Patients with high emergency department (ED) utilization account for a disproportionate number of ED visits. The existing research on high ED utilization has raised doubts about the homogeneity of the frequent ED user. Attention to differences among the subgroups of frequent visitors (FV) and highly frequent visitors (HFV) is necessary in order to plan more effective interventions.In the Netherlands, the incidence of high ED utilization is unknown. The purpose of this study was to investigate if the well-documented international high ED utilization also exists in the Netherlands and if so, to characterize these patients. Therefore, we assessed the proportion of FV and HFV; compared age, sex, and visit outcomes between patients with high ED utilization and patients with single ED visits; and explored the factors associated with high ED utilization.A 1-year retrospective descriptive correlational study was performed in two Dutch EDs, using thresholds of 7 to 17 visits for frequent ED use, and greater than or equal to 18 visits for highly frequent ED use.FV and HFV  attended the ED 2,338 times . FV and HFV were equally likely to be male or female, were less likely to be self-referred, and they suffered from urgent complaints more often compared to patients with single visits. FV were significantly older than patients with single visits and more often admitted than patients with single visits. Several chief complaints were indicative for frequent and highly frequent ED use, such as shortness of breath and a psychiatric disorder.Based on this study, high ED utilization in the Netherlands seems to be less a problem than outlined in international literature. No major differences were found between FV and HFV, they presented with the same, often serious, problems. Our study supports the notion that most patients with high ED utilization visit the ED for significant medical problems. Patients with high emergency department (ED) utilization, also called “frequent visitors” (FV) account for a disproportionate number of ED visits ,2. PatieThe existing research on high ED utilization has raised doubts about the homogeneity of the FV ,19,20. AIn the Netherlands, the incidence of high ED utilization is unknown. The purpose of this study was to investigate whether the well-documented international high ED utilization also exists in the Netherlands and if so, to characterize these patients.Therefore, we assessed the proportion of FV and HFV; compared age, sex, and visit outcomes between patients with high ED utilization and patients with single ED visits; and explored the factors associated with high ED utilization.A retrospective, descriptive, correlational study was performed in two ED locations in the Netherlands: a level one ED in an inner-city trauma centre and a level three ED in a hospital located in a small city. High ED utilization was defined according to thresholds developed by Doupe et al. : patientVariables collected from the hospitals’ database were based on previous research regarding high ED utilization and included age ,12,21, st-tests (age) and χ2 tests . The associations of visit characteristics with the presence of frequent ED use and highly frequent ED use were analysed using logistic regression. The variables included in the models were arrival time (reference: day shift), arrival with ambulance, self-referral, chief complaint , and triage level (reference: immediate/very urgent).The proportions of FV and HFV were summarized using descriptive statistics. Age, sex, visit outcomes, and triage level were compared between the patients with high ED utilization (FV and HFV) and patients with a single ED visit, using Two separate logistic regression models were developed, comparing frequent ED use with single ED use, and highly frequent ED use with single ED use. In both models, the ED visit was used as a unit of analysis. Adjusted odds ratios (ORs) are provided with their 95% confidence intervals (CI) to indicate the likelihood of frequent use or highly frequent use for each explanatory variable adjusted for the other variables. The calibration and overall discriminative capacity of the final models were assessed with the Hosmer-Lemeshow test and the area under the receiver operating curve (AUC ROC) analysis, respectively . Data weDuring the 1-year study period, 71,565 consultations were registered at the two EDs, of which 50,155 were at the level one ED and 21,410 at the level three ED. These visits were paid by 51,272 different individuals. Of these 51,272 patients, 38,959 patients visited the ED a single time during the study period.During the study year, 244 patients attended the ED 7 times or more Table : 95% of The remaining 12 patients attended the ED 18 times or more during the study year and were categorized as HFV (attending the ED on 263 occasions). FV and HFV  attended the ED 2,338 times .P <0.001). Among FV, most patients  were in the age category of 45 to 64 years (data not shown). No differences were found in sex and mean age and between the HFV and the patients with single visits.Results are presented for both EDs together, since no statistically significant differences in the results were found between the two EDs. Patient characteristics are shown in Table P <0.001). LWBS occurred more often during visits of FV. No differences in visit outcomes were found between HFV and patients with single visits. There was no mortality in the patients with frequent or highly frequent ED use.Visit outcomes are shown in Table Factors associated with high ED utilization are shown in Table Most of the FV and HFV arrived during the day shift or evening shift. However, when corrected for other variables, arriving during the night shift was indicative for high ED utilization. Self-referral was less likely to occur among FV and HFV compared to the patients with single visits. Several chief complaints were indicative for frequent and highly frequent ED use, namely shortness of breath, abdominal pain, urinary tract problems, and psychiatric disorders. HFV arrived by ambulance more often than patients with single visits.P <0.001).Both FV and HFV were assigned to the non-urgent or standard triage level significantly less often than patients with single visits .The Hosmer-Lemeshow goodness-of-fit test Since no univocal definition of high ED utilization exists in the literature , we usedWe assessed 244 individual FV and HFV , who presented to the two EDs on 2,338 occasions . FV and HFV together in this study are a low percentage (0.5%) compared to another study using the same thresholds, where FV composed 2.1% of users and 9.9% of ED visits, whereas HFV composed 0.2% and 3.6% of users and visits, respectively [FV were significantly older than patients with single visits (47.5 vs. 39.0 years), but HFV were not. Comparing our findings with other studies is difficult: we found a study indicating that the common FV is 35 years of age , as wellIn other studies, most frequent visits occurred during the evening or night shifts ,10. In oAlthough heterogeneity is assumed amongst the subgroups of FV and HFV, in our study, HFV were not very different from FV: they presented with the same problems and both were less likely to be self-referred compared with single visit patients. We found differences mainly in admission rates and ambulance-use; HFV were more often brought in by ambulance but less often admitted for further analysis. However, the validity of our conclusions regarding the HFV, being a very small group of patients (n = 12), may be unreliable and differences between FV and HFV, and between HFV and patients with a single visit should be further examined using a larger sample of HFV.Most studies indicate that patients with high ED utilization are usually sick patients with chronic illness associated with high admission rates ,2,7. To First, we were not able to account for some of the socio-economic factors that are known to influence high ED utilization, such as race, alcohol dependence, homelessness, and insurance coverage ,10,17,21Second, the finding that FV had higher admission rates than patients with single visits should be considered with great caution and warrants further investigation. When patients present for the third or fourth time within a few days, physicians tend to admit this patient for further analysis, so bias by frequency of presentation is possible.Third, we had no data on our patients’ medical diagnoses and thus we were not able to compare our findings regarding psychiatric co-morbidity with other studies. Instead of diagnoses, we used chief complaints based on the triage flow charts. The use of chief complaints was sufficient for our purpose (identifying factors associated with high ED utilization) and certainly more feasible than using medical diagnoses.Fourth, we had no data on patients’ use of other EDs in the surrounding area. FV and HFV are prone to use more than one health care service , so the Finally, our findings may have limited generalizability because of the cultural, social, and health care delivery characteristics of our population.Based on this two-ED study, in the Netherlands high ED utilization seems to be less of a problem than outlined in international literature. No major differences were found between FV and HFV. Both presented with shortness of breath, abdominal pain, urinary tract problems, or psychiatric disorders more often compared to patients with single ED visits. FV were more likely to be admitted than patients with single visits. Our study supports the notion that most patients with high ED utilization visit the ED for significant medical problems.AUC ROC: Area under the receiver operating curve; CI: Confidence interval; ED: Emergency department; FV: Frequent visitor; GP: General practitioner; HFV: Highly frequent visitor; LWBS: Leaving without being seen; OR: Odds ratio.The authors declare that there are no competing interests.CvdL had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept & design: CvdL, CvdB, NvdL, AR, and CB. Acquisition of the data: CvdL. Analysis and interpretation of data: CvdL and CvdB. Drafting of the manuscript: CvdL, CvdB, NvdL, and AR. Critical revision of the manuscript for important intellectual content: CvdB and CB. All authors read and approved the final manuscript.


