kit , 500 μL elution buffer was added into each tube by following the dried blood spots protocol for the kit. The K13 propeller gene , TA1 (Chr6), TAA42 (Chr5), TAA60 (Chr13), TAA81 (Chr5), TAA87 (Chr6), TAA109 (Chr6), pfPK2 (Chr12), Polyα (Chr4), and B5M2 (Chr7) were selected for analysis . The fira), number of effective alleles (Ne), number of private alleles (Np), and the expected heterozygosity (He) were evaluated using the GenAlEx 6 microsatellite plugin , number of different alleles (Ne plugin . He was He = [n/(n−1)] (1–∑3{2(n−2)[∑. The map was created by ArcGIS 10.1 . The Fisher's exact test was used to assess the differences in gene polymorphisms between indigenous cases and imported cases from abroad, as well as the temporal distribution of mutant alleles. Calculated The study was reviewed and approved by the ethical committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention . This study was carried out in accordance with the recommendations of Chinese National Antimalarial Resistance Surveillance Plan, China CDC. The protocol was approved by the NIPD, China CDC. All subjects gave written informed consent in accordance with the Declaration of Helsinki.A total of 285 isolates were collected: 123 isolates were collected from 18 counties in Yunnan, including 6 indigenous isolates and 117 imported isolates from Southeast Asia (116 isolates from Myanmar and 1 isolate from Laos). Another 162 isolates were collected from Guangxi, all of which were imported from African countries. Because 2 isolates were unable to be sequenced, therefore 283 effective isolates were finally obtained for analysis . All then = 28, 22.8%), Ruili , and Tengchong . Most isolates were from male patients  with the age of 16–35 years . The isolates were mainly collected in 2013 . Isolates from Guangxi were mainly from the counties of Shanglin , Bingyang , and Nanning . Similar to Yunnan, the isolates were mainly from patients aged 16–59 years  and most isolates were collected in 2013   .P. falciparum isolates were assayed and sequenced. K13 mutations were detected in 46 isolates (16.3%), with 33 of these from Yunnan  and 13 from Guangxi  ; the remaining 44 isolates were imported from abroad.To investigate genetic diversity in K13 propeller allele, all 283  13/160) . Two locp < 0.05). Additionally, N458Y, which is associated with ring-stage survival assay, was found in two samples from Myanmar. The mutant allele A578S was detected in 6 patients, 5 from Ghana and 1 from Myanmar (p < 0.05). Further, three isolates harboring the A676D allele were all found in patients returning from Myanmar  . F446I w Myanmar .p = 0.121).From 2012 to 2015, the temporal distribution of the F446I mutation rate exhibited no significant difference , and all were F446I. Furthermore, 31 of 115 isolates from countries in Southeast Asia exhibited K13 mutations (27.0%). Conversely, only 13 of 160 isolates from Africa harbored mutated sites (8.1%) (p < 0.05). With respect to the distribution of K13 mutant alleles in China, 33 isolates harboring mutated alleles were observed in Yunnan, mainly in Tengchong , Ruili , and Yingjiang  counties. In Guangxi, 13 isolates harbored K13 mutated alleles were mainly from Ghana , and there was one patient each from Sierra Leone, Cameroon, the Congo, and Equatorial Guinea , F446I allele type (n = 20), and other mutant allele types . Generally, no significant difference in allele frequency was observed between wild type and MT; the He value of F446I was lower than that of wild type and MT (He = 0.112 ± 0.007) had fewer alleles than wild type (He = 0.385 ± 0.028) and MT (He = 0.478 ± 0.036) in Myanmar isolates (He = 0.139 ± 0.012) and those from African countries (He = 0.603 ± 0.044) (We genotyped 60 isolates (including 17 from African countries and 43 from Southeast Asia) using analysis of 10 neutral microsatellite loci. According to the K13 single nucleotide polymorphism (SNP) sequencing data and sample size, and because F446I is the prominent mutant allele in the China–Myanmar border area, the microsatellite loci data were classified into three types: wild type (e and MT . Since misolates . With re± 0.044) .Anopheles mosquito remains present in the region  and dhfr  alleles (P. falciparum could be a multigenetic process and could also be combined with other molecular markers, such as pfmdr1, pfmdr2, pfatpse 6, and pfarps 10, all of which have been previously reported to be related to ART resistance (In K13 population genetic analysis, we conducted a microsatellite assay to assess and evaluate geographic evolution. F446I was seen to exhibit a lower e region . Thus, F alleles , 37. Thesistance –40. The For the limitation, of the malaria cases studied, only six cases were indigenous malaria. Considering that we intended to analyse the diversity of locally acquired infections and potential evolution of ART resistance in China, the number of samples were not adequate.He value than both wild type and MT at locus TAA109. As for imported cases from Africa, few K13 mutant alleles related to ART-resistance were found, with A578S predominating in African samples. Routine molecular surveillance and risk assessment should be conducted to monitor ART resistance throughout the study region. The present data will be helpful in development of a genetic molecular marker to trace malaria infection sources during the malaria elimination and post-elimination phases, to achieve the goal of eliminating ART resistance in P. falciparum throughout the GMS.In summary, the present data indicated a high prevalence of the F446I mutant allele, detected among both indigenous and imported malaria cases from Myanmar, especially for counties along the China–Myanmar border. In addition, a distribution pattern of K13 mutant alleles was observed that differed from other regions of the GMS or Africa. Microsatellite analysis indicated that the F446I allele had a much lower JF designed and conducted the study, and drafted the article. XK and HT carried out the statistical analysis. HY carried out the molecular experiment. DX carried out the epidemiology survey and recording. HZ and KL supported the study and performed sample collection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this study was to evaluate the impact of a single- vs. double-layer cementing technique on morphological cementation and the generation of microscopic cement layers or loose cement fragments in unicompartmental knee arthroplasty (UKA).UKAs were implanted in 12 cadaver knees. The specimens were divided into two groups of comparable bone mineral density. Six UKAs were implanted using a single-layer cementing technique (group A) and six UKAs were implanted using a double-layer cementing technique (group B). Morphological cementation was assessed on nine cuts through the implant–cement–bone interface in the frontal plane. Loose bone cement fragments and the microscopically quality of layer formation were evaluated.p = 0.126). The significant increase of areas without visible cement interlocking in the anteroposterior direction in group A (p = 0.005) was not evident in group B (p = 0.262). Penetration around the peg tended to occur more frequently in group B (67.5% vs. 90.6% p = 0.091). Scanning electron microscopy identified no evidence of fissure formations within the bilaminar cement mantle. Free bone cement fragments were documented in 66.7% in both groups with no difference concerning mass (p = 1.0).Contact between bone and prosthesis was observed in 45.4% of interfaces in group A and 27.8% in group B (This in-vitro study showed a tendency towards a more homogenous cementation of tibial UKAs using a double-layer cementing technique, although most of the differences did not reach the level of significance. However, theoretical downsides of the double-layer cementing technique such as an increased formation of free bone fragments or a microscopically fissure formation within the cement layer could not be detected either. With adequate patient selection, unicompartmental knee arthroplasty (UKA) has become an effective treatment for anteromedial osteoarthritis of the knee, leading to good long-term survival , 2 and bThe objective of this study was to evaluate the morphological effects of a double-layer cementing technique on the homogeneity of the cement