Although oxytocin (OT) has become a major target for the investigation of positive social processes, it can be assumed that it exerts its effects in concert with other neurotransmitters. One candidate for such an interaction is dopamine (DA). For both systems, genetic variants have been identified that influence the availability of the particular substance. A variant of the gene coding for the transmembrane protein CD38 (rs3796863), which is engaged in OT secretion, has been associated with OT plasma level. The common catechol-O-methyltransferase (COMT) val158met polymorphism is known to influence COMT activity and therefore the degradation of DA. The present study aimed to investigate OT × DA interactions in the context of an OT challenge study. Hence, we tested the influence of the above mentioned genetic variants and their interaction on the activation of different brain regions  during the presentation of social stimuli. In a pharmacological cross-over design 55 participants were investigated under OT and placebo (PLA) by means of fMRI. Brain imaging results revealed no significant effects for VTA or ventral striatum. Regarding the fusiform gyrus, we could not find any effects apart from those already described in Sauer et al. . Analyse In social neuroscience, the neuropeptide oxytocin (OT) has become a major target for the investigation of positive social processes  on the oxytocin receptor gene (OXTR) on the dopaminergic response to stress  is involved in the extraneuronal degradation of the transmitter and therefore influences its availability in the brain. A common variant on the gene coding for the enzyme, the val158met SNP (rs4860), is known to strongly influence the enzymatic activity of COMT . Only participants with no history of psychiatric or neurological diseases were included. All had at least 12 years of education, were non-smokers or smoked only occasionally and, except for one, all were right-handed. With respect to the CD38 SNP (rs3796863), 30 were homozygotic carriers of the C allele (CC), 23 were heterozygotes (CA) and 2 were homozygotic carriers of the A allele (AA). For subsequent statistical analyses, we pooled together AA and CA genotypes (A+) and compared them to the CC carriers (A−). Recent studies revealed the A− variant being associated with lower plasma OT levels and a higher risk for autism-spectrum-disorders compared to the A+ variant : Chi2 = 0.91, df = 1, n. s.).With respect to the COMT val158met SNP (rs4680), 11 subjects were homozygotic carriers of the met allele (met/met), 31 were heterozygotes  and 13 were homozygotic carriers of the val allele  which has been shown to lead to a higher COMT activity and lower DA levels in prefrontal cortex .Between the groups, there were no differences in age, education or order of substance application  and all participants gave written informed consent.The experimental design and procedures are described in detail elsewhere  with the following parameters for the functional MRI scans using EPI sequences: 30 axial slices à 4 mm, 1 mm gap, TR = 2 s, TE = 30 ms, FoV 192 × 192 mm, flip angle 80°. Anatomical data were obtained from a T1 weighted three dimensional MPRAGE sequence .http://www.fil.ion.ucl.ac.uk/spm). Preprocessing procedures included realignment to the first image, slice-time correction, spatial normalization into a standard stereotactic space with a voxel size of 2 × 2 × 2 mm using the Montreal Neurological Institute (MNI) template and smoothing with an 8 mm full width at half maximum (FWHM) Gaussian kernel.fMRI data were analyzed using SPM8  incorporating both substance conditions  as separate sessions was applied for each subject. For each session, five task regressors  and six motion parameters were included. On the first level we specified contrasts for both substance conditions separately  and for the comparison between substances (OT vs. PLA).Since we were interested in general aspects of social vs. non-social processing, we defined a contrast comparing all social stimuli to the control stimuli . We also defined contrasts comparing each social condition separately to the control condition . However, these contrasts did not reveal any additional aspects but the same results as the combined contrast. Therefore, we decided to only report results for the combined contrast since the single contrasts have a lower power to detect subtle gene effects and show only marginally significant results.Finally, we defined an interaction contrast by combining the task conditions with the substance conditions .http://fmri.wfubmc.edu). Masks for VTA and ventral striatum were created with the MARINA software tool  correction and an additional cluster size threshold of k = 10 contiguously activated voxels.On the second level, we performed multiple regression analyses for each first level contrast using the information of both polymorphisms independently and their interaction term as regressors. We also added substance order as a covariate of no interest to control for potential influences. To particularly address genetic effects on amygdala, VTA, ventral striatum and fusiform gyrus, we conducted ROI analyses. For the amygdala and the fusiform gyrus, we used anatomically defined Anatomical Automatic Labeling (AAL) masks provided by the Wake Forest University (WFU) PickAtlas software  for left and right amygdala separately. We then extracted mean parameter estimates from that voxels + 5 mm sphere for the PLA and OT conditions separately. Afterwards, we further analyzed these data using GLM repeated measures procedures implemented in IBM SPSS 20 . Models consisted of valence (positive vs. negative) and substance condition (OT vs. PLA) as within-subject factors, CD38 and COMT genotypes as between-subject factors and substance order as a covariate of no interest.For the analysis of behavioral effects, we analyzed the median of the response time (RT) for each condition. To control for baseline effects, we then computed differences between the social and the non-social conditions for both valences separately. These difference RTs were then incorporated into a GLM by means of IBM SPSS 20. Paralleling the analyses of amygdala activation, the GLM comprised valence and substance as within-subject factors, CD38 and COMT genotype as between-subject factors and substance order as covariate of no interest.DNA was extracted from buccal cells to avoid a selective exclusion of subjects with blood and injection phobia. Automated purification of genomic DNA was conducted by means of the MagNA Pure® LC system using a commercial extraction kit . Genotyping was performed by real time-polymerase chain reaction (RT-PCR) using fluorescence melting curve detection analysis by means of the Light Cycler System . The primers and hybridization probes used  were as follows:For CD38: forward primer: 5′-ACACTGAAGAAACTTGTCAGGTCTA-3′; reverse primer: 5′-CTTGGTTGCTGCTCCTACTGTT-3′; sensor hybridization probe: 5′-TTTGACCATCAGGTGGCA–FL -fluorescein-3′: anchor hybridization probe: 5′-LCRed640-GGATAGCTCCCCTCCCGACA-phosphate-3′.For COMT: forward primer: 5′-GGGCCTACTGTGGCTACTCA-3′; reverse primer: 5′-GGCCCTTTTTCCAGGTCTG-3′; anchor hybridization probe: 5′-LCRed640-TGTGCATGCCTGACCCGTTGTCA-phosphate-3′; sensor hybridization probe: 5′-ATTTCGCTGGCATGAAGGACAAG -fluorescein-3′.PCR runs comprised 55 cycles of denaturation , annealing  and extension  which followed an incubation period of 10 min to activate the FastStart Taq DNA Polymerase of the reaction mix . After amplification a melting curve was generated by holding the reaction time at 40°C for 2 min and then heating slowly to 95°C with a ramp rate of 0.2°C s-1. The fluorescence signal was plotted against temperature to yield the respective melting points (Tm) of the two alleles for both SNPs respectively. Tm for the CD38 C allele was 54.2°C and 61.5°C for the A allele. Tm for the COMT val allele was 59.00°C and 64.50°C for the met allele.x = −22, y = −5, z = −20): T(53) = 12.68, pFWE < 0.001; right amygdala: cluster-size = 202 voxels, peak voxel : T(53) = 17.57, pFWE < 0.001).As already reported in Sauer et al. , the comROI analyses of VTA and ventral striatum revealed no significant results, neither for the single SNPs, nor for their interaction. Regarding fusiform gyrus activation, we found no significant results in addition to those already reported in Sauer et al. .x = −18, y = −1, z = −12): T(50) = 3.54, pFWE(ROI) = 0.01; right amygdala: cluster-size = 21 voxels, peak voxel : T(50) = 3.25, pFWE(ROI) < 0.05). This interaction is exclusively driven by a significant gene × gene interaction occurring under PLA : T(50) = 3.8, pFWE(ROI) < 0.01; right amygdala: cluster-size = 23 voxels, peak voxel : T(50) = 3.68, pFWE(ROI) < 0.01).Analyses of amygdala activation revealed no gene main effect, neither for CD38 nor for COMT. Furthermore, there was no substance main effect and no gene × substance interaction for any variant. However, we found a significant gene × gene × substance interaction Figure  in the bF(2/48) = 5.42, p < 0.01; right amygdala: F(2/48) = 4.53, p < 0.02). As displayed in Figure Our analyses on the extracted data from the peak voxel sphere replicated the three-way interaction between substance and the two genotype groups on both sides  = 14.02, p < 0.001). As can be seen from Figure F(1/48) = 19.59, p < 0.001; right amygdala: F(1/48) = 5.31, p < 0.05, see Figure In addition, the GLM revealed a significant substance effect for the left amygdala  with reduced RTs during negative when compared to positive stimuli. In addition we found a significant substance × CD38 interaction  reflecting reduced RTs under OT specifically in the A− group. This effect was already reported in our previous publication (Sauer et al., Analysis of the RTs revealed a significant valence main effect [In the present study, we used an exploratory approach to test for OT and DA associated gene effects and gene × gene interactions on brain response to social stimuli as well as the modulation of this response by OT. First of all, as already reported in Sauer et al. , presentRegarding our main focus on the four different brain regions, we found neither effects of the single genotypes nor interaction effects on the VTA or the ventral striatum. At first glance, this is in contrast to the reported animal literature which showed strong evidence for an OT-DA interaction in these mesolimbic structures. However, there are aspects which could be responsible for our negative findings. First, most of the animal research focuses on OT-DA interactions in the context of sexual behavior which probably leads to a stronger activation of the endogenous OT and DA systems than our fMRI task. One could hypothesize that interaction effects in mesolimbic structures like the VTA or the ventral striatum can only be seen in a condition of sexual arousal. Second, the fMRI task we used neither robustly activates the VTA nor the ventral striatum which is a clear drawback for the identification of subtle gene effect. In a future study one should test this hypothesis using an appropriate fMRI task to address these mesolimbic structures.No additional effect could be found for the fusiform gyrus, either. Therefore, the effect of CD38 genotype on fusiform activation (Sauer et al., Regarding amygdala activation, we found no single gene effects but strong evidence for an epistasis effect of both genes reflecting an interaction between the OT and the DA system in the response to socially relevant stimuli. The effect of central OT secretion, as indexed by CD38 rs3796863 genotype, was strongly influenced by the COMT rs4860 genotype Figures , 3. WhilOur results could also be explained in the context of the warrior/worrier model (Goldman et al., However, this epistasis effect was exclusively present for the PLA condition while the application of intranasal OT completely diminished the observed interaction Figure . It coulIn general, when interpreting the results, we have to take into account that the direct biological pathways underlying are not clear. CD38 