The guidelines established by the National Comprehensive Cancer Network do not describe mucinous histology as a clinical factor that should influence the therapeutic algorithm. However, previous studies show conflicting results regarding the prognosis of colorectal mucinous adenocarcinoma. In this study, we described the clinicopathological features of mucinous adenocarcinoma in Japan, to identify optimal therapeutic strategies.144 patients with mucinous and 2673 with non-mucinous adenocarcinomas who underwent primary resection in two major centers in Yokohama, Japan were retrospectively evaluated for clinicopathological features and treatment factors. A multivariate analysis for overall survival followed by the comparison of overall survival using Cox proportional hazard model were performed.Patients with mucinous adenocarcinoma had larger primary lesions, higher preoperative CEA levels, a deeper depth of invasion, higher rates of nodal and distant metastasis, and more metastatic sites. A multivariate analysis for overall survival revealed a mucinous histology to be an independent prognostic factor. In the subgroup analysis stratified by stage, Patients diagnosed as StageIII and IV disease had a worse survival in mucinous adenocarcinoma than non-mucinous, while survival did not differ significantly in patients diagnosed as Stage0-II disease. In StageIII, local recurrence in rectal cases and peritoneal dissemination were more frequently observed in patients with a mucinous histology.Our study indentified that mucinous adenocarcinoma was associated with a worse survival compared with non-mucinous in patients with StageIII and IV disease. In rectal StageIII disease with mucinous histology, additional therapy to control local recurrence followed by surgical resection may be a strategical alternative. Further molecular investigations considering genetic features of mucinous histology will lead to drug development and better management of peritoneal metastasis Colorectal cancer is the third most common cancer and the fourth most frequent cause of cancer death worldwide . MucinouThe prognostic significance of MA is controversial. In previous studies, mucinous histology was reported not to be an independent prognostic factor for survival ,14. The The lack of a consensus may be the result of the low ratio of MA to all colorectal cancers and the limited detection power to clarify the differences between MA and NMA.We conducted a retrospective analysis of patients with colorectal cancer at two major centers to identify the clinicopathological features of MA, and also investigated the recurrence to establish an optimal therapeutic strategy for MA.The data from 2,817 patients with colorectal cancer in two major centers, Kanagawa Cancer Center and Yokohama City University Medical Center, between 2001 and 2010 were investigated. Written informed consent was obtained from the patient for publication of this report and any accompanying images. All patients initially underwent resection of a primary lesion followed by adjuvant chemotherapy when diagnosed as stage III disease. In tumor, node metastases (TNM) stage T3 to T4, lower-rectal cases, total mesorectal excision (TME) and lateral node dissection were routinely performed at initial resection. No patients were treated with neo- or adjuvant radiation therapy.The analyzed patients were diagnosed with MA, defined as tumors with more than 50% of the tumor volume comprising mucin, or with NMA, defined as tumors without any mucinous features, or with a less than 50% mucinous component . PatientThe covariates included in the study were gender, age, location of the tumor, size of the primary tumor, preoperative serum carcinoembryonic antigen (CEA) level, depth of invasion, lymph node metastasis, distant metastasis, operating facility, and histological type. The pathological tumor status was coded using the TNM classification system . The useIn the analysis of the survival rates, all cases were divided into 3 groups, that is, patients without any metastases (stage 0 to II ), patien2-test, or Fisher’s exact test where appropriate. Overall survival was calculated from the date of surgery for the primary lesion until death from any cause, or was censored at the last follow-up visit. Survival data were analyzed using the Kaplan-Meier method. A comparison of survival curves was carried out using the log-rank test. The prognostic significance was analyzed by multivariate Cox proportional hazard models. P-values < 0.05 were considered statistically significant, and all P-values correspond to two-sided significance tests.The two groups of patients (MA and NMA) were compared using 2 × 2 tables for binary factors using the χOf the 2,817 colorectal cancer patients, MA accounted for 5.1% (144) of the colorectal cancer cases. The distribution of the patients’ characteristics is shown in Table Table To clarify the prognostic factors for colorectal cancer, univariate and multivariate analysis were carried out. Mucinous histology was noted to be one of the independent prognostic factors for overall survival in univariate and multivariate analysis Tables  and 4. TP = 0.099)    to compare the overall survival in each group. There were no significant differences in 5-year survival in stage 0 to II based on the histological status  Figure . However) Figure , the MA P = 0.049), and local recurrence  were more frequently observed in the MA group. All of the locally recurrent cases in the MA group were rectal cases.The recurrence pattern in stage III was analyzed Table . The numThe treatment factors were compared in stage IV disease Table . HoweverIn this study, the data from 2,817 colorectal cancer patients who underwent surgery in two major medical centers in Yokohama city, Japan, were analyzed. In these 2,817 patients, the incidence of MA was 5.1%, which generally corresponds to the rate in other Asian countries, as reported by Chew (6.0% from Singapore)  and SafaIn the analysis of the patients’ characteristics, the MA group had worse clinical factors, including larger primary lesions, deeper invasion, higher rates of nodal and distant metastasis, and a larger number of metastatic sites compared to the NMA group. The previous reports showed that younger patients, larger tumors, higher rates of lymph node metastasis, and peritoneal metastasis were correlated with MA histology when compared to NMA histology -19, whicet al. demonstrated that the more malignant characteristics of MA may be partly due to the production of mucus under pressure, which allows the MA cells to gain access to the peritoneal cavity. Moreover, the fluid produced by MA is taken up by the lymphatic system, which might help to promote tumor spread into regional lymph nodes [The reason why MA patients have worse characteristics than NMA patients is not fully understood. Sugarbaker ph nodes .It remains unclear whether MA adversely affects survival in colorectal cancer patients. In some studies, MA has been shown to be a significant prognostic factor ,17,20-23Based on our findings, we also performed an investigation to establish an optimal therapeutic strategy for MA. To exclude the bias caused by stage, which is related to the status of lymph node metastasis and distant metastasis, we divided all cases into three subgroups and analyzed the relative survival rate in each group. Consequently, the prognosis of patients with MA was similar to that of patients with NMA in stage I/II, whereas it was significantly worse for MA patients in both stage III and stage IV. Some of the above-mentioned literature supports the relatively worse survival of MA patients when the subjects are limited to stage III and stage IV disease ,18,21, wIn the analysis of the recurrence pattern of stage III disease, the incidence of local recurrence and peritoneal metastasis was significantly higher in patients with MA than in those with NMA. In addition, all cases of local recurrence in the MA group were in patients with rectal disease. Differences in the rates of liver, lung, and distant lymph node metastases were not statistically significant.In stage IV disease, we compared the treatment factors, including the curability of the first resection, first chemotherapeutic regimen, and the need for additional resection (liver and lung resection). This analysis revealed that equivalent treatments were performed for both MA and NMA patients.et al. pointed out that the lymphatic drainage of the pelvis is more extensive and varied compared with that of the colon [Our results indicate that better management of the local and peritoneal recurrence of stage III disease may improve the survival of MA patients. In terms of local control, previous studies have shown that the difference in survival between MA and NMA patients was mainly related to tumors with a rectal location , becausehe colon .et al. showed that the five-year overall survival and the five-year locally controlling ratio in patients with T3 to T4 rectal cancer treated by TME plus lateral node dissection, was 79.7% and 92.0% respectively [In the NCCN guidelines for treatment of rectal cancer , it is rectively . ConsequConsidering our result, the application of pre- or postoperative radiation therapy -36 may bWe also found that peritoneal metastasis was a significant site of recurrence for MA in stage III disease. Metastasis to the peritoneum is regarded to be a fatal manifestation of gastrointestinal cancer, and is associated with a median survival time of 5.2 to 12.6 months ,38. In tKRAS) and the suppressor gene in colorectal carcinogenesis [et al., MA histology is characterized by a high frequency of KRAS mutations, and a high frequency of microsatellite instability [It is well known that there are several molecular pathways involving oncogene (for example ogenesis , and cettability , suggesttability . DetaileOur study indentified MA histology as an independent prognostic factor, and revealed that MA was associated with worse survival compared with NMA in patients with stage III and IV disease. The ability of MA to disseminate and infiltrate more aggressively than NMA appears to be responsible, at least in part, for the higher rate of failure in stage III and IV, which are the main reasons for the overall poorer prognosis of patients with MA. Controlling local recurrence and managing peritoneal metastasis are necessary to improve the overall survival in patients with MA.Besides considering radiation therapy after TME and radical lymph node dissection for locally advanced rectal cases, further investigations focusing on the genetic and molecular characteristics of MA, will help better management of MA histology.CEA: Carcinoembryonic antigen; 5-FU: 5-fluolouracil; IRI: Irinotecan; MA: Mucinous adenocarcinoma; NCCN: National Comprehensive Cancer Network NMA, non-mucinous adenocarcinoma; OXA: Oxaliplatin; TME: Total mesorectal excision; TNM: Tumor, node, metastases.There are no financial or non-financial competing interests to declare in relation to this manuscript.MN carried out the analysis of data and wrote the manuscript. MS, TW, HT, NY, SM, KW, TG, TO, SF, CK, NY, YR, MM, and MA made the database of patients. All authors read and approved the final manuscript.


Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings. The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young  Artemisia annua L. The plant has been part of traditional Chinese medicine for >2,000 years and used for a variety of ailments [in planta has been characteristically low and synthetic production is not yet economically feasible [in planta is largely not understood, regulation of this terpenoid still requires considerable investigation. To our knowledge this is the first report showing kinetic changes in transcription of the genes in the AN biosynthetic pathway in A. annua in response to sugars.Artemisinin combination therapy (ACT) is the most effective means of treating malaria . Artemisailments , schistoailments , and a vailments . Productfeasible . VariousArtemisia annua, is produced by the glandular trichomes and sequestered in their epicuticular sacs [via either the cytosolic MVA pathway or the plastidic MEP pathway and evidence suggests that both pathways may be able to supply the IPP necessary for AN production [in vitro as a photo-oxidative reaction, though this may not necessarily be occurring in vivo [AN, one of a diverse pool of secondary metabolites produced by lar sacs ,8. AN islar sacs ,8. IPP ioduction . Indeed,oduction ,11,12. Toduction ,13,14,15oduction , which ioduction . The con in vivo . Whateve in vivo . How theVitis and more recently in Arabidopsis, a number of anthocyanin genes have been shown to be upregulated in response to sucrose [Arabidopsis [Numerous primary and secondary metabolites have been shown to be sugar responsive including products of the glyoxylate cycle , anthocy sucrose ,24. The  sucrose . A key ebidopsis . There abidopsis .Arabidopsis sucrose has been shown to accelerate flowering [It can be difficult to determine the direct effect of sugars since not only is there significant crosstalk in sugar signals, the action of invertases readily converts sucrose to its component monosaccharides, glucose and fructose, further confounding interpretation of much signaling pathway work ,28. Whillowering  while gllowering .A. annua sugars were shown to significantly alter the levels of AN production when seedlings were grown on equimolar carbon supplied in the form of either sucrose, glucose, or fructose [In fructose . FurtherTo determine if sugars were affecting the transcription of genes in the artemisinic biosynthetic pathways, we used Real-Time PCR to measure the relative transcript levels of the following genes: HMGR DXS, DXR, FPS, ADS, and CYP. Similar to methods used in sugar signaling studies in Arabidopsis, transcript levels were measured in seedlings grown in all three sugars with equimolar amounts of carbon. Then the responses in glucose and fructose were compared to transcript levels from seedlings grown in sucrose. The early pathway genes, HMGR and FPS, showed differences in expression soon after exposure to the test sugar A. ComparGlucose also stimulated both DXS and DXR from the MEP pathway A. ComparCompared to growth in sucrose, only glucose-fed seedlings showed significant responses in their DXS and DXR transcript levels B, essentIn contrast to the other 4 genes, both ADS and CYP responded quite differently to the three sugars tested A. While The CYP mRNA levels were more variable A. Again,Compared to the sucrose-fed seedlings, only glucose-fed seedlings showed any significant changes in ADS and CYP B. The leThe amounts of artemisinin and the artemisinic metabolites, AA, AB, and DHAA, were also analyzed in these same tissues. DHAA is thought to be a direct precursor of AN, while AA and AB are also formed from the product of the ADS gene, but apparently through a distinct pathway . All areA. annua is the AA/AB and not the AN/DHAA chemotype  of hemotype . In conthemotype . At day AN levels showed a very different pattern of production compared to the other metabolites and, interestingly, were also reduced to levels below the limits of detection from days 3 through 7 suggesting that as the seedlings begin to develop true leaves, AN may be further metabolized . ConsistConsistent with its role as an AN precursor, DHAA levels declined in glucose fed-seedlings between day 1 and 2 as AN increased . At day -1) showed an 8 fold reduction in the mean length of their primary roots after 10 days of exposure  appears also to be inhibited , specifically singlet oxygen . AN has et al.  providedcultures . A. annua tissues was significantly reduced in the presence of a ROS scavenger, ascorbic acid [ROS are cytotoxic to cells, and singlet oxygen in particular, may act as an apoptogenic signal in plant cells; therefore, it is important that accumulation of these toxic molecules be minimized ,37. Solubic acid . The dif2 rather than from sugars in the medium. In plants grown in sucrose or glucose, AN content does increase during this time, which is consistent with this hypothesis.A primary source of ROS in plants is photosynthesis, which is intimately involved with sugar metabolism of the plant ,42. In avia these later enzymes is not at the level of transcript abundance. Early cytosolic and plastidic pathway gene transcript levels seem to show little to no correlation to artemisinin precursor levels. At early time points, however, these do seem to correlate with relative AN levels. In the case of HMGR, when compared to sucrose-fed seedlings, both fructose and glucose cause an initial increase in transcript levels. This pattern of transcript levels is consistent with a possible sucrose specific, down-regulation of expression that is rapidly mitigated by the extracellular inversion of sucrose to glucose and fructose. The pattern of expression observed for FPS, DXS, and DXR, on the other hand, differed from HMGR in that differences between fructose and sucrose were minimal while the effects of glucose resulted in about an eight fold up-regulation in expression on day 1 (day 2 in the case of DXS). The patterns of expression observed for FPS, DXR, and DXS correlate most closely with AN production, while the AN specific genes show far less correlation, suggesting that, in seedlings, control of AN production A. annua plants expressing an additional copy of the FPS gene showed 3-4 fold higher levels of AN than their wild type counterparts [et al. [A. annua produced less AN than the nontransgenic 001 line. In the case of FPS, these results appear consistent with other work indicating that terparts . Another [et al. , howeverAfter day 2, early pathway genes  showed no significant differences in response to different sugars suggesting that sugar composition is not responsible for the transcriptional regulation of these genes later in development and that the level of transcriptional activation of these genes may have little to do with the levels of production of artemisinic metabolites. Indeed, the differences observed at early time points may simply be due to the movement from a sugar free medium to one of our experimental conditions. After the plants adjust to the difference they return to steady state levels of expression, indicating that the difference in transcript levels of the early pathway genes is in fact due to specific experimental conditions. The marked response in late pathway genes  further demonstrates the complex patterns of signaling interactions and highlights challenges inherent with measuring responses to sugars that may act as both signal and substrate molecule.Artemisia annua L. were used in all experiments. For experiments that provided individual sugars to plants, seeds were surface-sterilized and synchronously germinated according to the protocol developed by Wang and Weathers [Seeds from the YU strain  of Weathers . SeedlinWeathers . Ten seeWeathers  at pH 5.-1 AN. Due to solubility issues, AN was first dissolved in 70% ethanol before being serial diluted and added to media. The final concentration of ethanol in each flask was 0.35% and this amount was also added to controls. After 10 days growth under continuous light, seedlings were harvested, root lengths measured, and shoot tissues (cotyledons and true leaves) were extracted for artemisininc metabolite analyses. It was necessary to wait 10 days in order to obtain adequate amounts of shoot tissues for extraction.In the case of artemisinin exposure, seedlings were sterilized as above and germinated to the two cotyledon stage. Germinated seedlings were placed into B5 media with 3% sucrose and augmented with 0-200 µg mLet al. [Artemisia annua available at NCBI. Relative fold changes in gene expression were calculated based on the 2-ΔΔCT comparative method [Methods are briefly described here, but can be found in more detail in Mannan et al. . After he method ,48,49.et al. [Using freshly harvested tissues, AN and its metabolites, AA, AB and DHAA, were extracted and measured by LC/MS as described in Mannan et al. . U test [All experiments were run in triplicate and fold change values were expressed as the mean ± SD. Results were averaged and compared against controls to determine statistical differences. Statistical analyses of real-time PCR data were performed using the non-parametric Mann-Whitney U test . StatistA. annua seedlings, cues from sugars can have dramatic effects on the biosynthesis of artemisinin. Plant growth and metabolism are in constant flux with respect to sugars and these data could prove useful in furthering our fundamental understanding of the regulation of not only AN, but also other terpenes in planta.This is the first study to show that sugars play a role in the accumulation of artemisinin related gene transcripts and in the production of artemisinic precursor molecules. While sugar composition clearly has profound effects on the transcript level of numerous key genes, how modulation of these transcript levels is integrated into the greater model of control of AN production is not clear. Also presented is preliminary evidence suggesting that AN may be acting as an inhibitor of its own biosynthesis. Clearly the role of sugars in AN production is complex and untangling it from confounding factors of growth and development is a challenge that remains. However, we have clearly shown that at least in


Loss of muscle mass and muscle strength are natural consequences of the aging process, accompanied by an increased prevalence of chronic health conditions. Research suggests that in the elderly, the presence of comorbidities may impact the muscle mass/strength relationship. The objectives of this study were to characterize the muscle mass/strength relationship in older adults in the USA and to examine the impact of a variety of comorbidities on this relationship.2, as measured by dual-energy x-ray absorptiometry. Muscle strength was assessed via isokinetic quadriceps strength (IQS) in newton as measured by a dynamometer. The relationship between aASM and IQS was assessed adjusting for age and gender. The effects of a variety of comorbidities on IQS and/or on the relationship between IQS and aASM were assessed using multiple regression models.Data were obtained from the National Health and Nutrition Examination Survey 1999–2002 databases. Subjects aged 50 years and older were included in the present study. Muscle mass was assessed by height-adjusted appendicular skeleton muscle mass (aASM) in kg/m2) was 7.3 (0.04), and the mean (SE) IQS (newton) was 365.0 (3.00). After adjusting for age and gender, the correlation coefficient between aASM and IQS was 0.365 (P < 0.001). Diabetes, coronary heart disease/congestive heart failure (CHD/CHF), and vision problems were significant predictors of lower muscle strength (P < 0.05) in the multiple regression models that adjusted for age, gender, and aASM, and obesity significantly modified the relationship between aASM and IQS (P < 0.05).This study included 2,647 individuals, with a mean age of 62.6 years and 52.9% of whom were female. The mean (SE) aASM  and/or modify the relationship between muscle mass and muscle strength . By the year 2020, there are projected to be almost 55 million Americans, in the USA, over the age of 65 and more than 6 million over the age of 85 . One conAlthough the positive correlation between muscle mass and muscle strength has been well established ,7-11, itA number of clinical conditions have also been shown to correlate with muscle mass and/or strength, and it is possible that heterogeneity among older adults with respect to these conditions, many of which are increasingly prevalent with age , may conIn the present study, we aimed to better understand the relationship between muscle mass and muscle strength in US older adults, and to examine the impact of a variety of comorbidities on muscle strength and the relationship between muscle mass and muscle strength. Our specific objectives were as follows: (1) to describe muscle mass and muscle strength, stratified by age and