mantle and the interlocking of bone cement with trabecular bone. Moreover, potential negative effects of this technique such as the formation of loose cement particles in the posterior aspect of the joint or a microscopic layer formation within the cement mantle were analyzed.The study received approval from the local ethics committee. Twelve tibial and femoral UKA components  were implanted in the medial compartment of fresh-frozen human cadaver knees using a minimally-invasive approach. In advance, CT-scans  of all specimen were obtained to determine bone density and to exclude specimens with osseous irregularities . Based on bone mineral density (BMD), two groups with comparable bone density were formed Table . The HouAll implantations were performed by the same experienced surgeon (PEM) following the guidelines of the manufacturer. The appropriate implant size was selected based on preoperative CT measurement and intraoperative findings. After tibial resection, the cancellous bone was conditioned with pulsed lavage  and saline solution. The tibial and femoral components were cemented in two stages using manually mixed bone cement . In group A, approximately 10 g of cement were applied manually to the underside of the prosthesis while the resected tibia remained cementless (single-layer cementing technique). In group B, approximately 10 g of cement were applied in equal parts by hand to the underside of the prosthesis plus the tibia. Each tibial component, was carefully placed on the tibia, beginning with the dorsal edge to prevent dorsal cement extrusion, and impacted using a special instrument . Manual pressure was maintained until the cement cured completely. The joint was then opened and free cement bodies were collected, conserved and quantified using an analytical balance  and sawed into 10 slices in the coronary plane. To ensure that slices of the different component sizes showed the same implant region, we virtually designed a masterplate for the cutting process  that was manufactured in a rapid-prototyping process , attached to the embedded implant and guaranteed that all 10 slices of a specimen had the same thickness.The cut surfaces were cleaned and tiff-images with a resolution of 100 pixel per mm were acquired of all interfaces . The further evaluation of both sides of the nine serial cuts through the implant–cement–bone interface of the 12 specimens was carried out using Adobe Photoshop CS6 .In order to adequately capture the special geometry of the tibial tray and the associated penetration behavior, the underside was divided into two areas. Area 1 comprised the horizontal part of the prosthesis. Area 2 related to the area around the anchoring peg . If fissure formations were apparent, they were defined for the section number, crack number, location of the crack, and length of the crack.In addition to the macroscopic assessment of conventional scans of the cut surfaces, a anterior (front of 8th slice), central (front of 5th slice) and posterior (back of 3rd slice) interface was analyzed by a scanning electron microscope  and the associated software , to approximate a quartering of the specimen. The respective cement-bone-interface was put on the stage of the specimen chamber facing up. A low-vacuum of 1.23 × 10p-value < 0.05 was defined to be statistically significant. For statistical evaluation a repeated-measures analysis of variance (ANOVA) was performed to test for significant differences in the morphologic cementation parameters. Prior to analysis, normal distribution was verified . Differences of the parameters between the groups were evaluated with a Bonferroni post hoc analysis. Additionally, the correlation between penetration and BMD and the two measurement techniques of bone density was determined using the Spearman’s rank correlation.The statistical analysis was performed using GraphPad Prism 5 . A 3 BMD in group A and 168.4 (SD 55.9) HU or 113.3 (SD 41.1) mg/mm3 BMD in group B, demonstrating good comparability  between the groups. The correlation between HU and BMD was rs = 0.90 (P < 0.0001). In group A, the mean HU was 169.8 (SD 73.6) in the anterior, 123.4 (SD 38.9) in the central and 170.5 (SD 53.1) in the posterior section (p = 0.203). In group B, the corresponding figures were 185.8 (SD 78.3) HU anterior, 131.0 (SD 49.4) HU central and 188.4 (SD 57.6) HU posterior (p = 0.191).The mean bone density was 154.6 (SD 48.3) HU or 103.3 (SD 26.0) mg/mmp = 0.126; Fig. In area 1, interruptions of the cement mantle, e.g. direct contact between the bone and the prosthesis were observed in 45.4% of all interfaces in group A and in 27.8% of all interfaces in group B p = 0.12; Fig. 6.p = 0.936). The corresponding overall length proportion of area 1 without visible cement intrusion at cement-bone-interface was 37.6% of (SD 10.5%) in group A and 31.0% (SD 21.3%) in group B (p = 0.818). However, in the anterior-posterior development, group A showed a proportion of 14.2% (SD 10.5%) of area 1 without visible intrusion in the anterior section, 42.6% (SD 16.7%) in the central section and 55.2% (SD 17.1%) in the posterior section (p = 0.005). The corresponding figures for group B were 20.2% (SD 16.8%), 33.3% (SD 23.8%) and 40.1%  in group A, rs = 0.66 (p = 0.18) in group B and rs = 0.50 (p = 0.10) with respect to all specimens.Areas without visible cement intrusion at cement-bone-interface were observed in 87.8% of all interfaces in group A and 72.2% in group B (p = 0.091).Visible cement penetration in area 2 was less frequently observed in group A with 67.5% than in group B with 90.6% Fig. . Howeverp = 1.0; Fig. Loose fragments of bone cement could be found in 4 out of 6 cases in both groups . Their average weight was 40.1 mg (SD 10.1 mg) in group A and 40.5 mg (SD 10.6 mg) in group B . Cement penetration into trabecular bone in area 2 was visible on 67.5% of all interfaces in group A and 90.6% in group B (p = 0.096). The significant increase of the length proportion of area 1 without cement interdigitation in the anteroposterior direction in group A (p < 0.005) was not evident in group B (p = 0.262). The latter might be important, as the posterior part of the joint is often regarded as a weak point of cemented fixation [Concerning the macroscopic examination, there was an overall trend in favor of the double-layer cementing technique, but no significant differences. The proportion of cement-bone-prosthesis interfaces with direct contact between the prosthesis and trabecular bone was 45.6% in group A („single layer“) and 27.8% in group B („double layer“) in area 1 . Their frequent occurrence in four out of six specimen in both groups is in accordance with a study by Hauptmann et al., according to which free cement fragments after minimally-invasive implantation of UKA represent an underestimated problem [Moreover, there was no indication of an increase in the mass of free cement fragments in the posterior aspect of the joint ( problem . Elmadag problem . Bhuta e problem . Our res problem , 33, 34.In summary, the significant advantage of two-layer cementation on cement penetration described in Sawbones could not be reproduced under minimally-invasive surgical conditions in human tibia. However, no indications of adverse effects of two-layer cementation were observed. These results are consistent with previous results, that described no differences between the single and double-layer cementing technique of unicompartmental tibial components with regard to failure load .This study is subject to potential limitations. First, the present in-vitro examination on cadaver legs did not simulate blood circulation. It was postulated from hip arthroplasty experiments that blood flow might have a negative influence on cement penetration , 36. ForThe present study does not provide any indications of negative effects caused by a double-layer cementing technique such as an increase in the formation of free bone fragments or a microscopically layer formation in tibial UKA. However, improvements in the morphologic homogeneity of the cement mantle in area 1 or the improved average penetration in area 2 do not reach the level of significance. Therefore, the effectiveness of a double-layer cementation technique should be investigated further in randomized controlled clinical trials.