is not specific for the OT system and the effect could theoretically be due to other effects of CD38 like its impact on insulin secretion (Kim et al., post-hoc according to subjects' genotype. Therefore, replication of our results is definitely needed. Nevertheless, our results are an interesting starting point for future research leading toward a better understanding of OT-DA interactions in humans. For example, it would be interesting to see how this epistasis effect reacts to other intentional changes of the OT-DA system, e.g., inhibition of DA signaling via neuroleptic drugs.One major shortcoming of our study is the low number of subjects in the extreme groups. This is mainly due to the fact, that group assignment was performed Taken together, to our knowledge this is one of the first studies demonstrating an OT × DA interaction in humans. While such interactions have been shown in animal models, mainly in the context of sexual and pair bonding behavior and in regions of the mesolimbic DA system (Baskerville and Douglas, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study is to expand existing knowledge about the CRC-associated microbiome among Han Chinese, and to further discover the variation pattern of the human CRC microbiome across all population.Roseburia (P = 0.017), and a concurrent decrease of both Microbacterium (P = 0.009) and Anoxybacillus (P = 0.009) in tumor tissue.Using pyrosequencing-based molecular monitoring of bacterial 16S rRNA gene from eight tumor/normal tissue pairs of eight Chinese CRC patients, we analyzed and characterized the basic features of the CRC-associated microbiome. Firstly, we discovered an increasing diversity among tumor-associated bacterial communities. Secondly, in 50% of Chinese CRC patients, we found a significant increase of Roseburia bacteria at tumor sites and the over-represented Microbacterium and Anoxybacillus bacteria away from tumor sites were both closely related in Chinese CRC patients. Across several populations reported in this study and previously, we observed both common and distinctive patterns of human CRC microbiome’s association with a high-risk of CRC.We discovered a novel CRC microbiome pattern in Chinese. Both the over-represented  Fusobacterium spp., Bacteroides fragilis, and Escherichia coli) are related to a high-risk of CRC across several different populations. These findings prompted us ask whether such associations between CRC and microbiome patterns are common or population-specific. Such a finding to this question is essential in developing personalized medicine strategies and treatment options for CRC patients.Bacterial infections play a potentially significant role in the pathogenesis of colorectal cancer (CRC) -5, thougThe aim of this study was to quantitatively evaluate the differences of bacterial communities and compositions between eight tumor/normal pairs from eight Chinese CRC patients, as well as to characterize both the common and various patterns of the human CRC microbiome among different populations, ultimately to contribute towards a preliminary understanding of the bacterial driving forces at play in CRC.Eight Chinese CRC patients from Kunming, China were used in this study with (56.9±14.4) (SD) average age, (22.97±1.56) body mass index (BMI), and (1:1) male/female ratio. Four patients were diagnosed as having rectal cancers while the other half had colon cancers . From the eight patients, we obtained a total 16 tissue samples, including eight cancerous tissues and their matched adjacent normal tissues obtained via colonoscopy following the Standard Operating Procedures at the First People’s Hospital of Yunnan Province of China, Kunming. Participants were enrolled in the study prior to colonoscopy, and written informed consent was obtained from the patient for publication of this report and any accompanying images. Samples were removed endoscopically and immediately transferred from the colonoscopy room to the pathology suite and subsequently evaluated by the pathologist. Where possible one fragment of healthy tissue and one of tumor were chosen and placed in a cryotube, then frozen immediately in liquid nitrogen. All specimens were stored in their original tubes at -80°C prior to DNA extraction. This study conformed to the ethical guidelines outlined in the 1975 Declaration of Helsinki as reflected by a priori approval from the Medical Ethics Board of the First People's Hospital of Yunnan Province of China.® DNA Mini Kit . DNA from tissue was eluted in a final volume of 200 ul elution buffer and stored at -20°C. Tubes containing only QIAamp® DNA Mini Kit extraction controls were included throughout lysis and PCR to serve as negative controls.While frozen, an aliquot (~25 mg) of each specimen was suspended in a solution containing 200 ul buffer ATL (QIAGEN Kit Buffer for tissue lysis) and 200 ul of a slurry of 0.1-mm-diameter zirconia/silica beads . The mixed sample was then lysed by mechanical disruption with a bead beater (BioSpec Products), set on high for 2 min (20°C), followed by extraction with the QIAamp454 Life Sciences primer B sequence (5′ - CTATGCGCCTTGCCAGCCCGCTCAG -3′) and the broadly conserved bacterial primer 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′). The reverse primer included the 454 Life Sciences primer A sequence (5′-CGTATCGCCTCCCTCGCGCCATCAG- 3′), a unique 10-nt barcode used to tag each PCR product, and the broad-range bacterial primer 338R (5′- TGCTGCCTCCCGTAGGAGT-3′). Using the primer pair described above, triplicate PCR reactions were performed on each sample. Each resulting 25 ul reaction contained 0.2 μM forward and reverse primers, 3 μl template DNA, and 2.5 ul 10X PCR buffer plus Mg2+ (TaKaRa), 2.0 ul dNTP (2.5 mM each) (TaKaRa), 0.75 ul DMSO (100%), 0.25 ul TaKaRa Taq™ (5 U/μl). Thermal cycling was conducted at 95°C for 10 minutes, followed by 30 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 90 seconds, with a final extension of 10 minutes at 72°C. Replicate amplicons were pooled and visualized on 1.5% agarose gels using EB stain in 0.5X TE. Amplicons were cleaned using MinElute® Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen). Using the amplicon pool, we carried out pyrosequencing using primer A and Titanium chemistry on a 454 Life Sciences Genome Sequencer FLX instrument (Roche) at the DNA Sequencing Facility of the Kunming Institute of Zoology, Chinese Academy of Sciences.The forward primer included the Sequences were processed and analyzed using Qiime 1.4 . SequencP < 0.05.General characteristics were expressed as median and mean or percentages. Comparisons were performed between tumor and normal samples using the Mann-Whitney rank sum test or t-test in SigmaPlot 12.0 . Statistical significance was set at Unifrac matrix  (n = 16) sequences per sample, after filtering raw data with our set of criteria (Methods). From the dataset, we identified a total of 410 OTUs, based on the conventional criterion of 97% sequence similarity , with an average of 138.9 ± 46.2 OTUs per sample (n = 16). Compared with normal results, the diversity index by both species richness (OTU number) and evenness (phylogeny-based Chao1) suggested an increasing trend of microbial diversity in tumors . The significant difference of bacterial diversity was further confirmed by distinctive structural segregations of all 16 samples using PCoA analysis based on phylogeny-based Roseburia, Microbacterium, and Anoxybacillus)  lower abundance in tumor than in normal tissue   [Roseburia bacteria at tumor sites should in fact be considered as ‘passenger bacteria’ for CRC  and the over-represented Microbacterium and Anoxybacillus bacteria away from tumor sites—i.e., adjacent non-malignant tissue—as ‘driver bacteria’ for CRC .Our observations over the course of the study suggest that we have discovered a novel pattern to the human CRC microbiome among Han Chinese. According to the bacterial driver-passenger model for CRC put forward by Tjalsma l (2012) , among 5Fusobacterium spp. in tumors among 87.5% of patients, though we did not discover a significant increase. Across several of the different, previously studied populations including Europeans, Americans, and Asians , a similar pattern was observed; such a consistent overrepresentation of Fusobacterium spp. in tumor tissue suggests that there exists a common mechanism of gut microbial disorder connected with CRC. This finding implies that it is crucial to begin defining an underlying association of the gut passenger bacteria Fusobacterium with an increasing risk of CRC for most populations, despite the lack of relative reports for many different populations to date.Consistent with several previous reports ,4,11-13,Roseburia, is potentially associated with an increased risk of CRC, due to the overrepresentation of Roseburia in tumor tissue among Chinese, as we noted in this study, and as previously reported among Dutch [et al study on another Chinese population [Roseburia and its potential association with CRC may be due to different sample types, as another study done by Chen et al indicated a different microbial structure between the intestinal lumen and cancerous tissue in Chinese CRC patients. The microbial structure difference between the intestinal lumen and mucosa tissue in healthy subjects was further confirmed by Eckburg et al[Roseburia for CRC among Chinese, as we observed consistent overrepresentation of Roseburia in tumor tissues between the Chinese and Dutch, two populations with higher divergence of diet and genetics than within the Chinese population. Accordingly, the potential role of candidate gut passenger bacteria Roseburia should be emphasized more heavily during the occurrence of CRC, regardless of the population (e.g. Chinese or Dutch) and further investigated.We also found another gut bacterium, ng Dutch . This fipulation  that comurg et al. AdditioFusobacterium and Roseburia described above, the ‘passenger bacteria’ role of Bacteroides for CRC was supported by studies on the Chinese [Bacteroides was likewise confirmed by two other studies that examined a similar pattern among the Spanish, American and Vietnamese [et al confirmed that gut bacteria Bacteroides fragilis enables the promotion of colon tumorigenesis [in vivo experiments of mice model, E. coli-induced colitis is a driving factor of colorectal cancer [Escherichia species for CRC among the Chinese was also implied by the study done by Wang et al.. Moreover, besides the common pattern of the human CRC microbiome represented by gut passenger bacteria Fusobacterium within and between populations, there exists a diversified pattern in the human CRC microbiome due to three possible factors. One may be due largely to high variation of the normal human gut microbiome [Similar to the relationship between CRC and gut bacteria  Chinese , Dutch [ Chinese , and Fre Chinese . And theetnamese ,4. Furthigenesis . Similarl cancer , and thecrobiome , potenticrobiome ,18, age crobiome , sample crobiome , host gecrobiome , or othecrobiome . Anothercrobiome . The lascrobiome ,4. FurthIn summary, in the present study we presented some initial findings the lead towards a deeper and more comprehensive view of the human CRC microbiome. The existing findings are suggestive of further research, and underscore the necessity of borrowing from both high-throughput meta-genomic or transcriptomic data and  model experiments that will better define and validate the association of high-risk microbial populations with occurrence of CRCs across different populations.CRC: Colorectal cancer; OTUs: Operational taxonomic units; rRNA: Ribosomal RNA.The authors declare that they have no competing interests.JG performed research, analyzed data, and wrote the manuscript; HZ, XT, and HF performed research; ZZ conceived the study, performed research, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.JG, HF, XT, HZ: Department of Gastroenterology, The First People’s Hospital of Yunnan Province, Kunming, China. ZZ: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.