gender; (2) to assess the degree to which muscle mass and strength are correlated, adjusting for the effects of age and gender; and (3) to explore the impact of a variety of comorbid conditions on muscle strength and/or the relationship between muscle mass and strength. For the latter objective, our general hypothesis was that certain factors, such as obesity and arthritis, might limit the degree to which higher muscle mass translates into greater muscle strength. Insights gained from these analyses could aid in the management of patients, who have muscle atrophy or wasting by providing norms and targets for rehabilitation and by raising awareness of the influence of other comorbid conditions.This was a cross-sectional, population-based study that used data from men and women 50 years and older who were enrolled in the National Health and Nutrition Examination Survey (NHANES) 1999–2002. The NHANES performs a continuous, nationally representative health survey of the civilian, non-institutionalized U.S. population and collect data on approximately 5,000 persons each year from in-person interviews, physical examinations, and medical tests. To produce nationally representative estimates, NHANES has adopted a complex, multistage, probability sampling design, and the survey oversamples certain ethnic minorities and individuals aged 60 years and older , which e2) ), in newtons . There i2). Consistent with the recommendation from Center for Disease Control and Prevention [2 (underweight), 18.5-24.99 , 25–29.99 (overweight), and ≥30 (obese).Comorbidity data were obtained from self-reported personal interview questionnaire data collected in the NHANES. Presence of a variety of health conditions, which potentially affect muscle mass and/or strength, was assessed using data from the medical conditions/history questionnaire, including the following: diabetes; coronary heart disease (CHD) /congestive heart failure (CHF);vision problems; obesity; arthritis (including both rheumatoid and osteoarthritis); asthma; osteoporosis; cancer ; and chronic bronchitis/emphysema. Body mass index (BMI) was also investigated and defined as weight (kg) divided by height squared , 75th and 95th percentiles. The Pearson partial correlation coefficient, controlling for age and gender, was used to examine the association between aASM and IQS.Distributions of aASM and IQS, by gender and age group , were presented using box plots. The box plots present the mean, 5Multiple regression models, using survey strata and weighting, were conducted to examine the effect of each comorbidity on IQS and on the relationship between aASM and IQS. We first constructed a linear regression model on IQS with aASM as an exploratory variable, adjusting for age and gender (Model 1). Next, for each comorbidity, we built two additional linear regression models on IQS, one with the studied comorbidity as an exploratory variable, adjusting for aASM, age and gender (Model 2), and another with all variables in Model 2, plus the interaction between aASM and the comorbidity of interest (Model 3). The regression coefficients for aASM and the studied comorbidity were presented, including the 95% confidence intervals (CIs), along with the significance level of the interaction term from Model 3. Where results suggested a significant interaction between aASM and a given comorbidity, the relationship between aASM and IQS was graphically presented via scatter plot by using the raw non-weighted data, along with the age- and gender-adjusted regression lines, via Model 3 and stratifying on comorbidity status. In addition, subgroup analyses of linear regression models (Model 1) were fit within comorbidity subgroups , adjusting for age and gender.All analyses were conducted using SAS®, version 9.2 survey procedures . All hypothesis tests were 2-tailed with a significance level of 0.05, and regression coefficients were deemed significantly different if the corresponding 95% CIs were non-overlapping.2, and the mean (SE) IQS was 365.0 (3.0) newtons.Population characteristics are presented in Table The distributions of aASM and IQS, stratified by gender and age, are presented in Figure 2) [2) increase in aASM. Age, gender, and aASM, together, explained 59.9% of the variance in IQS.The correlation coefficient between aASM and IQS, after adjusting for age and gender, was 0.365 (P < 0.001); in other words, the coefficient of partial determination was 0.133 0.3652) , which i , which Results from the multiple regression models that incorporated each of the comorbidities suggested that diabetes, CHD/CHF, and vision problems had significant negative main effects on IQS (P < 0.05), while corresponding interaction effects between the comorbidity and aASM were not statistically significant (P > 0.05) Table . SpecifiBody mass index, arthritis, and osteoporosis had both significant main effects and interaction effects on IQS, while asthma had a significant interaction effect, but an insignificant main effect on IQS, which made the corresponding regression coefficients less directly interpretable. Cancer and bronchitis/emphysema had neither significant main nor interaction effects.The relationship between muscle mass and IQS among subgroups with and without the comorbidities for which there were significant interaction effects  is shown visually in Figure In Figure In this cross-sectional, population-based study of older US adults, we described the distribution of muscle mass and muscle strength stratified by age and gender. We found that muscle mass and muscle strength were positively correlated after adjusting for age and gender. We also found that certain comorbidities had significant impacts on muscle strength and/or on the relationship between muscle mass and muscle strength.To our knowledge, this is the first study to characterize the distribution of height-adjusted appendicular skeletal muscle mass in a nationally representative sample of older adults in the US. Kelly et al. (2009) provided nationally representative reference data on DXA body composition reference values, but did not include reference data on lean mass exclusive of bone mineral content, which is considered a better representation of skeletal muscle mass . The disIn this study, we observed that both muscle mass and muscle strength declined with advancing age in older adults, and that women have a relatively lower muscle mass and strength compared with men of the same age, and these findings are consistent with previous research -10,28-30This study quantified the positive association between muscle mass and muscle strength in older adults, and found that muscle mass explained about 13% of variance in muscle strength, independent of age and gender. After adjusting for age and gender, every one unit increase in aASM was associated with a corresponding 31.6 newton increase in IQS. As these results are derived from a cross-sectional study, any causal inference is precluded. Nevertheless, these data underscore the need for further prospective research that evaluates the downstream effects of interventions, which are aimed at preserving muscle mass, on muscle strength and physical function.Our study also revealed that certain comorbidities impact muscle strength or the muscle mass/strength relationship. Diabetes, CHD/CHF, and vision problems were independent predictors of low muscle strength, which suggests that individuals with these comorbidities have, on average, lower muscle strength compared with individuals of similar age, gender, and muscle mass. Our finding regarding the effect of diabetes is consistent with work conducted by Park et al. (2006), wherein diabetics in the Health ABC Study were observed to have poorer muscle quality than non-diabetics . Older dWhile arthritis, asthma and osteoporosis all appeared to alter the relationship between muscle mass and muscle strength to some extent, interpretation of these effects is challenging due to the significant interaction effects observed. It is possible that these conditions are meaningful effect modifiers, but the results from our subgroup analyses  failed to establish this. It may be that these effects are relatively marginal or perhaps more complex , thus, requiring a larger sample size or more complex modeling approaches. Further research is warranted to better understand the effects of these comorbidities.These findings should be interpreted in the context of study limitations. First, the cross-sectional nature of the data limits our ability to do more than describe asso-ciations, and future longitudinal research is needed to better understand the causal nature of these associations. Second, comorbidities were based upon self-report, rather than on formal clinical assessment, which raises concerns about recall and other biases. Finally, comorbidities were examined individually; whereas, in many cases, multiple comorbidities may co-occur. Unfortunately, incorporating additional two-way or three-way interaction effects would have significantly impacted the power available to draw meaningful conclusions.In conclusion, among individuals aged 50 and older in the US, muscle mass and muscle strength are positively correlated, independently of the associations of age and gender with muscle mass and strength. Certain comorbid medical conditions serve as independent predictors of lower muscle strength  or modify the relationship between muscle mass and muscle strength .All authors are full-time employees and stockholders of Eli Lilly and Company.LC research project conception, organization, and execution; statistical analysis design; and manuscript drafting, review, and critique. DN data acquisition, statistical analysis design and execution, and manuscript review and critique. YZ research project conception and organization, and manuscript review and critique. ZC manuscript review and critique. JJ research project conception; statistical analysis design; and manuscript drafting, review, and critique. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/13/74/prepub


Mortality amenable to health-care services  has been defined as “premature deaths that should not occur in the presence of timely and effective health care” and as “conditions for which effective clinical interventions exist.” We analyzed the regional variability in health-care services using amenable mortality as a performance indicator. Convergent validity was examined against other indicators, such as health expenditure, GDP per capita, life expectancy at birth, disability-free life expectancy at age 15, number of diagnostic and laboratory tests per 1,000 inhabitants, and the prevalence of cancer and cardiovascular diseases.Amenable mortality rate was calculated as the average annual number of deaths in the population aged 0–74 years per 100,000 inhabitants, and it was then stratified by gender and region. Data were drawn from national mortality statistics for the period 2006–08.p = 0.002; female: r = −0.88, p <0.001) and with disability-free life expectancy at age 15 . Amenable mortality displayed a statistically significant negative relationship with GDP per capita, the quantity of diagnostic and laboratory tests per 1,000 inhabitants, and the prevalence of cancer.During the study period (2006–08), the age-standardized death rate (SDR) amenable to health-care services in Italy was 62.6 per 100,000 inhabitants: 66.0 per 100,000 for males and 59.1 per 100,000 for females. Significant regional variations ranged from 54.1 per 100,000 inhabitants in Alto Adige to 76.3 per 100,000 in Campania. Regional variability in SDR was examined separately for male and females. The variability proved to be statistically significant for both males and females . However, among men, we found a clear-cut divide in SDR values between Central and Southern Italy; among women, this divide was less pronounced. Amenable mortality was negatively correlated with life expectancy at birth for both genders (male: r = −0.64, Amenable mortality shows a wide variation across Italian regions and an inverse relationship with life expectancy and GDP per capita, as expected. The performance of health-care systems in terms of maximizing population health has been a major concern of policy makers -5. ExpliIn recent years, the concept of amenable mortality has been used as a proxy for performance of health-care systems by Nolte and McKee -9 and ToMortality amenable to health-care services  has been defined as “premature deaths that should not occur in the presence of timely and effective health care”  and as “Nolte and McKee carried out a comprehensive study  of amenaAnother recent study  has provAmenable mortality is also a useful indicator for measuring the performance of health-care systems. As a strategy to deal with shrinking resources while increasing local government accountability, efficiency, quality, and