Ullmann et al. recently published a pilot study in Translational Psychiatry in which they report failing to find a statistically significant reduction in either hair cortisol or hair cortisone levels in circumcised men as compared with genitally intact (noncircumcised) men. Based on such null findings, the authors purport to have "refuted the psycho-pathological long-term effects of circumcision" and that the lack of significant results, "add to the growing body of evidence in the literature that male circumcision is not likely psychologically traumatizing across the life-span." In addition, they claim that they have proven a "healthy functionality of the LHPA axis" in men subjected to circumcision during infancy or childhood. However, it is not possible to draw any such conclusions on the basis of a null finding, especially one derived from an underpowered study in which the trend in the data suggest, if anything, that an adequately powered study may have shown the opposite of what the authors claim.When combined with other weaknesses in study design, measurement, and interpretation, it becomes apparent that the authors' conclusions are not supported by their data. Regrett2.2.1The NHST procedure used by Ullmann et al., while commonly employed, has been strongly criticized by statistical experts, with some authors arguing it is invalid . But eveAs advised by methodologists Szucs and Ioannidis, any researchers who do choose to use the NHST approach, despite its shortcomings, must "justify its use, and publish pre-study power calculations and effect sizes" wherever feasible . Since UIn order to determine the minimum sample sizes needed in order to find any significant between-group differences, power analyses were conducted by the present author using two entirely different methods: the classical approach advocated by Cohen ,35, and 2.2.2A power calculation using Cohen's method (computed via G*Power 3.1) with moderate effect size = 0.50, power = 0.80, and a = 0.05, indicates that even without Bonferroni correction for the multiple dependent measures, at least 51 genitally intact men and 153 men in the 'mixed' circumcision group would be needed in order to have an 80% chance of observing any significant between-group differences on any of the dependent variables . When a 2.2.3(cdf) of the standard normal distribution , / is the desired precision , k is the number of comparison groups, and n is the requisite sample size , respectively." Thus, "a desire for stringent precision . necessitates large samples [in order] to have a respectable probability" of obtaining replicable findings . Even acAs Keppel stated, "it is not sufficient simply to fail to reject the null hypothesis to 'prove' it, but you must do so under conditions of high power . an experiment that is specifically designed to prove the null hypothesis usually requires a huge commitment of subjects" . Since t2.3.4.We have seen that Ullmann et al. attempted to draw inferences about PTSD-like symptoms in relation to circumcision status on the basis of inadequate sample sizes and negative findings. Without considering possible confounding of sampling precision, homogeneity precision, and measurement precision ,41, UllmCortisol :95% CI: 7.4 ±2.7 (4.70 to 10.10); and 99% CI: 7.4 ±3.6 (3.80 to 11.00). Cortisol (9 circumcised men):95% CI: 5.7 ±1.8 (3.90 to 7.50); and 99% CI: 5.7 ± 2.3 (3.40 to 8.00). Cortisone :95% CI: 17.3 ±7.4 (9.9 to 24.70); and 99% CI: 17.3 ±9.8 (7.50 to 27.10). Cortisone (9 circumcised men):95% CI: 14.2 ±2.4 (11.80 to 16.60); and 99% CI: 14.2 ±3.1 (11.10 to 17.30).Perceived Stress Questionnaire scores  were unacceptably wide, showing that the mean PSQ scores reported were unreliable. The respective confidence intervals are as follows:However, Ullmann et al. omitted to report confidence intervals for any of the five subjective self-report questionnaires. Examination by the present author of both the 95% and 99% CIs reveals that the confidence intervals for the 30-item PSQ (7 circumcised men):95% CI: 0.02 ± 0.16 (-0.14 to 0.18); and 99% CI: 0.02 ± 0.20 (-0.18 to 0.22).Since Ullmann et al. stated that, "The PSQ meets the highest national and international quality standards" , one canContrary to the authors' assertions that "circumcision does not alter long-term glucocorticoids' accumulation" (and despite not being significant due to insufficient sample size), the reported data from the Ullmann et al. study actually did show substantial reductions in circumcised men of 23% and 18% in objectively measured hair cortisol and cortisone levels, respectively, thereby making it difficult to rule out dysfunction of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis. But, only an adequately powered study could properly answer the research question that Ullmann et al. had put forward.3.The Ullmann et al. paper exhibits weaknesses beyond those described in the present analysis, including an overt misrepresentation of policy statements bearing on the subject of their investigation see Box 2). What is most fundamentally at issue, however, is the authors' unjustified assertion of having proven the null hypothesis with an obviously underpowered study. It is not possible to ' prove' the null hypothesis, but only to fail to reject it . What is. Such miBox 2. Misrepresentation of policy statementsnot outweigh the risks  health benefits are not great enough to recommend routine circumcision for all male newborns" . The CDChe risks .Moreover, despite the negative findings of Ullmann et al., the documented empirical evidence demonstrates unequivocally that circumcision  is highly stressful for infants and children and can be traumatic -49. GunnAlthough individual differences in personality trait characteristics (such as resilience) suggest that the same potentially traumatic incident may impact on individuals differently , availabIn the context of this discussion, it is important to recognize that negative results may sometimes be meaningful, and scientific journals do need to be more open to publishing such results as argued recently in this journal . But in


Pneumonia, the leading reason underlying childhood deaths, may be triggered or exacerbated by air pollution. To date, only a few studies have examined the association of air pollution with emergency department (ED) visits for pediatric pneumonia, with inconsistent results. Therefore, we aimed to elucidate the impact of short-term exposure to particulate matter (PM) and other air pollutants on the incidence of ED visits for pediatric pneumonia.2.5, PM10, and other air pollutant levels were measured at 11 air quality-monitoring stations in Kaohsiung City, Taiwan, between 2008 and 2014. Further, we extracted the medical records of non-trauma patients aged ≤17 years and who had visited an ED with the principal diagnosis of pneumonia. A time-stratified case–crossover study design was employed to determine the hazard effect of air pollution in a total of 4024 patients.PM2.5, PM10, nitrogen dioxide (NO2), and sulfur dioxide (SO2) on 3 days before the event increased the odds of pediatric pneumonia by 14.0% , 10.9% , 14.1% , and 4.5% , respectively. In two-pollutant models, PM2.5 and NO2 were significant after adjusting for PM10 and SO2. Subgroup analyses showed that older children (aged ≥4 years) were more susceptible to PM2.5 (interaction p = 0.024) and children were more susceptible to NO2 during warm days .The single-pollutant model suggested that per interquartile range increment in PM2.5 and NO2 possibly plays an important role in pediatric pneumonia in Kaohsiung, Taiwan. Older children are more susceptible to PM2.5, and all children