Mortality in female patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary angioplasty (pPCI) is higher than in men. We examined gender differences in the prevalence and prognostic performance of renal dysfunction at admission in this setting.A multicenter retrospective sub-analysis of the Belgian STEMI-registry identified 1,638 patients  treated with pPCI in 8 tertiary care hospitals (January 2007-February 2011). The estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI equation. Main outcome measure was in-hospital mortality.More women than men suffered from renal dysfunction at admission . Mortality in women was doubled as compared to men  = 2.12 (1.36-3.32), p<0.001). In-hospital mortality for men and women with vs. without renal dysfunction was much higher . In a multivariable regression analysis, adjusting for age, gender, peripheral artery disease (PAD), coronary artery disease (CAD), hypertension, diabetes and low body weight (<67 kg), female gender was associated with renal dysfunction at admission (OR (95% CI) 1.65 (1.20-2.25), p = 0.002). In a multivariable model including TIMI risk score and renal dysfunction, renal dysfunction was an independent predictor of in-hospital mortality in both men (OR (95% CI) = 2.39 (1.27-4.51), p = 0.007) and women (OR (95% CI) = 4.03 (1.26-12.92), p = 0.02), with a comparable impact for men and women (p for interaction = 0.69).Female gender was independently associated with renal dysfunction at admission in pPCI treated patients. Renal dysfunction was equally associated with higher in-hospital mortality in both men and women. It has been demonstrated that women with STEMI undergoing primary PCI (pPCI) have higher odds for in-hospital mortality than men. Some authors demonstrated that this difference is likely explained by their older age and baseline comorbidities  -5, whileData on gender differences in prevalence of renal dysfunction at admission in pPCI treated STEMI patients are scarce and have seldom been accounted for when evaluating gender differences in outcome, furthermore different definitions (serum creatinine levels vs. estimated Glomerular Filtration Rate (eGFR) values) and different methods of estimating creatinine clearance have been applied ,4,8,13.2 [x 1.159 if black], where Scr is serum creatinine, k is 0.7 for females and 0.9 for males, a is -0.329 for females and -0.411 for males. The eGFR could not be calculated in 11.9% of the cases, due to missing values. We found no differences in proportions of women or baseline characteristics between patients with and without missing values, except for the incidence of Cardio Pulmonary Resuscitation (CPR)  and in-hospital mortality .eGFR = 141 x min The primary endpoint existed of in-hospital mortality.t test was used for continuous variables with a normal distribution (presented as the mean ± SD), and the Mann Whitney-U test was used for continuous variables (presented as the median and interquartile range (IQR)) without a normal distribution. Multivariable logistic-regression analysis was used to determine the independent predictors of renal dysfunction at admission including following covariates in the model: gender, age, bodyweight <67 kg, history of coronary artery disease (CAD), history of peripheral artery disease (PAD), hypertension and diabetes. Separate logistic regression analyses were performed regarding in-hospital mortality for each gender, including TIMI risk score as a continuous and renal dysfunction as a dichotomous variable in the model. Multivariable logistic regression analysis was also used to assess a possible interaction between gender and renal dysfunction regarding in-hospital mortality adding renal dysfunction and gender as dichotomous variables and the product of gender and eGFR < 60 mL/min/1.73 m2 as an interaction term to the previous model. All multivariable analyses were based on complete patient records.We compared the baseline characteristics, including renal dysfunction at admission, and in-hospital outcomes of women with those of men. Distributions of categorical variables were compared by using the Fisher exact test. Continuous variables were evaluated for normality. The Student's Adjusted Odds Ratios (OR) with 95% confidence intervals (CI) are reported. Statistical significance was defined as p < 0.05 or 95% confidence intervals (CI) for OR that did not include 1.0. All statistical analyses were performed using the SPSS 19 statistical software.This study was approved by the central ethical committee of the Ghent University Hospital (2011/455). Informed consent was obtained from all patients or their legal representatives.During the period between January 2007 and February 2011, 1,638 patients were treated with primary PCI in eight participating centers. Of them 338 (20.6%) were female and 1,300  were male. Baseline patient characteristics are shown in Table 2 or a Chronic Kidney Disease (CKD) stage 3, 4 or 5 was more frequently observed in female patients  . Renal dysfunction, defined as an eGFR below 60 mL/min per 1.73 mThe in-hospital mortality rate for the entire cohort of pPCI treated patients was 5.7%. Mortality in women was doubled as compared to men (9.5 vs. 4.7%), OR = 2.12 . Mortality in patients with renal dysfunction at the time of hospital admission was much higher compared to those without . In-hospital mortality was comparable in men and women without renal impairment: 2.3% vs. 2.4%, while there was a non-significant trend for higher mortality in women with renal dysfunction compared to men with renal dysfunction   and renal dysfunction  were strong predictors of in-hospital mortality; there was a trend towards higher mortality in women .Adding renal dysfunction to the TIMI risk score in men and women separately in a multivariable logistic regression model demonstrated that renal dysfunction was an independent predictor of in-hospital mortality in both men (OR (95% CI) = 2.39 (1.27-4.51), p = 0.007) and women (OR (95% CI) = 4.03 (1.26-12.92), p = 0.02), the interaction test demonstrated that this impact was comparable for men and women (p = 0.69).2 or CKD class 3 or higher), assessed by the CKD-EPI formula, was a common finding and that more women (42.3%) than men (25.3%) suffered from this condition. As expected, a CKD class 3 or higher on admission was associated with in-hospital mortality, and this independently of the TIMI risk score. Although there was a trend towards higher mortality for women with renal dysfunction compared to men with this condition, we could not demonstrate a gender difference in the impact of renal dysfunction on in-hospital mortality.In a sub-analysis of the Belgian STEMI registry, including 20,3% of Belgian STEMI patients undergoing pPCI for STEMI, we found that renal dysfunction at the time of hospital admission  acute kidney injury (AKI) and bleeding are the most likely to occur . By implToday it is not clear whether the prognostic performance of the currently most used simple and bedside risk scores could improve by adding eGFR to the model. Kidney function, represented by various cut-offs for serum creatinine but not by eGFR, is already incorporated in the GRACE risk score . Kidney 2, the use of the CKD-EPI equation was appropriate.This study is unique as it represents the first dataset that links gender, renal dysfunction, assessed by the CKD-EPI equation, and outcomes in a subgroup of PCI-treated STEMI patients included in the Belgian STEMI registry. As 71.2% of this Belgian STEMI cohort presented with an eGFR > 60 mL/min/1.73 mThe study has some limitations. First, we only studied patients who underwent pPCI in a subgroup of 8 tertiary care centers that participated in the Belgian STEMI registry; since it has been demonstrated that patients with renal failure have less access to invasive therapy, a selection bias is not excluded . Second,2 was common in a subgroup of patients undergoing primary PCI for STEMI and female gender was independently associated with this condition. Renal dysfunction, independent of the TIMI risk score, was associated with higher in-hospital mortality rates in both men and women. There was no gender difference in the prognostic impact of renal dysfunction regarding in-hospital mortality. Glomerular filtration rates should routinely be assessed at baseline in these patients and these and not serum creatinine values should be accounted for when evaluating gender differences in outcome after STEMI. Whether eGFR, assessed by the CKD-EPI equation, could improve the prognostic performance of currently used bedside risk stratification models remains to be elucidated.Renal dysfunction, defined as an eGFR < 60 mL/kg per 1.73 mThe authors declare that there are no disclosures related to the content of this article.SG designed the study. Data were collected by all authors but DDB. Data-analysis was performed by SG and DDB. The article was written by SG, MC and DDB and critically reviewed by all other authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/62/prepub