innovation in the health-care sector, the process of decentralization of powers from national to regional levels has been widely implemented across European health-care systems . TherefoIn Italy, the central government is responsible for national health planning and annual funding. It also has the exclusive power to set the so-called essential levels of care , an explicit, publicly funded health-benefit package to which all citizens are entitled. Regions have virtually exclusive responsibility for the organization and administration of publicly financed health care . TherefoThe aim of this study was twofold. The first was to analyze the regional variability in health-care services using amenable mortality as a performance indicator. The second was to examine the convergent validity of amenable mortality against other indicators, such as health expenditure, GDP per capita, life expectancy at birth, disability-free life expectancy at age 15, number of diagnostic and laboratory tests per 1,000 inhabitants, and the prevalence of cancer and cardiovascular diseases.A cross-sectional study was carried out on individual data from national mortality statistics for the period 2006–08 using data from the Italian National Institute of Statistics (ISTAT), where the causes of death are coded using the ICD-10 classification. Nolte and McKee -9 and ToAmenable mortality rate was calculated as the average annual number of deaths over the population aged 0–74 years per 100,000 inhabitants, and it was then stratified by gender, region (19 regions and the two autonomous provinces of Trentino and Alto Adige) and 10 disease categories defined by Gay et al. in an OECD report , the SDR in Italy was 62.6 per 100,000 inhabitants: 66.0 per 100,000 for males and 59.1 per 100,000 for females. Figures 006–08, tFigure Cancer and cardiovascular diseases were the leading causes of amenable mortality  and accounted, respectively, for 36.5% and 49.0% of overall amenable mortality. Figure p = 0.002; female: r = −0.88, p < 0.001)     Figure a, b 19]p = 0.002p = 0.267)    Figure ; however) Figure  and betw) Figure .Evidence from the most recent studies indicates that amenable mortality can be used to assess health-care performance across countries over time. Italy showed the third-lowest amenable mortality in 2005 among 31 OECD countries, indicating a good performance of health-care services .Our study shows that for the period 2006–08, the SDR was 62.6 per 100,000 inhabitants, though there were regional differences with a clear-cut divide being evident between Central and Southern Italy. In Italy, disparities in the geographical distribution have been documented for educational level (percentage of the population with only elementary education: 22.5% in the North and 27.2% in the South) , and povMoreover, the average SDR was lower in women  than in men . This may result from women’s higher life expectancy at birth and gender-specific help-seeking patterns. The divergence in SDR values between Central and Southern Italy was more evident among men than in women. Pinkhasov et al.  found diIn our study, cancer and cardiovascular diseases proved to be the leading causes of amenable mortality, accounting for 85.5% of the overall indicators. A greater regional variability was found for cardiovascular diseases than for cancer. This finding may suggest that Nolte and McKee’s list is especially sensitive to conditions for which effective and appropriate health care is essential, such as cardiovascular diseases.The convergent validity of amenable mortality with some health indicators was partially supported by a high negative correlation between SDR and life expectancy at birth and disability-free life expectancy at age 15 for both genders.Our study shows that amenable mortality is related to GDP per capita, but not to health expenditure per capita. A possible interpretation of this finding is that SDR is influenced by organizational and care delivery models and different priority settings—not by the amount of dedicated resources. Recently, Nagy et al.  also repSeveral caveats should be noted in the use of amenable mortality as an outcome indicator of health-system performance. First, it is important to note that the categorization of a condition as amenable is essentially based on a judgment about the effectiveness of medical interventions in treating different conditions and preventing death . FurtherMoreover, we chose to use Nolte and McKee’s list of causes of death amenable to health-care. Unlike Tobias and Yeh’s list, Nolte and McKee’s list included among death causes adverse events to patients during surgical and medical care, which are strongly related to quality of care. Last, Tobias and Yeh’s list included among the death causes chronic obstructive pulmonary disease (age >45 years), whose prognosis is more related to lifestyle than to health care, and bladder and thyroid cancer, where treatment and surgical interventions are moderately effective.Second, the prevalence of diseases whose deaths are amenable to health-care may vary across regions. For instance, if the prevalence of cancer is substantially higher in one region, this region will need to devote more resources to avoid deaths from this disease category . To addrThird, health-service performance may depend on the quantity of resources available to provide effective interventions. However, as previously noted, we discovered no relationship between SDR and per capita public health expenditure.Keeping these limitations in mind, the present study provides, in the context of decentralization of powers in health care, an easily calculated, valid indicator for monitoring the performance of health-care systems as a basis for evidence-based policy decision making. This indicator could be useful not only for a comparison between systems but also to detect variations in a health-care system over time. And the indicator could be a positive element in continuous efforts to improve the policies, organization and quality in a decentralized health-care system.Our results highlight that, in line with other health-care performance indicators, amenable mortality is lower in North and Central regions of Italy than in Southern regions. We argue that this indicator can be used to inform policy decision-making processes in decentralized health-care systems and monitor their effectiveness and equity. Amenable mortality exhibits an inverse relationship with life expectancy, prevalence of cancer and cardiovascular diseases, and socioeconomic and resource consumption indicators. Further investigation is warranted to analyze the trend of SDRs at a subnational level over time.The authors declare that they have no competing interests.MPF, GDa, and WR contributed to the conception of this paper. MPF and GDa conceived the study design; LF and AB provided data sources and participated in the study design. GF provided the acquisition of data; JL and CR conceived the statistical methodology and performed statistical analyses. GDa, MPF, GF, and JL drafted the manuscript. MPF and JL had full access to all of the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. GDo critically revised the draft and contributed to the final writing of the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/310/prepubTable S1. Nolte and McKee’s list of causes of death arranged into 10 disease categories.Click here for file


Nitrate and nitrite are widespread contaminants of vegetables, fruits, and waters. The levels of these compounds are increased as a result of using organic wastes from chemical industries, domestic wastes, effluents, nitrogenous fertilizers, and herbicides in agriculture. Therefore, determining the nitrate and nitrite levels in biological, food, and environmental samples is important to protect human health and the environment. In this context, we set this study, in which we report the effect of production system  on the accumulation of nitrates and nitrites in fresh baby-leaf samples. The average levels of the nitrate ( Western countries are consuming “organically” grown foods with increasing quantity and frequency , red leaf lettuce (Lactuca sativa var. romana), and watercress . Additionally, we extended this work on rucola (Rocket)  Thell., Brassica eruca L.), chard (Beta vulgaris var. cicla L.), and corn salad , but only on samples from a conventional production system, due to the lack of this kind of vegetables in local organic farmers. The vegetables selected for this study are some of the most commonly consumed vegetables all over the year. All the commercial samples were obtained in original package, within the shelf life of up to 8 days, as declared on labels. After purchase, the samples were transported to the laboratory of phytochemicals at University of Trás-os-Montes e Alto Douro (UTAD) and 150 g were taken and freeze-dried , grounded in a blender , and weighed. The dry matter was determined.Samples of fresh baby-leaf salads were randomly acquired from supermarket  and directly from local farmers (organic production), in Northern Region of Portugal, and included green leaf lettuce (N-(1-naphthyl)-ethylenediamine dihydrochloride to form a highly colored azo dye that is measured at 540 nm. The nitrite present in the sample is determined by analyzing without the reduction step. The nitrate is calculated as the difference between the total nitrite content after reduction and the initial nitrite concentration  was used for statistical analysis. All experiments were performed in triplicate, and the results were presented as the mean ± SEM (standard error of the mean). The data were analyzed using one-way analysis of variance (ANOVA). The differences between the mean values were separated using Duncan's test at a significant level of t-test was used for determination of significant differences between the mean values.A single-laboratory validation was carried out following the standard protocol adopted in the laboratory. The following parameters were evaluated: linearity, working range, detection and quantification limits, precision, recovery, and accuracy. Calibration curve was plotted, and linearity was evaluated by the values of determination and variation coefficients of the method after the application of several statistical tests. The calibration curves were obtained using a series of nitrate and nitrite standard solutions. All calibration curves were linear with correlation coefficients from the linear regression ranging from 0.992 to 0.999. Method performance data for nitrate and nitrite determination in baby-leaf salads are shown in Table To our knowledge, this is the first report that compares the nitrates/nitrites levels of fresh and baby-leaf salads in two types of agriculture. Individual results obtained for nitrate and nitrite levels are shown in Tables P < 0.05) in conventional produce, and as expected, the averages levels of nitrites were lower when compared with nitrates. The average levels of nitrites, except red lettuce, were very similar (P > 0.05) in both organic and conventional agriculture system (Tables Asteraceae (lettuce) and Brassicaceae (watercress) – the families with the highest average levels. This result is in agreement with Santamaria , Chenopodiaceae , Asteraceae (lettuce), and Apiaceae  are usually, among the vegetables, the plant families with highest nitrate contents. This tendency was confirmed in the present study.The results showed a considerable significant variation in the average levels of nitrate contents between the two production systems. The average levels of nitrates were higher  recommended by European Union regulations (Regulation [EC] No. 1258/P < 0.05) were observed. For green lettuce and watercress, there were no significant differences. Nevertheless, the average nitrite content was higher in organic produce. Compared with literature, our average values are very similar to those reported in fresh vegetables (González et al. One important aspect particularly to human health is related to contamination with nitrites. It is well accepted that when nitrates is reduced to nitrites, nitrite may react with amines or amides to form carcinogenic compounds (Savino et al. Chung et al.  and PrasTo conclude, based on our results, it seems that baby-leaf salads produced in organic and conventional systems in Northern Portugal have low levels of nitrates and nitrites. Therefore, they are toxicologically safe, and their consumption might be incremented without risk for health. The differences between organically and conventionally cultivated plants are less but exist. Nevertheless, it does not represent any type of risk for human health. These results are important not only for adults but particularly for children, in which the toxicological aspects related with nitrates and nitrites accumulation assume more importance.