are more susceptible to NO2 during warm days.Short-term exposure to PM Partineumonia . Epidemiarticles .10 (PM having an aerodynamic diameter of < 10 μm), PM2.5, NO2, and O3 are related to lung and systemic inflammation [2.5 on emergency hospitalizations for pneumonia [Pneumonia, an inflammatory lung condition, is the leading cause of death in children, accounting for approximately 1.3 million deaths among children aged < 5 years in 2010–2011 . Air polammation –10. The ammation , populatammation , the proneumonia  was greaneumonia . For chineumonia , 17. Howneumonia , 19. Fur2.5, especially in individuals with pre-existing disease.Over a 7-year period, in South Taiwan, we collected data of pediatric patients who presented at the ED from a tertiary academic medical center due to pneumonia. Using a case–crossover design, the data were analyzed with respect to weather and air pollution parameters. The study aimed: (1) to evaluate correlation between increase in short-term exposure to air pollutants and events of pediatric pneumonia and (2) to evaluate the potential triggering effects of PMKaohsiung is located in the southwestern part of Taiwan and has a tropical monsoon climate. It is the leading industrial city with the largest commercial harbor in Taiwan. The industry clusters include basic metals, nonmetallic mineral products, transportation equipment manufacturing, food and agricultural products, chemical products, machinery and repairs, and power equipment and repairs.This was a retrospective observational study conducted in an urban tertiary medical center, which has 72,000 ED visits on an average every year. The study period was from January 1, 2008 to December 31, 2014. We retrospectively reviewed the electronic medical records, and from the ED’s administrative database, extracted data of non-trauma patients who were ≤ 17 years old and had visited an ED with a documented pneumonia diagnosis . We abstracted the following characteristics from the electronic charts: age, sex, and underlying conditions, including respiratory diseases (such as chronic respiratory failure and restrictive lung), cerebral palsy, asthma, and epilepsy.This study was approved by the institutional review board of our hospital (no. 201801301B0) and has been performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. For this type of study, informed consent from the subjects was not required.2 (using ultraviolet fluorescence), sulfur dioxide , PM10 (using beta-ray absorption), PM2.5 (using beta-ray absorption), and O3 (using ultraviolet photometry), as well as weather condition, such as temperature and humidity. Missing data accounted for less than 1% of the total data.In 1994, Taiwanese Environmental Protection Administration, a government agency, had constructed 11 air quality monitoring stations in Kaohsiung City. The commercial monitoring instruments of the stations were designated by the US Environmental Protection Agency as equivalent or reference instruments and manufactured by US Thermo Environmental Instruments, Inc. . The automatic stations routinely monitor several “criteria” pollutant levels, including NOFrom all the monitoring stations, we collected air pollution data on an hourly basis, and collected addresses of pediatric pneumonia patients from medical records. In addition, we collected recordings of mean temperature and humidity on a daily basis. Finally, from the nearest monitoring station, we computed the 24-h average pollutant levels.2.5 mass and each air pollutant were estimated. Subgroup analyses including sex, age, and underlying diseases in the most susceptible groups were also performed. Exposure levels to air pollutants were included into the pollutant models as continuous variables. Each model was adjusted for meteorological variables, such as average daily temperature and humidity on the same day and during lag intervals. Our analysis was conducted in two steps. First, conditional logistic regression analysis was performed using the SPSS version 25.0 software. The baseline model included a linear expression that included air pollutants and confounding factors, such as temperature and humidity. Second, we examined nonlinear effects by introducing temperature and humidity separately in the model and comparing the goodness-of-fit using the Akaike information criterion (AIC). The second step was performed using the SAS macro lgtphcurv9 (in SAS version 9.4), which implements natural cubic spline methodology to fit a potentially nonlinear response curve in conditional logistical regression models for matched case–control studies. With temperature, the AIC value for the linear model  was better than that for the spline model , and the test of curvature (nonlinear relationship) was nonsignificant (p = 0.30). Similarly, with humidity, the AIC value for the linear model  was better than that for the spline model , and the test of curvature was nonsignificant (p = 0.59). As a result, we used the linear model for the entire conditional logistic regression analysis.To analyze pediatric pneumonia events, we employed a time-stratified case–crossover study design , 21 as a2.5, PM10, NO2, SO2, and O3 exposure. The significance criterion was set at p < 0.05. All statistical analyses were performed with SPSS version 25.0 .The ORs were calculated based on interquartile range (IQR) increments in PMIn total, 4625 pediatric pneumonia cases were recorded in Kaohsiung over the 7-year study period. Of these, 601 patients were excluded because they were not residents of Kaohsiung City; whereas the other 4024 patients were included in the study. Table 2.5 and PM10 concentrations over the study period were 41.1 and 72.8 μg/m3, respectively. The average NO2, SO2, and O3 levels were 19.1, 6.4, and 29.1 ppb, respectively.Table 2.5 was highly correlated with PM10  and NO2 ; and moderately correlated with SO2  and O3 .Table 2.5, PM10, NO2, and SO2 levels on lag 3 were associated with increments of 14.0% , 10.9% , 14.1% , and 4.5%  in the odds of pediatric pneumonia ED visits, respectively. Meanwhile, the IQR increase in O3 level was associated with a 6.6% , not significantly related to pediatric pneumonia ED visits.Figure 2.5 was significantly related to ED visits for pediatric pneumonia after adjustment for PM10  and SO2 . An IQR increase in NO2 was significantly associated with ED visits for pediatric pneumonia after adjustment for PM10  and SO2 . Associations with pneumonia ED visits were no longer significant when PM2.5 was adjusted for NO2  or when NO2was adjusted for PM2.5  in two-pollutant models.A two-pollutant model was used to determine which individual contaminant influences the number of pediatric pneumonia ED visits independently of other pollutant effects. In accordance with the results obtained from the single-pollutant models, the multi-pollutant models were fitted with different pollutant combinations (with up to two pollutants per model) to assess the stability of the effects of PM. The results obtained are presented in Table 2.5 and NO2 on pediatric pneumonia according to different seasons, temperature, and underlying diseases on lag 3, after adjustment for temperature and humidity. As shown in Fig. 2.5; an increase in the IQR for PM2.5 was associated with increases in the odds of ED visits for pneumonia of 21.7%  for older children and 1.7%  for younger children (aged < 4 years). The children were more susceptible to NO2 during the warm days (≥26.5 °C) than during the cool days (< 26.5 °C)  in the odds of ED visits for pneumonia, respectively. There were no significant differences with respect to the effects of PM2.5 or NO2 on pediatric pneumonia