The overall mean ± SD age was 12.0 ± 7.6 months, 38.5% (no difference between case and controls). The mean ± SD WHZ of cases and controls was −3.24 ± 1.01 versus −0.74 ± 0.95 (P < 0.001), respectively. Logistic regression analysis revealed that children with acute malnutrition were more likely than controls to be older (age > 1 year) (adjusted OR (AOR): 3.1, P = 0.004); have an undernourished mother (body mass index < 18.5), ; have a father with no or a low-paying job ; come from a family having a monthly income of <10,000 taka, (1 US$ = 80 taka) ; and often have stopped predominant breastfeeding before 4 months of age . Improved understanding of these characteristics enables the design and targeting of preventive-intervention programs of childhood acute malnutrition.To assess the risk factors for acute malnutrition (weight-for-height  Despite recent advances in prevention and management of childhood malnutrition in Bangladesh, 16% of children under 5 years of age are acutely malnourished (WHZ < −2) [One of every five children aged less than 5 years in low-income, developing countries is malnourished. Globally, undernutrition is associated with more than one-third of all deaths in this age group . Acute mHZ < −2) . ChildhoIt is well documented that poverty and malnutrition, regardless of location, are highly intertwined. Although risk factors for malnutrition have been identified, individual factors potentially change in specific areas over time and a current characterisation of risk factors provides the basis for preventative intervention programmes.The study was conducted in the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research  situated in Dhaka, Bangladesh, a metropolitan area  with a total population of ~15 million. Each year, the Dhaka Hospital provides care and treatment for over 120,000 patients with diarrhoea, with or without other associated health problems. The hospital also conducts research on enteric and other common infectious diseases as well as undernutrition and provides training on case management of diarrheal diseases, management of malnutrition, and research methodology. Under-five children constitute about 60% of the total patient population and most (~60%) are from poor socioeconomic communities in urban and periurban areas of Dhaka.z-score (WHZ), weight-for-age z-score (WAZ), height-for-age z-score (HAZ), and body mass index-for-age z-score (BMI-AZ) of all studied children were calculated. Children with moderate or severe wasting (WHZ < −2) but without bilateral pedal edema were considered cases. Children with a WHZ ≥ −2 and without bilateral pedal edema were enrolled as control children. Verbal consent from the attending guardian, usually the mother of the child, was obtained.A nonmatched case-control study design was used to assess and identify potential risk factors associated with acute malnutrition/wasting among 6–59-month-old children. Children in this age group without any congenital anomaly or other chronic conditions causally associated with malnutrition , who were admitted to the Dhaka Hospital of icddr,b from June to September 2012, were enrolled. Using the contemporary growth standards of the World Health Organization (2006) and ANTHRO software , weight-One of the investigators and/or a research assistant interviewed the mother/caregiver using a pretested, structured questionnaire. Data recorded from the interviews included age and sex of child, birth order, number of total and under-five siblings, feeding and immunisation history, type of house hold latrine, marital status of mother, monthly family income, and parental age, education, and occupation. Children's nude weight using a frequently standardised digital scale with 10 g precision  and recumbent length to the nearest mm using a calibrated, locally constructed length board were obtained. Mother's (if present) weight and height were measured using standard procedures [t-tests. For continuous variables not normally distributed, the Mann-Whitney U test was performed. Differences in proportions were compared by the chi-square test or Fisher's exact test if the expected number in any cell was ≤5. A probability of less than 0.05 was considered statistically significant. The strength of association of selected associated/risk factors for acute malnutrition was determined by estimating odds ratios (ORs) and their 95% confidence intervals (CIs). All independent variables, for example, birth order, number of siblings, socioeconomic status, parental characteristics, child feeding, and immunisation history, were analysed initially in univariate models and the attributes that were significantly associated with wasting (dependent variable) and biologically plausible were included in logistic regression models.Data were entered using SPSS software for Windows (version 11.5) . For normally distributed continuous variables, means were compared using unpaired P < 0.001). As dictated by the study design, the cases had significantly lower anthropometric values than the control children (P = 0.004), have an undernourished mother (body mass index (BMI) < 18.5), , have a father with no or a low-paying job , come from a family having a monthly income of less than 10,000 taka, (1 US$ = 80 taka) , and have shorter period of predominant breastfeeding   and 271 were controls. Their overall mean ± SD age was 12.0 ± 7.6 months (cases were on an average 2 months older), and 38.5% were female without any group difference between case and control children. The mean ± SD WHZ and BMIAZ of case and control children were −3.24 ± 1.01 versus −0.74 ± 0.95 and −3.21 ± 1.04 versus −0.77 ± 0.96, respectively (children . Cases hchildren . Logisti= 0.013) .The aim of this study was to identify risk factors associated with acute malnutrition  in our population of 6–59-month-old children. Our study shows that the major associated/risk factors for acute malnutrition among these children were older age of the child, undernourished mother, jobless father or father with a low-paying job, low total family income, and poorer breastfeeding practices. Some of these factors may operate in synergy to increase the risk of acute malnutrition.Older age as a risk/associated factor for acute malnutrition of children in our study might reflect a selection bias. However, Jeyaseelan and Lakshman from Tamil Nadu, India , and KikSimilar to other studies from Bangladesh –11 and AThe fathers of most 84%) of the wasted children in our study were rickshaw pullers or day laborers. Likewise, a study from South India  also fou4% of theOur finding of improper/inadequate breastfeeding as an associated factor with acute malnutrition is in accordance with the findings of several other studies , 19, 20 One of the possible limitations of the present study is that the same personnel who obtained the anthropometric measurements of the children and mothers also conducted the interviews, so interviewer biases could be there. However, most of the variables identified as risk/associated factors for acute malnutrition in our study were objective in type. The other limitation was the cross-sectional nature of the present study, which did not allow us to state the identified associated factors as definite causally related risk factors.The children suffering from acute malnutrition often need supplementary food. In this regard it is worthwhile to mention that icddr,b has developed ready-to-use foods using locally available food ingredients. These foods can be used to prevent and to treat moderate wasting in children living in food-insecure communities. Moreover, the associated factors identified for acute malnutrition in this study can be incorporated into the design and targeting of preventive interventions. Factors such as breastfeeding practices are potentially modifiable. Interventions that motivate behaviours more consistent with recommended infant and young child feeding practices would be expected to have a positive impact. Certain factors and possible causes of acute malnutrition are complex and involve societal and broad-based preventive programs.