There have been no prior population-based studies of variation in performance of hospitalists.To measure the variation in performance of hospitalists.Retrospective research design of 100 % Texas Medicare data using multilevel, multivariable models.131,710 hospitalized patients cared for by 1,099 hospitalists in 268 hospitals from 2006–2009.We calculated, for each hospitalist, adjusted for patient and disease factors (case mix), their patients' average length of stay, rate of discharge home or to skilled nursing facility (SNF) and rate of 30-day mortality, readmissions and emergency room (ER) visits.In two-level models , there was significant variation in average length of stay and discharge location among hospitalists, but very little variation in 30-day mortality, readmission or emergency room visit rates. There was stability over time (2008–2009 vs. 2006–2007) in hospitalist performance. In three-level models including admissions, hospitalists and hospitals, the variation among hospitalists was substantially reduced. For example, hospitals, hospitalists and case mix contributed 1.02 %, 0.75 % and 42.15 % of the total variance in 30-day mortality rates, respectively.There is significant variation among hospitalists in length of stay and discharge destination of their patients, but much of the variation is attributable to the hospitals where they practice. The very low variation among hospitalists in 30-day readmission rates suggests that hospitalists are not important contributors to variations in those rates among hospitals. Hospitalists are physicians who specialize in the care of hospitalized patients. There are advantages and disadvantages to the “hospitalist” model. The potential advantages stem from greater efficiency and expertise from physicians concentrating just on inpatient care.We have used 5 % national Medicare data to describe the growth of hospitalists from 1996 through 2006,Variation in outcomes and quality of care that cannot be explained by illness severity, patient preference, or “unwarranted variation”, indicates an opportunity to decrease the cost or improve the effectiveness of healthcare.https://www.cms.gov/Medicare/Medicare-Fee-for-Service-Payment/AcuteInpatientPPS/index.html).Claims from the years 2005 to 2009 for 100 % of Texas Medicare beneficiaries were used, including Medicare beneficiary summary files, Medicare Provider Analysis and Review (MedPAR) files, Outpatient Standard Analytical Files (OutSAF), and Medicare Carrier files. Diagnosis related groups (DRG) associated information, including weights, Major Diagnostic Categories (MDC), and geometric mean length of stay, were obtained from Centers for Medicare & Medicaid Services  who had at least 100 evaluation-and-management (E&M) billings in a given year and generated at least 90 % of their total E&M billings in that year from inpatient services.This process is outlined in Table We categorized beneficiaries by age, gender and ethnicity using Medicare beneficiary summary files. We used the Medicaid indicator as a proxy of low income. Information on weekday vs. weekend admission, emergent admission, and DRG were obtained from MedPAR files. Elixhauser medical conditions were identified using the claims from MedPAR, Carrier and OutSAF files in the year prior to that of the admission of interest.Hospital length of stay was obtained from MedPAR files. For each admission, we calculated a difference in length of stay by subtracting the geometric mean length of stay for that DRG obtained from the Center for Medicare and Medicaid Services from the actual length of stay. This measure intrinsically controls for case mix among hospitalists, because geometric mean length of stay differs for each DRG. We excluded outliers more than three standard deviations from the norm in order to approximate the normal distribution and analyze with a hierarchical general linear model, leaving 129,491 admissions, and 1,099 hospitalists.Mortality within 30 days of admission was calculated from date of death in the Medicare beneficiary summary file. These analyses included all 131,710 admissions and 1,099 hospitalists in the cohort. We chose mortality within 30 days of admission rather than from discharge to avoid biases in different hospital length of stay among hospitalists. However, our analyses of 30-day post discharge mortality produced almost identical results.We calculated the rate of admissions discharged home and the rate discharged to a Skilled Nursing Facility (SNF), obtained from MedPAR files. We excluded those who were discharged dead, transferred to another acute care hospital or had stayed in a nursing facility any time in the three months prior to the admission of interest, leaving 99,522 admissions and 990 hospitalists.ER visits were identified by CPT E&M codes 99281-99285 and 99288 from Carrier files. To study readmissions and ER visits within 30 days of discharge, we excluded those who were discharged dead or transferred to another acute care hospital, or died in the 30 days post discharge without an event (readmission or ER visit), leaving 108,547 admissions and 1,019 hospitalists in the study cohort for 30-day readmission, and 108,226 admissions and 1,018 hospitalists for 30-day ER visits. Readmissions and ER visits were not mutually exclusive; i.e., most readmissions also had an ER visit.Multilevel analyses were used to account for the clustering of patients within hospitalists and hospitalists within hospitals. For differences in length of stay, a hierarchical general linear model was used. For other outcomes, we used hierarchical generalized linear models with binomial distribution. The hospitalist-specific estimates were derived from two-level models adjusted with patient characteristics and then plotted by rank, and from three-level models including hospitals. Patient characteristics included age, race/ethnicity, gender, Medicaid eligibility, emergency admission, weekend admission, DRG weight, MDC, Elixhauser medical condition , number of hospitalizations, number of physician visits and having a PCP in the year prior to the admission of interest. For the model analyzing differences in length of stay, DRG weight was not adjusted because it was a within-DRG comparison. Because some hospitalists cared for admissions at more than one hospital in the three-level models, we assigned hospitalists to the hospital in which > 50 % of their E&M charges occurred, and excluded admissions by those hospitalists to other hospitals. All analyses were performed with SAS version 9.2 . The threshold models for the partitioned variances were performed with MLwiN version 2.02.th and 75th percentiles were 54 and 156. The characteristics of those admissions are summarized in Table The final sample was 131,710 admissions cared for by 1,099 hospitalists. The median number of Medicare admissions cared for by each hospitalist was 98, and the 25Our primary interest was variation in patient length of stay, discharge location and 30-day outcomes, at the level of each hospitalist. For this, we conducted a series of two-level models, controlling for the characteristics in Table Figure We evaluated the stability of hospitalist performance over time by assessing the average adjusted length of stay for hospitalists in 2008–2009 compared to their length of stay in 2006–2007. We ranked the 633 hospitalists with > 30 admissions in both 2006–2007 and 2008–2009 by quintile of their average adjusted length of stay in 2008–2009 (Table The two-level models described above do not account for the fact that hospitalists cluster within hospitals. Therefore, to distinguish variation at the hospitalist level from variation among hospitals, we constructed three-level models examining the contribution of patient, hospitalist and hospital to variations in length of stay, discharge destination and 30-day outcomes. Table In all the analyses above, if a patient had multiple hospitalizations in a given year, we selected one at random to avoid clustering at the patient level. However, this method would tend to lower estimates of the rehospitalization rate. Accordingly, in supplemental analyses we included all admissions. This had almost no effect on the estimates of the ICC for readmission.The purpose of this study was to assess the extent of variation in the care provided by hospitalists by measures of their practice: average adjusted length of stay, discharge destination, 30-day mortality, and rates of readmission and ER visits within 30 days of discharge. We found significant variation in length of stay and discharge destination among hospitalists, a variation that was stable over time. However, much of the variation among hospitalists was because of clustering of hospitalists within hospitals. We found little to no variation among hospitalists in 30-day mortality, rehospitalization or ER visit rates, either in the two-level or three-level models.The variations in lengths of stay and discharge destination, between hospitalists and between hospitals, suggest underlying variations in hospitalist practice styles and hospital-based systems of care. The relative lack of variation at the hospitalist level in 30-day outcomes is not likely due to insufficient power, given the large number of hospitalists and substantial number of admissions per hospitalist. This suggests that the hospitalist practice styles that lead to variations in length of stay and discharge destination do not have a noticeable impact on mortality, readmission rates or ER visit rates. Several prior studies have also suggested a weak link between care in hospital and readmission rates.Hospitals are being held accountable for readmissions of patients they discharge.Our study has limitations. It is an observational study and susceptible to bias and confounding. We studied patients with fee-for-service Medicare who received care in a single large state in the USA over a two-year period. It is possible that our results may not apply to a younger population, those in other states, or during a different time period. In particular, there are substantial variations in hospital readmission rates among different regions of the USA.As hospitalist care continues to increase in prevalence in the USA, so does the ability of hospitalists to impact the cost and quality of hospital care.