between the male and female patients, between those with or without underlying cerebral palsy, epilepsy, respiratory disease, or asthma, and between different seasons or temperatures.Figure 2.5 and NO2 possibly play important roles in pediatric pneumonia events in Kaohsiung, Taiwan. Of all pollutant exposures included in the analysis, the odds of pediatric pneumonia following PM2.5 exposure was greater in older children. Additionally, the effect of NO2 on pediatric pneumonia was more significant during warm days.In this study, we estimated the effects of PM and other air pollutants on pediatric pneumonia and found that PM2.5 and human health, especially in respiratory and cardiovascular diseases. Several previous studies demonstrated a positive association between PM2.5 and pediatric respiratory disease, such as upper respiratory infection and asthma [2.5 and pediatric pneumonia. Lv et al. demonstrated increased risk of hospital admissions for pediatric pneumonia for PM2.5 on lag 4 [2.5, were associated with pediatric ED visits for respiratory tract infections on lag 0–3 [2.5 concentrations on pediatric pneumonia [2.5 and pneumonia. Our study demonstrated a positive correlation between PM2.5 and pediatric pneumonia ED visits on lag 3; Lv et al.’s study on lag 4 and Xiao et al.’s study on lag 0–3 had the same result [2.5 may have different health effects. Darrow et al. observed that the carbon fraction of PM2.5, particularly organic carbon, was positively associated with pediatric pneumonia, but the total PM2.5 showed a negative association [3 and PM2.5 components SO42−, NO3−, and NH4+, and found the result was significantly associated with increased odds of pediatric pneumonia [Recently, many studies have focused on the association between PMd asthma , 24. Somon lag 4 . Xiao et lag 0–3 . On the neumonia , 27. Thee result , 25. Strociation . Xiao et2.5 has been reported to contribute to varying human health effects in different age groups. A case–crossover study found people of advanced age were more susceptible to the adverse effects of PM2.5 on OHCA [p values were not calculated in the study [2.5 exposure was greater in older children . One possible reason for this result was that children of different ages might spend different amounts of time outdoors, leading to varying air pollution exposure. Furthermore, air pollutants seem to have varying health effects on different age groups. Hassanvand et al. found that PM was not associated with increased high-sensitivity C-reactive protein (hsCRP) in healthy young adults, whereas in elderly subjects, hsCRP increased with PM2.5 exposure [PM on OHCA . Few stuhe study . Lv et ahe study . The preexposure . Using uexposure .2.5 exposure was associated with increased levels of DNA lesions in mouse lungs [2.5 interferes with alveolar macrophages, and pulmonary epithelial cells stimulate the release of a variety of cytokines and lead to inflammation [2.5 was linked to elevated inflammation and coagulation of blood markers [2.5 exposure was associated with airway inflammation in school children [2.5 and PM10 exposure was associated with increased instances of pediatric pneumonia [2.5 and pediatric pneumonia ED visits.Several studies tried to identify the mechanism by which PM contributes to pneumonia. An animal study found that PMse lungs . Throughammation . Hassanv markers . Zhang echildren . A revieneumonia . The pre2 has been found to be associated with all-cause mortality [2 might damage macrophages, natural killer cells, and CD4 to CD8 ratios in the respiratory tract, leading to decreased mucociliary clearance and vulnerable respiratory epithelium [2 and pediatric pneumonia remains controversial. Some studies demonstrated the acute effect of NO2 exposure leading to ED visits for pediatric pneumonia [2 and pediatric pneumonia [2was associated with an increase in hospital admissions due to pediatric pneumonia [2 might play an important role in pediatric pneumonia ED visits.NOortality , cardiovortality , hospitaortality , and admortality . Toxicolithelium . Howeverneumonia , 37. On neumonia . A systeneumonia . The cur2 and SO2 on daily ischemic stroke counts were stronger in the cold season than in the warm season [2.5 and pneumonia with septicemia ED visits during the warm season [2.5 mass correlated with increased all-cause mortality, especially in transitional seasons rather than in summer and winter [p-value was not calculated in that study. The present study revealed that ORs for NO2were higher during the warm days (p = 0.011). One possible reason for this result is that variable concentrations of air pollutants and each PM component may relate to seasonal differences in human health [Seasonal variation seems to play an important role on air pollutants and human health. Szyszkowiczet al. demonstrated that the associations between air pollutants and respiratory health outcomes were stronger during the warm season . Dong etm season . Cheng em season . Ueda etd winter . Few stud winter ; but sead winter . In addid winter , the intn health , 41. Sean health . In addin health . The comThere are certain limitations to our study. First, the study was conducted in an industrial city having a tropical monsoon climate; the mixture of air pollutants and seasonal effects may be different in other regions. Second, as an exposure estimate for the entire population, we analyzed air pollution data from fixed monitoring sites assuming the exposure to be homogenous across the whole area. Factors such as personal protective equipment use and time spent outdoors may affect personal exposure. Third, individuals were identified in a single tertiary medical center, which limited the sample size. Moreover, individual susceptibility might vary due to ethnic differences. Thus, further studies should be conducted in more regions with larger samples and include seasonal constituent analysis.2.5 and NO2 possibly play important roles in pediatric pneumonia events in Kaohsiung. Older children were found to be more susceptible to the adverse effects of PM2.5. Additionally, the health effects of the different air pollutants varied with temperature and patients were more susceptible to NO2 during the warm days.We found that PM


Actinobacillus pleuropneumoniae CVCC 259, a piglet tissue cage (TC) infection model was established. After A. pleuropneumoniae populations maintained at 108 CFU/mL in TCs, piglets were treated with various doses of danofloxacin once daily for 5 consecutive days by intramuscular injection. Both the concentrations of danofloxacin and the population of vial cells were determined. Changes in susceptibility and resistance frequency were monitored. Polymerase chain reaction (PCR) amplification of quinolone resistance-determining regions (QRDRs) and DNA sequencing were performed to identify point mutations in gyrA, gyrB, parC, and parE genes. Furthermore, the susceptibility of mutants to danofloxacin and enrofloxacin was determined in the presence or absence of reserpine to assess whether the mutants were caused by efflux pumps. The MICs and resistant frequency of A. pleuropneumoniae both increased when danofloxacin concentrations fluctuated between MIC99 (0.05 μg/mL) and MPC . As for PK/PD parameters, the resistant mutants were selected and enriched when AUC24h/MIC99 ranged from 34.68 to 148.65 h or AUC24h/MPC ranged from 4.33 to 18.58 h. Substitutions of Ser-83→Tyr or Ser-83→Phe in gyrA and Lys-53→Glu in parC were observed. The susceptibility of mutants obtained via danofloxacin treatment at 1.25 and 2.5 mg/kg were less affected by reserpine. These results demonstrate that maintaining the value of AUC24h/MPC above 