Stevia rebaudiana Bertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently, Stevia extracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies on Stevia and steviol glycosides in vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercial Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt. Furthermore, Stevia extracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis that Stevia extracts could mimic insulin effects modulating PI3K/Akt pathway.Extracts from  Stevia rebaudiana Bertoni is a weak perennial shrub belonging to Asteraceae (Compositae) family, native to subtropical regions of Brazil and Paraguay. Its leaves have been used as a sweetener since ancient times and for many other medicinal purposes in Latin America and the Orient for centuries [enturies , 2. The enturies , 4.Glycemic control is fundamental to the management of diabetes since it is associated with significantly decreased rates of retinopathy, nephropathy, neuropathy, and cardiovascular disease, the most common cause of death in diabetic patients. The effort to achieve near-normoglycemia through the key strategy of glycemic control includes recommendations for prevention and control of diabetes, for example, monitoring carbohydrate intake and limiting the consumption of sugar-sweetened beverages .Stevia leaves and extracts are natural noncaloric sweeteners that can substitute sucrose. The main sweet components in leaves, approximately 200–400 times sweeter than sucrose as shown by organoleptic tests [ic tests , 6, are ic tests   approved the use of steviol glycosides as food additive [in vitro and in vivo animal studies and some human tolerance studies), steviol glycosides are considered not carcinogenic, genotoxic, or associated with any reproductive/developmental toxicity. Joint Expert Committee on Food Additives (JECFA) established an accepted daily intake (ADI) for steviol glycosides  of 4 mg/kg bw/day [Steviol glycosides have been recently authorised as commercial sweeteners. US Food and Drug Administration (FDA) has allowed the use of additive , 9. Consg bw/day , 11.Stevia has also been used to help control weight in obese subjects [in vivo animal studies and, at a lesser extent, in humans. Results suggest that stevioside and related compounds affect plasma glucose modulating insulin secretion and sensitivity, which increase glucose removal from the plasma [Stevia and steviol glycosides, very little is reported concerning the cellular and molecular mechanisms underpinning these effects.Besides sweetness, steviol glycosides, in particular stevioside, have been shown to possess beneficial effects on human health , 12, 13.subjects ; moreovesubjects , 16. Stee plasma , 18. In e plasma , 20. Thee plasma , 22. Des+-coupled glucose transporters (SGLT) and the facilitative glucose transporters (GLUT). The human GLUT family is integral membrane proteins widely distributed in probably all mammalian cells that regulate the movement of glucose between extracellular and intracellular compartments maintaining a constant supply of glucose available for metabolism [In the present study, we examined the role of steviol glycosides on cellular glucose transport in cultured cells. Glucose is a polar molecule and requires specific carrier proteins, located in the plasma membrane, to cross the lipid bilayer and enter the cell. Glucose is transported into the cells through two different types of membrane associated carrier proteins, the Natabolism . To datetabolism , 24. GLUtabolism , 26, whetabolism  and, fortabolism . We havetabolism , 30. Mortabolism , 32.Stevia extracts, in order to evaluate a possible effect of these compounds on glucose transport and to clarify the molecular mechanism of action. Starting from this knowledge and this background, we chose the neuroblastoma SH-SY5Y and the promyelocytic leukaemia line HL-60, both expressing insulin and insulin-like growth factor-1 (IGF-I) receptors , 33 to t2DCFDA, dichlorofluorescin diacetate), 2-deoxy-glucose (DOG), phloretin, CelLytic M, mammalian protease inhibitor mixture, primary antibody to β-actin, methylglyoxal (MG), hydrogen peroxide, bovine serum albumin (BSA), rebaudioside A standard (StReb), stevioside standard (StStev), and all other chemicals of the highest analytical grade were purchased from Sigma-Aldrich. Roswell Park Memorial Institute (RPMI) 1640 medium  was purchased from PAA. 2-Deoxy-D-[Dulbecco's modified Eagle's medium (DMEM), foetal calf serum (FCS), penicillin/streptomycin, 3--2, 5-diphenyltetrazolium bromide (MTT), staurosporine, 2′,7′-dichlorodihydrofluorescein diacetate (HStevia rebaudiana Bertoni were kindly supplied by Eridania Sadam SpA.Extracts from Stevia extracts has to be at least 95% (w/w), and rebaudioside A plus stevioside must be at least 75%. The four extracts tested differ by the relative content of rebaudioside A and stevioside. In particular, according to the certificates of analysis of each sweetener, Reb A (R97) contains 97-98% rebaudioside A, Stevia RA60 (R60) contains about 60% rebaudioside A and about 20% stevioside; Steviol Glycosides SG95 (SG) contains 50% rebaudioside A and at least 25% stevioside; Truvia (TRU) contains a mixture of steviol glycosides not analytically quantified. According to FDA and EFSA –11, tota2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin, as reported in [μg/mL streptomycin, at 37°C in a humidified atmosphere maintained at 5% CO2.SH-SY5Y, human neuroblastoma cells, were grown at 37°C in a humidified incubator with 5% COorted in . HL-60, 2) to ensure complete lysis. The absorbance at 570 nm was measured using a multiwell plate reader .Cells were treated with different concentrations of steviol glycosides (0.5 to 5 mg/mL) or 1 mM (corresponding to 1 mg/mL) StReb or 1 mM (corresponding to 0.8 mg/mL) StStev for 24 h. Cell viability was evaluated by the MTT assay as reported in . SH-SY5YStevia extract for 24 h. Lactate dehydrogenase (LDH) release from cells was monitored by collecting aliquots of medium. LDH activity was assayed by a spectrophotometric method based on the reduction of pyruvate to lactic acid coupled to NADH oxidation. The decrease in absorbance at 340 nm was monitored at 37°C. 100 μM H2O2 for 30 minutes was used as a positive control.SH-SY5Y and HL-60 cells were incubated with 1 mg/mL of each 2, PerkinElmer); excitation and emission wavelengths were 360 nm and 460 nm, respectively.Caspase 3 activity in the cell lysates was measured using a colorimetric assay kit by following the instructions from the manufacturer (Sigma), as described in . Cells wμg/mL) was used as an apoptosis inducer (positive control).The concentration of the AMC released was calculated using an AMC standard curve. Caspase 3 activity was expressed in nmole of AMC released per min per mL of cell lysate and normalised for total protein content in the lysate. Results are reported as percentage with respect to the control. Staurosporine (1 2DCFDA). SH-SY5Y and HL-60 cells were incubated with 5 mg/mL of each Stevia extract for 1 h and then subjected or not to oxidative stress generated by 100 μM H2O2 for 30 minutes. Successively, cells were washed twice in PBS and incubated with 5 μM H2DCFDA for 20 min at 37°C. H2DCFDA is a small nonpolar, nonfluorescent molecule that diffuses into the cells, where it is enzymatically deacetylated by intracellular esterases to a polar nonfluorescent compound, that is oxidised to the highly green fluorescent 2′,7′-dichlorofluorescein (DCF). The fluorescence of oxidized probe was measured using a multiwell plate reader . Excitation wavelength was 485 nm and emission wavelength was 535 nm. Fluorescence values were reported as the percentage of intracellular ROS with respect to control.ROS intracellular level was evaluated by using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (HμCi/assay) and 1.0 mM unlabeled glucose analogue, under conditions where the uptake was linear at least for 20 min. The transport was stopped by adding phloretin , a potent inhibitor of glucose transport activity. Radioactivity was measured by liquid scintillation counting .Glucose transport assay was performed as described in , 38. Celμg/lane) were electrophoretically transferred to nitrocellulose membrane  in Tris-glycine buffer at 110 V for 90 min. Membranes were then incubated in blocking buffer containing 5% (w/v) albumin in Tris-buffered saline (TBS)/Tween to avoid nonspecific binding and incubated overnight at 4°C with primary antibodies . Nitrocellulose membranes were then washed 3 times with TBS/Tween and incubated with secondary antibodies in TBS/Tween containing 5% albumin for 60 min at room temperature and successively washed with TBS/Tween. The results were visualized by chemiluminescence using ECL Advance reagent according to the manufacturer's protocol . Images of the blots were obtained using a CCD imager . Bands were acquired and analysed by using Image Lab analysis software.After treatments, cells were washed with ice-cold PBS and lysed on ice using CelLytic M containing mammalian protease and phosphatase inhibitor mixture. The resulting lysed cells were left on ice to solubilize for 45 min. The lysates were centrifuged at 5000 g for 5 min at 4°C to remove unbroken cell debris and nuclei. Cell lysate protein concentration was determined by the Bio-Rad Bradford protein assay (Bio-Rad Laboratories). Samples were kept at 95°C for 5 min prior to separation on 10% SDS-PAGE Mini-Protean TGX precast gels using a Mini-Protean apparatus (Bio-Rad Laboratories). Proteins  . In braiIt is also recognised that cancer cells frequently overexpress the GLUT family members, due to the uncontrolled proliferation requiring elevated energy, and they often express GLUT isoforms not present in normal conditions. Moreover, large hypoxic areas into the tumour cause an increase in glucose utilization by cancer cells through glycolysis. The requirement for energy is satisfied by an augmented sugar intake, realised by an increase in GLUT expression and an increment in the translocation of the transporters to the plasma membrane . For theStevia extracts on cellular viability, assessed by MTT assay. SH-SY5Y and HL-60 cells were treated with different concentrations  of Stevia extracts for 24 h. Data reported in The first aim of our paper was to evaluate the effect of four different Cytotoxicity was also evaluated by lactate dehydrogenase (LDH) assay, which indicated that cell membrane integrity was not compromised and excluded cellular necrosis .Stevia extracts for 1 hour, 6 hours (data not shown), or for 24 hours . This lack of antioxidant activity is in contrast with the data reported by other authors [Stevia leaves, likely responsible for Stevia antioxidant activity.Since antioxidant properties of escribed , 16, thecent DCF . As show authors , 16, proStevia extracts are largely used as a noncaloric high-potency biosweetener substitute for sugar. The effect of four Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia cells, expressing principally GLUT1, the basal glucose transporter, and in SH-SY5Y human neuroblastoma cells, expressing also GLUT4, the insulin-sensitive one. Stevia extracts.Since the increase in glucose uptake obtained with standards is consistent with that shown by the whole extracts, we can hypothesize that this effect is due to rebaudioside A and stevioside, the two major components of Stevia extracts on glucose transport activity was compared to the effect of 100 nM insulin –dependent mechanism similar to the mechanism described in muscle or adipose tissues [Stevia extracts or insulin was assessed by Western blot analysis on cell lysates. Stevia extracts is similar to that obtained by insulin stimulation. These results are in accordance with those observed in the evaluation of glucose transport activity.It is well known that insulin induces the translocation of GLUT4 from cytosolic storage vesicles to the plasma membrane, enhancing glucose transport. Recently, this phenomenon was observed also for neural cells  tissues . Translo tissues ; doxorub tissues ; L-cyste tissues ; growth  tissues –49. The Stevia extracts enhance glucose transport, the phosphorylation status of PI3K and Akt was evaluated following Stevia extract treatment and insulin stimulation. Insulin activates the PI3K/Akt pathway, critical for neuronal survival and growth, synaptic plasticity and development, and learning [Stevia extracts, indicating a possible similar mechanism of action or, at least, a common signalling pathway.To clarify the molecular mechanism by which learning , 51. Indlearning , 53, prolearning , 55. Higlearning . Immunobin vivo. Advanced