Motor impairment in old age is a growing public-health concern, and several different constructs have been used to identify motor impairments in older people. We tested the hypothesis that combinations of motor constructs more strongly predict adverse health outcomes in older people.In total, 949 people without dementia, history of stroke or Parkinson's disease, who were participating in the Rush Memory and Aging Project , underwent assessment at study entry. From this, three constructs were derived: 1) physical frailty based on grip strength, timed walk, body mass index and fatigue; 2) Parkinsonian Signs Score based on the modified motor section of the Unified Parkinson's Disease Rating Scale; and 3) a motor construct, based on nine strength measures and nine motor performances. Disability and cognitive status were assessed annually. A series of Cox proportional-hazards models, controlling for age, sex and education, were used to examine the association of each of these three constructs alone and in various combinations with death, disability and Alzheimer's disease (AD).r = 0.50, all P < 0.001), and when considered individually in separate proportional-hazards models, were associated with risk of death, incident disability and AD. However, when considered together, combinations of these constructs more strongly predicted adverse health outcomes.All three constructs were related (mean Physical frailty, parkinsonian signs score and global motor score are related constructs that capture different aspects of motor function. Assessments using several motor constructs may more accurately identify people at the highest risk of adverse health consequences in old age. Loss of motor function is a common consequence of old age, and is associated with adverse health consequences -5. The sCurrently there is no single scale that can be used to assess motor impairments. Several constructs based on assessments of different motor abilities have been used to document motor impairments in old age, including presence of sarcopenia, based on muscle strength and bulk;  physicalWe used data from 949 older people participating in the Rush Memory and Aging Project, a longitudinal study of common chronic conditions of aging, to examine the associations of three constructs that have been used in previous studies: physical frailty, parkinsonian signs score, and a composite motor score estimating the risk of death, incident disability and incident Alzheimer's disease (AD). We hypothesized that although all three constructs assess motor function, each construct captures aspects of motor abilities not appraised by the other constructs. Thus, we predicted that combinations of these three motor constructs would more strongly predict adverse health outcomes in older people compared with analyses using each motor construct alone.All participants were from the Rush Memory and Aging Project, which was conducted in accordance with the latest version of the Declaration of Helsinki, and approved by the institutional review board of Rush University Medical Center .Clinical evaluations for the study started in 1997, and at the time of these analyses, 1237 people had completed their baseline clinical evaluation. We excluded 95 people with dementia, 128 people with history of stroke, and 15 people with Parkinson's disease (PD) at baseline. Of the remaining 999 people, another 35 were excluded because follow-up was not possible . This left 964 participants at baseline who were eligible for follow-up, and 949 (98.4%) of these had completed one or more follow-up evaluations. These participants had an average of nearly five follow-up examinations (mean ± SD 4.7 ± 2.4). The participants' baseline characteristics are shown in Table Subjects underwent a uniform structured clinical evaluation including medical history, clinical examination, and cognitive performance testing as previously described . Clinicaet al. is a widely used construct [et al. to remove physical activity, in order to examine its association with physical frailty and other constructs related to motor abilities [Physical frailty is an evolving concept, with numerous definitions proposed. The categorical measure proposed by Fried onstruct . We modibilities -18. Secobilities , and offbilities . As descbilities ,21. Physbilities . Fatiguebilities ,11,20.A modified form of the motor section of the Unified Parkinson's Disease Rating Scale (mUPDRS) was used for each patient. This assesses four parkinsonian domains  as previously described . Each si®; Lafayette Instruments). Motor performances were tested in both arms and legs, using seven performance-based tests of leg function and two tests of motor performance of the arms, as previously described [Portable hand-held dynamometers  were used to assess arm abduction, flexion and extension, hip flexion, knee extension, plantar flexion, and ankle dorsiflexion. Grip and pinch strength were measured bilaterally using a hydraulic dynamometer .We scored each of the performance measures so that higher scores were associated with better performance. To ensure the same directionality in all the performance measures, we first reciprocated the recorded values for five variables . A score of 0 was recorded if a participant was unable to perform a particular task. Each score was then scaled by dividing by the sex-specific median value of the non-zero values at baseline -26. The z scores . The corBasic activities of daily living were assessed using a modified version of the Katz Index, a self-report measure that assesses six activities: feeding, bathing, dressing, toileting, transferring, and walking across a small room . ParticiDemographic information including date of birth, gender and years of education were collected via participant interview. The number of hours of physical activity per week was assessed using questions adapted from the 1985 National Health Interview Survey . We summParticipation in the Memory and Aging Project includes the patient agreeing to donation of brain, spinal cord, and selected muscles and nerves at the time of death, and the study has an autopsy rate of >80%. At the time of these analyses, vital status was available for all participants.t-tests were used to compare baseline characteristics. Statistical significance was considered to be P < 0.05.Pearson correlations were used to examine the bivariate associations between motor constructs and demographic variables, and The goal of these analyses was to test the hypothesis that models using combinations of the three motor constructs would provide additional explanatory power for the health outcome of interest compared with models using the individual motor constructs alone. We used a set of eight proportional-hazards models to estimate risk of death associated with demographic variables , and with the three motor constructs alone and in combination.First, we determined the contribution of the demographic variables age, gender and education to the risk of death (core model) Figure , row 1. P-values that are spuriously large. The deviance tests are extensions of partial F tests to generalized linear models [k additional variables. The coefficients are estimated by maximizing a partial likelihood function, and the maximum of the likelihood function for M2 will always be larger than that for M1. Then, under the null hypothesis that M1 is the true model, twice the difference of the logarithm of the likelihood for the two models (a statistic known as the deviance between the two models), follows an approximate χ2 distribution with k degrees of freedom. If M1 is not the true model, the deviance test will tend to be larger.Using the Wilks or deviance test, we compared the three models of each of the motor constructs alone with the four models consisting of combinations of these motor constructs. We chose this approach because Wald's method for obtaining observed significance levels for terms in a proportional-hazard model can result in r models . This apThese different comparisons are summarized in Figure We used a similar series of models for discrete (tied) data to examine the relationships of the three motor constructs with the risk of developing incident disability Figure  and thenGrip strength and timed walk were components common to both global motor score and physical frailty, thus to avoid duplication, we removed these two components from the global motor score before analyzing combinations of these constructs. In a final analysis, we added back the two components of grip strength and timed walk that had been removed from the global motor score measure (see above), and reran the set of models above.® System for Linux; SAS Institute Inc., Cary, NC, USA) [Models were examined graphically and analytically, and assumptions were judged to have been met adequately. The models for this paper were programmed using SAS/STAT software  .r = 0.33, P < 0.001) and education , and men were more frail than women .Physical frailty ranged from -1.71 to 1.90 (mean ± SD -0.054 ± 0.55), with higher scores indicating a greater degree of physical frailty or impairment. Physical frailty was related to age  and education  and did not differ by sex .Parkinsonian signs score ranged from 0 to 49 (mean ± SD 8.46 ± 6.88), with higher scores indicating more parkinsonian signs. The score was related to age  and education , and did not differ by gender .Global motor score ranged from 0.16 to 2.03 (mean ± SD 1.01 ± 0.27), with higher scores indicating better motor function. Global motor score was related to age  and global motor score . Parkinsonian signs score was also related to global motor score .As expected, the three constructs were all related to one another. Physical frailty was related to both parkinsonian signs score , 253 participants (26.7%) died. In separate proportional-hazard models controlling for age, sex and education, we found that physical frailty, parkinsonian signs score parkinsonian signs score and global motor score were all individually associated with risk of death  for age = 1.10; 95% CI 1.07-1.12), resulting in about a 10% increase in the risk of death for each year of age at baseline. Comparison of the estimates for physical frailty and age showed that the risk of death associated with a 0.6-unit (1 SD) increase in the level of physical frailty at baseline was comparable with a participant being >4 years older at baseline . A similar comparison would show that a seven-point (1 SD) increase in parkinsonian signs score at baseline was comparable with the risk associated with being about 5 years older at baseline, and a 0.27-unit (1 SD) decrease in global motor score was comparable with being 9 years older at baseline.We examined a set of proportional-hazards models that included all possible combinations of these three constructs to determine if the combinations with two or more constructs showed a significant reduction in model deviance  compared with the individual constructs alone. In these analyses Figure , a signiIn further analyses, we added back grip strength and timed walk, which had been removed from the global motor score measure, and the results were unchanged (results not shown).We restricted the next analysis to the 841 people (88.6% of 949 people) without Katz disability at baseline. Over an average follow-up of almost 5 years (mean ± SD 4.9 ± 2.39 years), 295 of 841 participants (35.1%) developed Katz disability. We examined the associations of each of these three constructs with incident disability by fitting a set of proportional-hazard models that controlled for age, sex and education. Physical frailty, parkinsonian signs score and global motor score were all associated with incident disability .During an average follow-up of >4 years (mean ± SD 4.2 ± 2.35), 155 of the 919 participants (16.9%) developed AD. We examined the associations of each of the three motor constructs with the risk of AD by fitting a set of proportional-hazard models that controlled for age, sex and education. Physical frailty, parkinsonian signs score and global motor score were each associated with risk of AD .In a cohort of 949 older people without dementia, history of stroke or PD, we found that three well-studied motor constructs - physical frailty, parkinsonian signs score and global motor score - were all associated with adverse health consequences including mortality, incident