18.58 h may produce a desirable antibacterial effect and protect against A. pleuropneumoniae resistance to danofloxacin.To evaluate the relationship between pharmacokinetic/pharmacodynamic (PK/PD) parameters and changes in susceptibility and resistance frequency of  Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a severe respiratory disease that is a global problem in pig production. The acute form of this disease is highly contagious and often fatal, resulting in considerable economic losses to pig producers  infection model has been used as a feasible system in exploring the relationship between PK/PD parameters and antibacterial effects  of gyrA, gyrB, parC, and parE genes. Finally, the relationship between PK/PD parameters and changes in susceptibility and resistance frequency of A. pleuropneumoniae was analyzed. We aimed to demonstrate that this model could elucidate the relationship between emergence of resistant A. pleuropneumoniae and PK/PD parameters associated with danofloxacin.In the present study, a standard A. pleuropneumoniae standard strain, CVCC259, was purchased from the Chinese Veterinary Culture Collection Center. Danofloxacin mesylate standard (>99%) and enrofloxacin standard (98%) were kindly supplied by Guangdong Dahuanong Animal Health Products. Pentobarbital sodium was purchased from Jian Yang Biotechnology Co., Ltd. Procainamide hydrochloride was supplied by Xin Zheng Co., Ltd., Tianjin Pharmaceutical Group. Tryptic Soy Broth (TSB) and Mueller–Hinton agar (MHA) were purchased from Guangdong Huankai Microbial Technology. Nicotinamide adenine dinucleotide  was purchased from MYM biological technology company limited (Beijing). Newborn bovine serum was provided by Guangzhou Ruite Biotechnology Ltd. Compound aminopyrine injection was purchased from Shandong Zhengmu Biological Pharmaceutical Co., Ltd.The Actinobacillus pleuropneumoniae was grown in TSB or on MHA supplemented with 4% newborn bovine serum and 1% NAD at 1 mg/mL. The MIC was tested by an agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) reference methods . For exact MPC, the concentrations of danofloxacin in the agar decreased at a linear trajectory by 10%, which was based on MPCpr approaching 1/2 MPCpr. Then, we repeated the method for the MPCpr test. The lowest antibiotic concentration at which no colonies grew on an agar plate was defined as the MPC.For MPC, approximately 10ad libitum. All the experimental protocols were approved by the South China Agricultural University Committee on Animal Ethics .Healthy castrated crossbred piglets (Duroc × Landrace× Yorkshire), weighing 20–25 kg, were obtained from Guangzhou Fine Breed Swine Farm. They were housed in individual cages and fed antibiotic-free fodder (guangchubao premix feed for pig from the Guangzhou Zhongwang Feed Company) twice a day. Water was available Tissue cages were made using food grade silicone tubes and the size of the TCs were the same as those described previously . Implant10 CFU/mL) was added to each TC. Two days after infection, 0.5 mL of TCF was extracted from each TC for bacterial enumeration. The TCFs containing a bacterial concentration exceeding 108 CFU/mL were used for the experiment.One milliliter of logarithmic growth phase bacterial suspension  were randomly allocated to one control group and seven study groups. The control group (two piglets and four TCs) was treated with 1 mL sterile physiological saline. The study groups were treated with danofloxacin at 0.4, 0.6, 0.8, 1.25, 2.5, 3.5, and 5 mg/kg (four TCs for each group) of body weight for 5 days, once daily by IM injection. TCFs (0.3 mL) were collected from the TC at 2, 4, 6, 8, 10, and 24 h after each administration. Samples were clarified by centrifugation at 3000 × g for 10 min. Two-hundred microliters of clear supernatant and 800 μL water were mixed and then transferred to an HPLC vial. The HPLC was applied with an Agilent TC-C18 column  and the mobile phase was triethylamine phosphate (pH 2.4): acetonitrile  with a flow rate of 0.8 mL/min. The injection volume was 20 μL. A calibration curve was determined using nine danofloxacin concentrations (0.001–0.5 μg/mL). The mean relative recovery (RR) of danofloxacin in TCF samples was 96.9 ± 9.83% (mean ± SD).Danofloxacin concentrations in TCF were determined by high-performance liquid chromatography with fluorescence detection  and AUC24h (the area under the concentration–time curve over 24 h), were calculated by the non-compartmental model using WinNonlin software .Pharmacokinetic parameters, including CMultiple TCFs (0.5 mL) were collected from the TCs before, during, and after the treatment (after every administration) at 24 and 48 h after the termination of treatment. To quantify the numbers of surviving bacteria and resistant mutants, each sample was serially diluted with sterile saline and 20 μL was inoculated in triplicate on to drug-free MHA or MHA containing 1 × MIC of danofloxacin. After incubation 18–20 h, the resultant bacterial colonies were counted. The detection limit was 50 CFU/mL. The time-kill curves were depicted as the number of bacteria on drug-free MHA, while the recovery curves of resistant mutants were drawn as the populations grown on MHA containing 1 × MIC of danofloxacin.A. pleuropneumoniae on to drug-free MHA every 24 h for 5 days. MICs were tested as described above. To evaluate the contribution of efflux, the susceptibility to both danofloxacin and enrofloxacin was then determined in the presence or absence of reserpine at 20 μg/mL.Loss of bacterial susceptibility in TCF was examined at before danofloxacin administration, during the treatment (after every administration), 24 and 48 h after the termination of treatment. The stability of mutants was determined by consecutive passage of To detect the resistant frequency of mutants, each sample was plated on to MHA containing 1 × MIC of danofloxacin (detection limit 50 CFU/mL). The definition of resistant frequency was expressed by the ratio of bacterial numbers counted in the presence of antibiotics to that in the absence of antibiotics.24h/MIC99, AUC24h/MPC, %T > MIC99 (the percentage of the time that drug concentration remains above the MIC99), %T > MPC (the percentage of time that drug concentration remains above the MPC), Cmax/MIC99, and Cmax/MPC were calculated using WinNonlin program . Fisher’s exact test was used for statistical analysis of the relationship between PK/PD indices and the changes in susceptible. Control group (two piglets and four TCs) were used as a control. P < 0.05 was considered to be statistically significant.Pharmacokinetic/pharmacodynamic parameters such as AUCgyrA, gyrB, parC, and parE genes were determined as previously described  amplification. The nucleotide sequence of the QRDRs of the escribed . The rea99, and MPC were 0.06, 0.05, and 0.4 μg/mL, respectively. All experiments were performed in triplicate on different occasions.The values of MIC, MICFigure 1 and exhibit the antibacterial effect of danofloxcin against A. pleuropneumoniae CVCC259 in TCF after different doses were administered. For the control group, bacterial populations remained constant (approximately 108 CFU/mL). Compared to the control group, administration of danofloxacin at 0.4 mg/kg slightly decreased bacterial numbers. Bacterial numbers were reduced in response to the first 4 administrations of danofloxacin at 0.4, 0.6, and 0.8 mg/kg, although there was re-growth of bacteria after the last treatment. For the danofloxacin dosages at 1.25 and 2.5 mg/kg, bacterial numbers were obviously reduced after the five administrations, although there was re-growth at 48 h after the last