glycation end products (AGEs) have been implicated in development and progression of several diseases, including diabetes and its associated vascular complications, renal failure, cirrhosis, aging, and recently also in diabetic neuropathy and in Alzheimer's disease [To better characterize the mechanisms of glucose uptake induction by steviol glycosides, we used methylglyoxal (MG) as an inhibitor of the insulin receptor/PI3K/Akt pathway. MG is a reactive ketoaldehyde, product of many metabolic pathways, primarily glycolysis, which is considered the most relevant and reactive glycation agent  disease . Further disease .Stevia extracts or insulin is able to raise the glucose uptake, corroborating the hypothesis that Stevia extracts could act via PI3K/Akt pathway similar to insulin. As MG is a reactive aldehyde toxic to cells, MTT assay was performed to verify that the MG concentration (0.1 mM) used to inhibit glucose transport did not influence cell viability (data not shown).Stevia rebaudiana Bertoni leaves, on glucose transport activity in two different cell lines. We demonstrated, for the first time to our knowledge, that rebaudioside A and stevioside, the major glycosides in Stevia extracts, are able to enhance glucose uptake in both SH-SY5Y neuroblastoma and HL-60 myeloid leukaemia human cells, the raise being similar to that induced by insulin. Our data suggest that steviol glycosides act by modulating GLUT translocation through the PI3K/Akt pathway. Although further experiments are needed, these results support the hypothesis that steviol glycosides and insulin could share a similar mechanism in regulating glucose entry into cells. In conclusion, Stevia extracts, commercialised as zero-calorie natural sweeteners, are involved in insulin regulated glucose metabolism. These findings suggest that the use of Stevia extracts goes beyond their sweetening power and may also offer therapeutic benefits, supporting the use of botanicals dietary supplements to improve the quality of life.In this study, we evaluated the effects of steviol glycosides, extracted from


Jacaranda decurrens subsp. symmetrifoliolata Farias & Proença (Bignoniaceae) is a species traditionally used for the treatment of inflammatory and infectious diseases. Previous findings from our group reported scientifically that J. decurrens has anti-inflammatory efficacy. However, more toxicological studies are needed to support and ensure its safe use. The present study was carried out to evaluate the toxic effects of a prolonged treatment with hydroethanolic root extract of J. decurrens (EJD) on hematological, biochemical, and reproductive parameters in adult male rats. The animals received by oral gavage 0; 250; 500; or 1000 mg/kg body weight of EJD for 28 days. After the treatment, biochemical, hematological, histopathological, and reproductive parameters were analyzed. The EJD treatment did not cause adverse effects on body weight gain, feed and water consumption, hematological and biochemical profiles, or histopathological analysis of liver and kidney. Similarly, there were no statistically significant differences in reproductive parameters, such as sperm production, number of sperm in the epididymis, and sperm morphology. These results demonstrate the absence of subacute toxicity as a result of the oral treatment with EJD for 28 days in adult male rats. However, other studies should be performed to evaluate the total safety of this plant. In recent years, natural health products have been increasingly used worldwide, particularly herbal products . One reaJacaranda decurrens subsp. symmetrifoliolata Farias & Proença (Bignoniaceae), traditionally known as “carobinha-do-campo,” “carobinha,” or “caroba,” is an endemic species found in the Brazilian states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, and São Paulo [ão Paulo . Accordião Paulo . Jacaranda genus have antioxidants [J. decurrens extract has anti-inflammatory properties in adult rats [Pharmacological evaluations have reported that species from oxidants , antimicoxidants , and cheoxidants . Recent ult rats . J. decurrens can be found in literature. After performing acute toxicity tests, no signs of toxicity were observed at doses of 2000 or 5000 mg/kg of leaves [J. decurrens extract [J. decurrens roots administered to pregnant rats interfered with the onset of reproductive development in male offspring, anticipating the age of testicular descent and reducing absolute and relative weight of testis and epididymis of the animals at 60 days of age [Although studies have demonstrated their therapeutic effects, few toxicological studies conducted on f leaves  or 500,  extract  in mouses of age , which rJ. decurrens extract, the aim of this study is to evaluate the toxic effects of hydroethanolic root extract of J. decurrens in adult male rats through hematological, biochemical, and reproductive parameters.Due to the scarce information in the literature about the toxicological profile after subacute exposure and evidence of reproductive disturbances in male offspring after maternal exposure to  J. decurrens subsp. symmetrifoliolata roots (EJD) were collected (April 2010) in the Medicinal Plant Garden of the Federal University of Grande Dourados. A voucher specimen was identified by Dr. Rosana Farias Singer and deposited (register: W. G. Garcia 14.008) in the Herbarium of the Department of Botany at the Biology Institute of the State University of Campinas. J. decurrens were dried at a temperature of ±30°C. The dry roots (560 g) of J. decurrens were extracted with 3.5 L of ethanol : water (70 : 30 v : v) at room temperature. Each extraction was performed with 700 mL by seven days, amounting to 3500 mL. The procedure was performed in quintuplicate. Extracts were united, filtered, and concentrated under vacuum and lyophilizer. During the treatment, the extract was dissolved in a hydroethanolic solution. The roots of μL of methanol, filtered through a membrane with pores of 0.22 μm, and analyzed using high performance liquid chromatography (HPLC). The compounds were employed as standards quercetin, rutin, myricetin, myricetrin, luteolin, and quercetrin. The fractions were analyzed in an analytical LC (Varian 210) system with a ternary solvent delivery system equipped with an autosampler, a photodiode array detector (PDA) monitored at λ = 200–800 nm. The LC column was a C-18 , with a small precolumn (2.5 cm × 3 mm) containing the same packing, used to protect the analytical column. In each analysis the flow rate and the injected volume were 1.0 mL/min and 20 μL, respectively. All chromatographic analyses were performed at 25°C. Elution was carried out using the following solvent-gradient program : water : methanol  by 25 min, taking 15 min to reach 100% methanol, then returning in exactly 5 min to the initial conditions. The identification was made by comparison of retention times and UV spectra of peaks of the samples with the peak standards.The EJD (1.14 g) was dissolved in water (0.2 L) and fractionated by XAD-2  resin column chromatography (30 × 3 cm) eluted with water (0.3 L), followed by methanol (0.2 L) and with ethyl acetate (0.2 L). The water and ethyl acetate fractions were concentrated in employing nitrogen. The fractions obtained were dissolved with 5000 n = 32) from the Federal University of Mato Grosso do Sul were maintained under controlled temperature (23°C), with a constant 12 h light-dark cycle and free access to food and water. The experimental procedures were in accordance with the Ethical Principles in Animal Research and approved by the Committee for Ethics in Animal Experimentation at the University Center of Grande Dourados (Protocol number: 334/10). Adult male Wistar rats —Guideline 407 and ANVISA guidelines  , 13. AduThe animals were weighted daily and observed for clinical signs of systemic toxicity. Behavior parameters, death, their weight, and the amount of water and feed were analyzed . δ-GT, creatinine, and urea) [After treatment, the experimental group animals were weighed and anesthetized . Next, blood samples were collected from the renal vein, with and without anticoagulant . The blood samples were used to determine the hematology parameters , and the nonanticoagulated serum samples were used for biochemical analysis  , 14. TheThe reproductive organs  of the experimental group animals were removed and weighed (absolute and relative to body weight). For standardization, the right testis and epididymis were used for the germ cell counts, and the left organs were fixed for histopathological evaluation, using the same method described earlier for the vital organs. Homogenization-resistant testicular spermatids (stage 19 of spermiogenesis) and sperm in the caput/corpus and cauda epididymis were enumerated as described previously by Robb et al. , with adFor evaluation of sperm morphology, the interior of the left vas deferens of mature rats was washed, by the aid of a syringe and needle, with 1.5 mL were prepared of saline solution, after which histological slides. Two hundred spermatozoa (heads only or intact sperm) per animal were evaluated for head and/or flagellar defects by phase-contrast microscopy  in wet preparations .P < 0.05. The statistical analyses were performed by GraphPad InStat (version 3.02). For comparison of results among the experimental groups, statistical tests for analysis of variance were utilized—ANOVA, with the “a posteriori” Tukey-Kramer test. Differences were considered significant when J. decurrens roots in adult male rats. Toxicological evaluations after repeated exposures are required by regulatory agencies to characterize the toxicological profile of any substance , 13. HowJ. decurrens roots did not exhibit clinical signals of toxicity at the doses tested. The subacute oral toxicity study also showed no significant changes in the amount of water and feed between the experimental groups (data not shown). Additionally, the absolute and relative weight of the vital organs   . Male rand lung)  showed nThe hematopoietic system is one of the targets more susceptible to toxic substances and is an important parameter for assessing the physiological and pathological status in humans and animals . After tδ-GT) [δ-GT, creatinine, and urea) in the present study did not differ significantly in the experimental groups  , 19 and δ-GT) . The biol groups . Similarl groups  or regarReproductive toxicity studies must be regarded as part of an herbal safety evaluation process which supplements toxicological systemic tests . TherefoIn this study, no significant differences were observed in absolute and relative weight of the most reproductive organs  of animals exposed to EJD . HoweverJ. decurrens extract [Previous findings from our group indicated that the oleanolic and ursolic acids (triterpenoids) were isolated in the methanolic fraction of the EJD roots . In othe extract . In the Eugenia jambolana, was shown to have antifertility effects in male rats [α-reductase and to have antimale hormone activities [Flavonoids and triterpenoids are substances naturally found in a large variety of vegetarian foods and medicinal plants. Despite its beneficial effects on health and its relatively low toxicity , some isale rats . Oleanoltivities . Thus, ttivities . A toxictivities . Chromolaena odorata, at doses of 250 and 500 mg/kg, evidencing its antiandrogenic activity, since there was a reduced number of sperm in the male rats exposed orally for 14 days. In the same way, Arena et al. [J. decurrens. Controversially, in the present study, after sperm counts in the testis and epididymis, no changes were observed in the sperm production or in the epididymal sperm number (n = 8) and abnormal forms were similar among the groups. In the same way, the testis and epididymis analysis by light microscopy did not reveal any morphological changes related to the treatment (data not shown). Conflicting results may be due to differences in the experimental protocol of these two studies, such as the treatment period (during pregnancy or adult life) and the vehicle (aqueous or hydroethanolic) used in the extract preparation. Yakubu et al.  demonstra et al.  observedm number . MorpholThese results demonstrate the absence of subacute toxicity due to oral treatment with EJD for 28 days in adult male rats. Furthermore, in this experimental model, EJD not interfere with the male reproductive system of rats, since the evaluated parameters were not affected. However, other studies based on protocols elaborated by regulatory agencies should be performed  in order to evaluate the total safety of this plant.