disability and incident AD. Furthermore, when considered together in the same models, combinations of these three constructs substantially improved the prediction of adverse health outcomes relative to the constructs considered alone. Thus, these three related motor constructs capture, in part, different aspects of motor function, therefore assessments using more than one motor measure might more accurately identify older people at risk for adverse health consequences.Loss of motor function in old age is common, and is associated with adverse health consequences. There are currently about 40 million people over the age of 65 years in the USA, and by 2030, this will have increased to more than 70 million . The groThe current study extends on previous findings in several important ways. First, in contrast to previous studies, which usually focused on a single construct, the current study examined three well-studied constructs in the same population, and confirmed that when each of these three constructs is considered alone in separate models, they each predict the likelihood of death, disability and AD. Second, direct comparison showed that these three constructs are related to one another. Thus, reports about use of these three constructs in old age should be considered related literature that inform on one another. Finally, the current study showed that combinations of two or more of these constructs had separate effects on adverse health outcomes, and that the associations varied by outcome. For example, parkinsonian signs score and global motor score were most strongly related to risk of death and incident disability, whereas physical frailty and either of the other two constructs most strongly predicted incident AD. Together, these findings have important translational consequences for the assessment of motor impairments in old age, because they suggest that a battery of motor measures may be necessary to understand age-related motor decline more fully and to identify more accurately the people at risk for negative health outcomes in our rapidly aging population.Localized brain lesions , specific diseases  and localized musculoskeletal disease  may selectively impair distinct motor abilities while sparing others -34. ThesThe findings in the current study are supported by recent advances in neuroscience, which have begun to characterize the complex brain processes necessary to ensure accurate movements. Integration of a wide range of sensory and visuospatial information  is essential for accurate movements , and difSeveral factors increase confidence in the findings from this study. Cognitive and motor functions were evaluated as part of a uniform clinical evaluation, and incorporated three widely accepted motor measures which are reliable and available. Strength testing was performed for all four limbs, and motor performances were tested in both the arms and the legs. The aggregation of multiple measures of motor function into composite motor measures is likely to yield a more stable measure of motor function, and may increase the power to identify adverse health consequences of motor decline in aging. In addition, a relatively large number of older people were studied, with high rates of follow-up participation, so that there was adequate statistical power to identify the associations of interest while controlling for potentially confounding demographic variables and reducing bias.The limitations of this study include the selected nature of the cohort, thus replication of these results in a population-based study is important. Further, although the findings from this study suggest that assessment of motor function may have clinical implication, additional work is needed to determine which measures are most clinically relevant. Finally, because people with stroke or PD at baseline were excluded, the results almost certainly underestimate the magnitude of the association between motor function with adverse health outcomes.In a cohort of nearly 1000 community-dwelling older people free of dementia, PD or history of stroke, who were followed up for up to 12 years, we found that three different motor constructs  were all related, and were all associated with death and the subsequent development of disability and AD. Most importantly, these results extend those of previous studies by showing that combinations of these three constructs in the same models more strongly predict adverse health outcomes than the individual constructs alone. Thus, a battery of motor measures may be necessary to understand age-related motor impairments more fully, and thereby identify at-risk people more accurately in order to design interventions that decrease the growing burden of motor impairment in our rapidly aging population.The authors declare that they have no competing interests.ASB had full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis, and affirms that everyone who contributed significantly to the work has been listed. ASB was involved with study concept and design, analysis and interpretation of data and preparation of the manuscript. SEL, PAB, JAS, SEA and DAB were involved in study concept and acquisition of data, assisted with the analysis and interpretation of the data, and critically revised the manuscript for important intellectual content. All authors have seen and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/42/prepub


Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection.Canine leishmaniosis (CanL) due to Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection.One hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.Although other cases of CanL have previously been described in the country, this is the first reported study of canine  Leishmania, which parasitise several mammalian species, including humans. Leishmania infantum (syn. Leishmania chagasi) is the agent of zoonotic visceral leishmaniosis, in southern Europe, North Africa, the Middle East, Central and South America, Central Asia and China; while Leishmania donovani is the agent of anthroponotic visceral leishmaniosis mainly in the Indian subcontinent and East Africa [The leishmanioses are diseases caused by protozoa of the genus t Africa .L. infantum is a global zoonosis regarded as endemic in more than 70 countries in Europe, Africa, Asia and America, with dogs being the main reservoir for the human infection [Phlebotomus are the vectors of Leishmania spp. in the “Old World”, i.e. Europe, Africa and Asia [Canine leishmaniosis (CanL) due to nfection -5. CanL nfection ,7. Phleband Asia .Leishmania spp. infection in dogs and humans is scarce. A small number of human visceral leishmaniosis cases have been described in the scientific literature [Epidemiological surveys in dogs and clinical cases of CanL have been reported in North Africa -12, but terature -21 and oLeishmania infection in dogs from Luanda, Angola.The objective of this study was to perform a serological and molecular survey of One hundred-and-three dogs presented to a veterinary medical centre in the city of Luanda Figure  were assLeishmania followed a previously described protocol [7 promastigotes per milliliter . Plasma twofold dilution series ranging from 1:100 to 1:6400 were tested. Results are expressed as an antibody titre, i.e. the reciprocal of the highest dilution at which agglutination (large diffused blue mats) is still clearly visible after 18 h of incubation at room temperature. A cut-off titre of 400 was chosen for seropositivity [The direct agglutination test (DAT) for titration of immunoglobulin G antibodies specific to protocol , using asitivity .L. infantum rRNA operon was amplified by real-time polymerase chain reaction (PCR) using primers ITS-219 F (5′-AGC TGG ATC ATT TTC CGA TG-3′) and ITS-219R (5′-ATC GCG ACA CGT TAT GTG AG-3′) and then evaluated by high resolution melt (HRM) analysis [L. infantum and DNA from colony-bred dogs negative by PCR for vector-borne pathogens were used as positive and negative controls, respectively.DNA was extracted from concentrated blood samples with commercial kits , according to the manufacturer’s instructions. A 265 bp fragment within the internal transcribed spacer 1 (ITS1) region of the analysis . The PCRhttp://www.ncbi.nlm.nih.gov/BLAST/).PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), at the Center for Genomic Technologies, Hebrew University of Jerusalem, Israel. DNA sequences were evaluated with the ChromasPro software version 1.33 and compared for similarity to sequences in GenBank, using the BLAST 2.2.9 program . No other dog was found to be PCR-positive, leading to a molecular prevalence of 1.0%.Two dogs were seropositive out of the 103 tested, with DAT titres of 800 (dog 27) and ≥6400 (dog 29), which corresponds to a seroprevalence of 1.9% in the studied population. Dog 29 was also found to be PCR-positive and confirmed to be infected with Both dogs 27 and 29 were considered clinically sick, presenting clinical signs compatible with leishmaniosis. Dog 27 was an intact Bullmastiff female aged 6 years that lived exclusively outdoors. On clinical history and physical examination, it was presented for anorexia, weight loss, vomiting, diarrhoea and generalized lymphadenomegaly. Although no external parasites were detected at the time of consultation, a previous infestation with ticks was reported by the owner. Dog 29 was a 4-year old intact mongrel female weighing 29 kg that also lived exclusively outdoors. On clinical history and physical examination, it had anorexia, weight loss, fever, skin lesions, bilateral conjunctivitis and generalized lymphadenomegaly and had a recent episode of tick infestation. Dog 27 had been imported from Portugal as a puppy; while dog 29 had never left Angola.Leishmania infection in the overall population of dogs from Angola might be higher.The prevalence of canine infection in the studied population was apparently low (i.e. 1.0% by PCR and 1.9% by serology), but the studied dogs were only client-owned animals and presumably well cared for. Due to this fact, the prevalence of Dog 29 was born in Angola, the same as its parents and co-habitants, which suggests an autochthonous infection. Dog 27 was imported from Portugal at approximately 3 months of age, and since then it had never left Angola; therefore, it is not clear whether the infection occurred in Angola or in Portugal, a country where CanL is endemic ,25. The Both dogs were clinically sick at the time of presentation and had clinical signs compatible with leishmaniosis. Although, according to the results of the present study, leishmaniosis is apparently not hyperendemic, it should be considered as a differential diagnosis in dogs with compatible clinical signs in Angola.L. infantum [Phlebotomus spp. of the Synphlebotomus group, were detected in southwestern Angola [L. infantum for humans, and canine infection with this parasite might also be regarded as a public health problem in Angola.Cases of human visceral leishmaniosis have been reported from Angola, some of them with a presumed autochthonous origin  and one infantum . In addin Angola . WorldwiLeishmania are needed to better characterize infection and disease in this country.Further investigation, including a larger number of dogs, canine populations from other cities and provinces of Angola, as well as studies in other vertebrate species, including humans, and in potential vectors of Leishmania infection in the country, as well as the first one to characterize L. infantum at the molecular level in dogs. Infection among client-owned dogs is apparently not high, but prevalence in the overall canine population of Angola and specific populations might be higher.Other cases of CanL were described in Angola in the 1960s and 70s, but this is the first population study on canine The authors declare that they have no competing interests.HV supervised the study, analysed data and wrote the manuscript; SG collected samples, performed DAT and assisted in drafting the manuscript; ACO co-supervised the study, performed clinical examination and collected samples; HDFHS provided DAT antigen and analysed data; YNB performed PCR and sequencing, and analysed data; LC performed DAT, analysed data and revised the manuscript; GB performed PCR and sequencing, analysed data and revised the manuscript. All authors read and approved the final version of the manuscript.