administration. Administration of danofloxacin at 3.5 and 5 mg/kg caused bacterial numbers to reduce throughout treatment and they remained low during the growth recovery phase.The time-kill curves are depicted in Figure 2 when the piglets were administrated danofloxacin at 0.8, 1.25, and 2.5 mg/mL. As a result, the danofloxacin concentrations were located between the MIC99 and MPC. Both the numbers of total and resistant bacteria are listed in Figure 2. The total bacterial populations reduced during treatment and then gradually increased. However, resistant bacteria numbers were initially constant or slightly reduced before amplification after several administrations. At last, the number of mutant and total bacteria were almost equal.Three representative recovery curves are shown in Figures 3A1–A7. Determined by trapezoidal rules, the average values of AUC24h ranged from 0.96 ± 0.34 to 18.94 ± 3.34 μg⋅h/mL. The average maximum concentration (Cmax) ranged from 0.05 ± 0.01 to 1.13 ± 0.15 μg/mL. The detailed values for AUC24h and Cmax are listed in Table 1. The AUC24h and Cmax values in the TCF increased in a non-linear fashion with increasing doses and the correlation coefficients (R2) were 0.95 and 0.91, respectively. After various dosages of danofloxacin were administered, the mean concentrations in the TCFs were ranged from MIC99 to MPC: almost completely below MIC99 (A1), across the MIC99 (A2), completely between MIC99 and MPC (A3–A5), across the MPC (A6), and above the MPC (A7).Danofloxacin concentrations collected at various time points during the treatment are depicted in A. pleuropneumoniae in the TCFs was examined after administration with different doses of danofloxacin . The MICs gradually increased  when the drug concentrations were partially or completely located between MIC99 and MPC . The significant increase in MICs  were observed when the concentration of danofloxacin fluctuated between the MIC99 and MPC . When danofloxacin concentrations were maintained either below the MIC99  or above the MPC , MIC did not increase, either during or after treatment .Susceptibility of Figures 3C1–C7. Dramatic increases  were observed when the drug concentration located between MIC99 and MPC . When drug concentrations were mostly below the MIC99  or exceeded the MPC , the resistant frequencies slightly increased and then gradually decreased .The resistant frequencies are depicted in 99- and MPC-related PK/PD parameters are listed in Table 2. Relationships between PK/PD indices, determined as steady-state values after the fifth dose, and changes in susceptibility are shown in Table 3. For fluoroquinolones, the AUC24h/MIC99 index is most commonly associated with restriction of susceptible bacterial growth . Loss of bacterial susceptibility occurred in 10 of 12 TCs when AUC24h/MIC99 was between 34.68 and 148.65 h . Only one of eight TCs lost susceptibility when the AUC24h/MIC99 exceeded 148.65 h . As for AUC24h/MPC, mutant enrichment was observed, where 10 of 12 TCs lost susceptibility, when the AUC24h/MPC was between 4.33 and 18.58 h . Only one of eight TCs occurred loss of susceptibility  when AUC24h/MPC > 18.58 h.Pharmacokinetic/pharmacodynamic parameters provide an empirical way to relate antimicrobial dose to favorable treatment effects associated with bactericidal agents . The MICl growth . Only twTable 3. Mutants were selected by enrichment when the Cmax/MIC99 values were between 1.09 and 8.42 or Cmax/MPC values were between 0.14 and 1.05. Resistant bacteria were recovered from 12 of 20 TCs when the administration time of danofloxacin concentration was above the MPC for <29.63% of the dosing interval.Statistically significant correlations with selection of resistance for other PK/PD indices are also listed in Table 4). Mutants obtained from 1.25 and 2.5 mg/kg dosages were less affected by reserpine (Table 4). Mutations in the QRDR target genes are listed in Table 4. No mutant genes were observed in gyrB and parE. All mutants had a (Lys-53→Glu) substitution in parC. When the dose was 0.6 and 0.8 mg/kg, no substitution was founded in gyrA. When the dose was 1.25 and 2.5 mg/kg, the mutants had a (Ser-83→Tyr) substitution in gyrA. When the dose was 3.5 mg/kg, the mutants had a (Ser-83→Phe) substitution in gyrA.Mutants selected from danofloxacin dosages of 0.6, 0.8, and 3.5 mg/kg tended to be non-topoisomerase mutants that exhibited increased efflux. This was confirmed by adding an efflux inhibitor (reserpine), which could decrease the MIC for danofloxacin and enrofloxacin  in the present study. This phenomenon was also observed by other researchers . Another reason that could explain the increase in resistance frequency may due to gene mutations that arise in bacteria  in vivo. This group considered that resistant mutants were not enriched at a value of AUC24h/MPC > 15 h  in parC. In a previous study , four different substitutions in ParC , and five different substitutions in ParE .In Gram-negative bacteria, fluoroquinolone resistance occurs mainly by interplay of three mechanisms. This is realized by stepwise accumulation of mutations in the QRDRs of DNA gyrase and topoisomerase IV, active efflux of fluoroquinolones, and the presence of plasmid-borne resistance genes (qnr) protecting the target topoisomerase . In our us study , more muA. pleuropneumoniae, a lung infection model may be preferable for the study of PD and PK information in future studies.Although we successfully established a piglet TC infection model to evaluate the relationship between MIC- and MPC-based PK/PD parameters and the emergence of resistant mutants, there are some limitations to our study. First, because of the limited number of piglets, the sample size of resistant mutants is not enough to generalize. Larger datasets should be considered in future research. Second, although a TC infection model was suitable for exploring the relationship between PK/PD indices and antibacterial effects, there are still obvious differences between TCF and clinically infected organs in animals. Therefore, for A. pleuropneumoniae after different dosages of danofloxacin. After analyzing the relationship between MIC- and MPC-based PK/PD parameters and the emergence of resistant mutants, we suggest that danofloxacin concentrations should be maintained above the MPC or AUC24h/MPC > 18.58 h, which could maintain effective antibacterial activity and minimize the emergence of resistant A. pleuropneumoniae.We successfully established a piglet TC infection model and investigated the changes in susceptibility and mutant frequencies of LZ and HD contributed to the methodology, software, validation, formal analysis, data curation, writing , writing (review and editing), visualization, and the project administration. LZ, ZH, ZK, and LY contributed to the investigation. LZ, QC, XS, and HD contributed to the resources. XG and HD contributed to the supervision. HD contributed to the funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nanoparticles can be considered as a useful tool for improving properties of poorly soluble active ingredients. Hydrochlorothiazide  was chosen as a model compound. Antisolvent precipitation-solvent evaporation and emulsion solvent evaporation methods were used for preparation of 18 samples containing hydrochlorothiazide nanoparticles. Water solutions of surfactants sodium dodecyl sulfate, Tween 80 and carboxymethyl dextran were used in mass concentrations of 1%, 3% and 5%. Acetone and dichloromethane were used as solvents of the model compound. The particle size of the prepared samples was measured by dynamic light scattering. The selected sample of hydrochlorothiazide