Acute kidney injury in patients with acute pancreatitis carries a poor prognosis. Hemolytic uremic syndrome (HUS) is characterized by non-immune hemolytic anemia, thrombocytopenia, and renal failure caused by platelet thrombi in the microcirculation of the kidney, and though rare in adults it is associated with high mortality and a high rate of chronic renal failure.Herein, we report a case of alcohol-induced acute pancreatitis in a 38-year-old Chinese female complicated by HUS. Her renal function progressively deteriorated in 2 days, and daily continuous renal replacement therapy (CRRT) was thus performed for a total of 13 treatments. She also received intermittent transfusions of fresh frozen plasma. Her renal failure was successfully managed, with subsequent return of normal renal function. She was discharged 1 month after admission and follow-up at 3 months revealed normal urea and creatinine.CRRT was shown to be useful for the treatment of HUS following acute pancreatitis. Prior case reports and our case should remind clinicians that HUS is a possible complication of acute pancreatitis. This study highlights the importance of early diagnosis and prompt initiation of CRRT to prevent mortality and improve outcomes. The incidence of acute kidney injury in patients with acute pancreatitis ranges from 14-42%, and carries a poor prognosis [A previously healthy 38-year-old female, non-smoker and non-alcoholic, was hospitalized following acute alcohol intoxication. She had drunk more than 500 ml of alcohol in about 2 hours and subsequently lost consciousness. Prior to losing consciousness she complained of severe epigastric pain and frequent vomiting. On admission, serum creatinine, blood urea nitrogen (BUN), hemoglobin, white blood cell (WBC) count, electrolytes, and liver function tests were within normal ranges. Serum amylase was 1728 IU/L . Ultrasound revealed the body and tail of the pancreas to be edematous, the biliary tree was not dilated, and there was sludge in the gallbladder. Abdominal computed tomography (CT) revealed an edematous pancreas with necrotic tissue in the left parieto-colic area. The diagnosis was alcohol-induced acute pancreatitis.3 , her platelet count was 32 × 109/L , and haptoglobin was 0.05 g/L . Peripheral blood smear revealed schistocytes, and the Coombs’ test was negative. The level of fibrin degradation products was normal, and her prothrombin time international normalized ratio (PT-INR) was 1.5-2.5. Early in the disease course plasma LDH is elevated in patient , and LDH levels gradually decline with CRRT treatment and mitigation of pancreatitis. Her clinical symptoms of acute kidney injury and her treatment reaction were not consistent with a diagnosis of DIC. Renal biopsy was not performed because the patient refused invasive examinations. A diagnosis of HUS was made, and she was transferred to the intensive care unit (ICU).During the next 2 days her renal function progressively deteriorated, and her urine output decreased to < 400 ml per 24 hours. On the third day of admission her urea and creatinine were 12.8 mmol/L  and 420 μmol/L , respectively. Her hemoglobin level had dropped from 14.9 g/dL to 8.8 g/dL, with a reticulocyte count of 75,000/mm9/L. A year and 3months later, she gave birth to a healthy boy.She received daily continuous renal replacement therapy (CRRT) by hemofiltration on the third through the fifth hospital days, and then every other day for a total of 13 treatments. CRRT was administered at a dosage of 40 ml/kg/h for a duration of 6–8 hours, and the volume of fluid removed varied from 500 to 2500 ml. She also received intermittent transfusions of fresh frozen plasma. Her urine volume increased beginning of the fifth hospital day, and her serum amylase level returned to the normal range by hospital day 7. Her renal function progressively improved, and she was transferred from the ICU to the general ward on day 20. She was discharged 1 month after admission in good condition, and follow-up at 3 months revealed normal urea and creatinine , and her platelet count was 129 × 10E. coli O157:H7 infection and 80-90% of cases resolve without squelae [Hemolytic uremic syndrome is characterized by non-immune hemolytic anemia, thrombocytopenia, and renal failure caused by platelet thrombi in the microcirculation of the kidney and other organs . The dis squelae . HUS is  squelae ,3.The etiology of HUS following pancreatitis is not clearly understood, as there are only about 20 cases reported in the literature . Our casThe treatment of HUS following acute pancreatitis is primarily supportive. Fresh frozen plasma infusion may improve the renal outcome in adult HUS , and excIn renal replacement therapy (RRT) performed via hemofiltration, solute movement is governed by convection and unlike in dialysis a dialysate is not used . PositivIn summary, we have presented a case of adult HUS secondary to acute pancreatitis successfully managed by prompt diagnosis and rapid initiation of CRRT via hemofiltration before severe kidney injury or renal failure had occurred. Prior case reports and our case should remind clinicians that HUS is a possible complication of acute pancreatitis. Early institution of CRRT may be an effective treatment to prevent mortality and improve outcomes.Written informed consent was obtained from the patient for publication of this Case report. A copy of the written consent is available for review by the Editor of this journal.The authors declare that they have no competing interests.PF: guarantor of integrity of the entire study; study concepts; study design; definition of intellectual content; data analysis; statistical analysis; manuscript preparation; manuscript editing; manuscript review. AHY: literature research. CHW: clinical studies. XL: clinical studies. HYW: data acquisition. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/15/1/prepub


The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria™ RDT (AccessBio®) which is currently used in Cambodia, and real-time PCR (as “gold standard”).Plasmodium falciparum and non-P. falciparum were with 20–30 min reading times  and were similar to those for the CareStart Malaria™ test.The best performances of the VIKIA Malaria Ag Pf/Pan™ test for detection of both This new RDT performs similarly well as other commercially available tests , and conforms to the World Health Organization’s recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas. Plasmodium falciparum parasites[Now that artemisinin derivatives in combination with partner drugs (artemisinin combination therapy (ACT)) are being used, parasitological confirmation has become essential before treatment in routine malaria case management, in most countries endemic for malaria,2. This arasites, particuarasites-7.Microscopic examination of blood films still remains the gold standard for malaria diagnosis despite the requirement for high-quality microscopes and equipment, and for qualified personnel. HoweverP. falciparum and detect the histidine-rich-2 protein (HRP-2). Others combine HRP-2 detection with the detection of antigens common to all species, such as lactate dehydrogenase or aldolase (combo RDTs). Combo RDTs can diagnose infections by P. falciparum or by non-P. falciparum malaria parasites [The malaria RDTs available are all based on the same principle and like other lateral flow immune-chromatographic tests, there are various formats . They contain antibodies conjugated to colloidal gold particles, which bind specifically with parasite antigens. Some of them are specific for alariae).© is a company which aims to address public health requirements by developing reliable diagnostic tools responding to the constraints associated with use in the field for populations in developing countries at affordable prices[IMACCESSe prices. The comIn this context, the objective of the study presented here was to assess the performances of this new immunochromatographic test by following a conventional field evaluation design including microscopic examination (thick and thin blood film) and molecular detection  as gold standard methods for malaria diagnosis of febrile, malaria-suspected patients seen at health centre level. In addition, the VIKIA Malaria Ag Pf/Pan™ test was also compared to another RDT widely used in Cambodia and elsewhere .©, Ref. 41 2499, Lot Number: V110727601, Lyon, France), containing one package insert, 25 test cassettes in individual sealed pouches with a disposable specimen pipette and a dessicant and one vial of running lysis buffer, was used according to the manufacturer’s instructions. Briefly, a 5 μL aliquot of blood drawn from patients was added to the sample well. Five drops of lysis buffer were then dispensed into the buffer well  was also performed according to the manufacturer’s instructions and used to diagnose patients as malaria infected.The VIKIA Malaria Ag Pf/Pan™ test kit  were selected Figure: Veal VePlasmodium species all co-exist in the Cambodia and P. falciparum is the most frequent cause of malaria infections . The distributions of Plasmodium species have been changing over recent years, with a particularly significant increase in the incidence of P. vivax malaria cases (from 8% in 2000 to 23% in 2007). In low transmission areas, a proportion of infections that are P. vivax is commonly up to 50%[Malaria remains a public health problem at these sites, and is mainly linked to the forests. Forest villagers in the eastern and northern provinces are at high risk of malaria, whereas elsewhere in the country malaria is an occupational disease with particular groups being at high risk, including forestry workers, new settlers and mobile/migrant populations moving into forested areas, and soldiers and their families serving in the forests. The prevalence is between 15% and 40% in villages near or in forested areas, and between 0% and 3% in the plains and rice field areas. The five human p to 50%.P. falciparum malaria according to the definition of the World Health Organization (2001) were excluded[In the four sites, patients with clinical symptoms of malaria (fever ≥37.5°C) and with a prescription for malaria diagnosis from a health care worker, agreeing to be enrolled in the study (by signing the consent form), were included. Pregnant women and patients with signs of severe and complicated nization 01 were eBlood samples were collected by finger-prick and used for malaria testing . In addition, two drops of blood were spotted onto Whatman 3 M filter paper™ and individually stored in an envelope and a plastic bag. Slides for thick and thin microscopic examination were also prepared, and stored in appropriate boxes. Clinical and biological information were recorded on an anonymous form to ensure patient confidentiality. Filter papers and slide boxes were sent to the Institut Pasteur du Cambodge (IPC) in Phnom Penh every week.At IPC, thick and thin blood smears were stained with 3% Giemsa for 30 min and analysed by light microscopy by two experienced technicians without reference to RDT results. A minimum of 200 consecutive fields was checked in the thick blood film before classifying a slide as negative. Parasites in thick blood films were counted against 200 white blood cells. The parasite density was estimated assuming 8,000 white blood cells/μL of blood. One in 10 slides selected at random, and all slides corresponding to discordance between the first two readings, were read a third time by a third expert microscopist.