nanoparticles stabilized with carboxymethyl dextran sodium salt with particle size 2.6 nm was characterized additionally by Fourier transform mid-infrared spectroscopy and scanning electron microscopy. It was found that the solubility of this sample was 6.5-fold higher than that of bulk hydrochlorothiazide. H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, is a thiazide diuretic that is frequently used in antihypertensive therapy in combination with antihypertensive agents. HCTZ affects the distal renal tubular mechanism of electrolyte reabsorption: it increases excretion of sodium and chloride ions [Hydrochlorothiazide HCTZ, , 6-chloride ions ,4,5,6,7.ide ions . This faide ions . Drug NPide ions . HCTZ beide ions . Drugs oide ions . Some naide ions .The aim of this study was the preparation of stabilized HCTZ NPs via antisolvent precipitation with the following solubility experiment. It was supposed that HCTZ NPs would have improved bioavailability  and carboxymethyl dextran sodium salt in combination with acetone . On the other hand, carboxymethyl dextran sodium salt in 5% concentration in combination with dichloromethane or acetone formed samples with the smallest particle sizes (2.6 nm of sample 5c and 2.8 nm of sample 6c). Sodium dodecyl sulfate in combination with dichloromethane provided particle size near 20 nm (samples 1a and 1c). Other prepared samples contained NPs in the size range from 4.2 to 13.6 nm, mostly near 10 nm.The investigated particles showed good particle size stability throughout the light scattering measurements, except for sample 1a and 5b  were eliminated.The dispersity is a measure/degree of the homogeneity/heterogeneity of sizes of particles in a mixture/system. The uniformity of dispersed systems is expressed as polydispersity index (PDI), see 5c with the smallest particle size and low PDI, where HCZT was stabilized with 5% carboxymethyl dextran sodium salt, was chosen for additional characterization and solubility test. 5c. Scanning electron microscopy (SEM) shows bulk HCZT, see 5c. The surface and structure of nanoparticles were uniform with no roughness.Sample 5c) was tested; the results of this investigation are presented in The solubility of bulk HCTZ (as a control sample) and HCTZ NPs  was dissolved in water (10 mL), and three solutions with mass concentrations 1%, 3% and 5% were prepared. HCTZ (0.1 g) was dissolved in dichloromethane or acetone (10 mL), i.e., 1% solutions were prepared. The solution of HCTZ in dichloromethane/acetone was slowly dropped (2 mL/min) to the aqueous solutions of excipients, and the solutions were stirred at 600 rpm for 15 min at 25 °C, after which the mixtures were transferred to the ultrasonic bath in the fume chamber, where they were mixed again for 20 min for homogenization of the samples. Finally, the solvent was evaporated.The model compound hydrochlorothiazide (HCTZ) and the excipients sodium dodecyl sulfate, Tween 80 and carboxymethyl dextran sodium salt were purchased from Sigma-Aldrich . All compounds were of analytical grade. HThe particle size was determined using a Brookhaven dynamic light scattering system BI 9000  with a goniometer SM-200 and an argon gas laser . Scattered intensity was registered at scattering angle 90° and temperature 25 °C. All the samples were dispersed by sonication and additionally filtered directly before the measurement through syringe filters with 0.45 μm pore size to remove mechanical impurities. Five independent recordings of the autocorrelation function were done for each investigated excipient concentration. The particle size was calculated from the translational diffusion coefficient using the Stokes-Einstein formula. The translational diffusion coefficient was obtained based on the cumulant expansion of the autocorrelation function up to the second cumulant. The presented particle sizes are reported as the mean values taken of the set of five independent measurements. The results are summarized in −1 with the resolution of 4 cm−1 and 32 scans.Fourier transform mid infrared (FT-MIR) spectra  were meaThe morphology of the sample was studied using scanning electron microscopy (SEM). The sample was attached to a microscopic glass, dried at 25 °C and then sputter-coated with thin film of gold. An electron microscope Quanta 450FEG  was used for imaging of the sample at the following working conditions: accelerating voltage 10.0 kV, working distance 8.0 mm and probe current 100 mA. Structures of the samples are illustrated in 5c were separately added to beakers with buffer under continuous stirring (250 rpm). The beakers were covered by Parafilm®. When the turbidity occurred, the content of the beaker was stirred for additional 24 h. If the turbidity did not disappear, the samples were centrifuged and analyzed. Analysis of samples was performed using a high performance liquid chromatograph Agilent 1200 Series  equipped with a DAD, a quarter pump, and an automatic injector. The HPLC separation process was monitored and evaluated by the ChemStation software. A chromatographic column Gemini 5 µm C6-Phenyl 110A, 4.6 mm × 250 mm  was used. Isocratic elution by a mixture of MeOH p.a. (35%) and formic buffer  as a mobile phase was used. The total flow of the column was 1.0 mL/min, injection 10 μL, column temperature 40 °C, and sample temperature 22 °C. The detection wavelength of 273 nm was chosen. The retention time of HCTZ was 4.19 ± 0.05 min, the limit of detection (LOD) was 0.01 µg/mL, and the limit of quantification (LOQ) of HCTZ was 0.04 µg/mL. The results are presented in Two beakers filled with 10 mL of phosphate buffer (pH 6) were placed on magnetic stirrers and tempered to 36 °C ,41,42. D5c of HCTZ NPs stabilized with 5% concentration of carboxymethyl dextran sodium salt  was chosen as the sample with the smallest particle size for following characterization and analysis. SEM was used for surface identification, and FT-MIR spectroscopy was used for verification of the sample composition. Finally, the solubility test confirmed the enhanced solubility of HCTZ NPs in comparison with the solubility of bulk HCTZ (6.5-fold higher).Eighteen samples of HCTZ NPs were prepared by the antisolvent precipitation-solvent evaporation and the emulsion solvent evaporation techniques. Three different excipients were used as stabilizers in 1%, 3%, and 5% aqueous mass concentration. Dynamic light scattering was used for particle size analysis. The polydispersity index (PDI) was determined. Low PDI values of the samples confirmed narrow size distribution and the uniformity of the prepared systems. Sample


Cancer is considered a family disease as the caregivers (CG’s) role extends beyond providing care as they can also help facilitate treatment decisions. While much has been reported in the literature about patient (PT) goals of care (GoC), little is known about discordance between PT and CG GoC and the impact of PT age. The variables of interest were PT and CG identified GoC using a 100-point visual analog scale (VAS) with anchors of quality of life (0) and survival (100). Discordance was defined as a > 40 point difference on the VAS. The GoC data reported here were those obtained at enrollment and prior to subject’s death. A sample of 235 PTs and CGs of PTs diagnosed with advanced cancers were included in the study. Mean age for the PTs was 64.7  with 54% being > 65. At enrollment, 28.7% of the PT-CG pairs of those PTs 65 years (X2 (1)=1.06, p=.304). At death, 61.8% (X2 (1)=31.04 <.001, Φ=.49) with discord at enrollment had discord at death. For patients who were older, 66.7% who had discord at enrollment also had discord at death and for patients