©, Marnes la Coquette, France), according to the manufacturer’s instructions. Molecular detection and identification of Plasmodium parasites involved two steps: Plasmodium was detected by a “screening real-time PCR” with primers targeting the Plasmodium cytochrome b gene. Secondly, DNA samples identified as positive for Plasmodium were analysed for malaria species by PCR with several pairs of specific primers  targeting the same gene . From August to December 2011, 1,000 patients  with an age range of one to 70 years  were recruited. The male/female ratio was 2.1/1. The mean ± SD axillary temperature was 38.1 ± 0.9°C (range = 37.5–41.5°C) and the mean ± SD parasitaemia density was 13,455 ± 52,694 parasites/μL .Plasmodium falciparum was present in 39.5%, P. vivax in 58.8% and both P. falciparum and P. vivax (mixed infections) in 1.5% of the positive specimens. A total of 92 discordant results (9.2%) were found between microscopy and real-time PCR . In addition, 59 samples were misclassified by microscopy: 54 were classified as single infections by microscopy  but found to be mixed infections by real-time PCR ; three cases classified as mixed P. falciparum/P. vivax infections were identified as single P. falciparum infections by real-time PCR; two cases classified as single P. vivax infections were found to be single P. falciparum infections by real-time PCR.Microscopy results showed that 389 (38.9%) of the 1,000 patients had malaria. Plasmodium spp. by real-time PCR, microscopy, CareStart Malaria™ and VIKIA Malaria Ag Pf/Pan™ tests at several reading times  are shown in TablePlasmodium spp. parasitaemia are summarized in TableP. falciparum infections and FigureP. falciparum infections.Details concerning patients positive for vs 42.2%), CareStart Malaria™ test  or VIKIA Malaria Ag Pf/Pan™ test . A similar trend was also found for a greater sensitivity of microscopy by expert microscopists than malaria RDTs: 2.0% more positive samples by microscopy than CareStart Malaria™ test (38.8% vs 38.1%) and 2.8% (60 min reading time) to 20.8% (10 min reading time) more than with the VIKIA Malaria Ag Pf/Pan™ test .This study reports the first evaluation of the performance of the VIKIA Malaria Ag Pf/Pan™, a new immunochromatographic test developed by IMACCESS®, using a combination of microscopy and real-time PCR as reference methods for classification of samples. This approach had the advantage of combining the high sensitivity  and specificity (to correctly identify the parasite species) of real-time PCR, with the possibility of estimating parasite density by microscopy. As expected, more positive samples were found using real-time PCR than microscopy  to 94.2% for P. falciparum detection and 82.7% for non-P. falciparum detection (60 min). The specificity  decreased with reading time from 99.3% for P. falciparum detection and 99.5% for non-P. falciparum detection (10 min) to 98.1% for P. falciparum detection and 98.9% for non-P. falciparum detection (60 min).The sensitivity  of the VIKIA Malaria Ag Pf/Pan™ test increased with reading time, from 89.7% for P. falciparum and non-P. falciparum detection were observed at 20–30 min . Using these reading times, the performance of the VIKIA Malaria Ag Pf/Pan™ test was similar to those of the CareStart Malaria™ test or those found in previous studies[The best performances of the VIKIA Malaria Ag Pf/Pan™ test for both  studies-26.P. falciparum, the sensitivity of the both tests started to decrease at levels of parasitaemia < 500 parasites/μL (100% above 500 parasites/μL) and for non-P. falciparum, the sensitivity of the CareStart Malaria™ test and the VIKIA Malaria Ag Pf/Pan™ test started to decrease below 1,000 parasites/μL or below 10,000 parasites/μL, respectively. False negative results for both malaria RDT were only found in P. falciparum samples containing <250 parasites/μL and in non-P. falciparum samples with <1,000 parasites/μL.Moreover, as has been previously reported, the sensitivities of both malaria RDT decreased with the level of the parasitaemia,26-30: fAccording to WHO recommendations for RDT performances, sensitiIn conclusion, this new RDT performs similarly well as other commercially available tests , and conforms to WHO recommendations for RDT performance. It appears to be a satisfactory alternative tool for the diagnosis of malaria in endemic areas.The authors declare that they have no competing interests.MC, SK and DM contributed to the design and coordination of the study, assisted with data entry and interpretation and prepared the manuscript. SK supervised the field study. NK, SC, SS, DD and LC were involved in laboratory work. MC, SK, CN and DM helped to write the manuscript and gave constructive advice. All authors read and approved the final manuscript.


Streptococcus pneumoniae is the leading cause of community–acquired pneumonia in Africa. Antimicrobial resistance of S. pneumoniae to penicillin and other commonly used antibiotics has increased worldwide. However, prevalence data from the African region are sparse, especially with regard to adults.In this study, adult patients presenting at an urban referral hospital in central Mozambique were screened for pneumococcal pneumonia during an 8-week period in 2010: Patients with a respiratory syndrome underwent chest radiography and a sputum sample was collected for pneumococcal culture and antimicrobial susceptibility testing. A urine sample was tested for the presence of pneumococcal antigen.177 patients with a respiratory syndrome were included. Overall, 41/177 (23%) patients fulfilled criteria for definite or probable pneumococcal pneumonia and in the group of patients with a positive chest x-ray this concerned 35/86 (41%) patients. 166 sputum cultures yielded 16 pneumococcal strains. One mg oxacillin disc testing identified potential penicillin resistance in 7/16 (44%) strains. Penicillin minimal inhibitory concentrations (MICs) were measured for 15 of these strains and ranged from <0.016-0.75 mg/L. No MICs >2 mg/L were found, but 3/15 (20%) pneumococcal strains had MICs >0.5 mg/L. All pneumococci were sensitive to erythromycin as measured by disc diffusion testing, whereas 44% was resistant to trimethoprim-sulfametoxazole.S. pneumoniae strains tested were penicillin resistant, standard penicillin dosing for pneumonia may be insufficient given the observed range of pneumococcal penicillin MICs.The proportion of pneumonia cases attributable to pneumococcus appeared to be high. Whilst none of the  Streptococcus pneumoniae is the leading cause of community-acquired pneumonia around the world and in Africa [n Africa . Severe n Africa ,3.S. pneumoniae against the most commonly used antimicrobial drugs is increasing worldwide, principally affecting β-lactam, macrolide and sulfonamide sensitivity [Antimicrobial resistance of sitivity . In Afrisitivity ,6.S. pneumoniae in adults with pneumococcal pneumonia presenting in an urban hospital in central Mozambique.We investigated the prevalence of penicillin resistance of Hospital Central da Beira: HCB), a 932-bed governmental referral health facility with 260 internal medicine beds, located in the coastal capital of the central Sofala province of the sub-Saharan African Mozambique. The HIV prevalence among adult women in this region was found to be 22.9% in 2009 [Our study was conducted at the Beira Central Hospital  presenting at the HCB emergency room with a respiratory syndrome. A respiratory syndrome was defined as fever (axillary body temperature ≥37.5°C) in combination with one or more of the following complaints: cough with or without sputum production, dyspnoea and chest pain. Study participants were recruited prospectively during a study period of 8 weeks, from April to June 2010. A posterior-anterior chest x-ray (CXR) was made immediately after presentation and subsequently digitalized for review by an experienced, external pulmonologist, who was blinded to the study participant’s clinical data and pneumococcal test results.Written informed consent was obtained from each patient before study entry. Ethical approval was obtained from the Mozambican National Committee of Bio-ethics, through its sub-committee seated in Beira (ref.: 007/10/SBCE).Sputum samples were collected from all patients for pneumococcal culture and sensitivity testing. Patients were carefully instructed about how to provide a good-quality sputum sample in a standardized way and study staff directly observed the sputum production process for all study participants. Sputum was inoculated on selective Columbia CNA sheep blood agar plates . Plates were incubated overnight at 35–37°C in a candle jar after placement of a 5 μg optochin disc . Colonies suspected for pneumococcus, on the basis of colony morphology and optochin susceptibility as per manufacturer’s instructions, were subcultured on sheep blood agar plates . The Dryspot Pneumo latex agglutination test  was used as per manufacturer’s instructions for final identification.Antimicrobial susceptibility of pneumococcal isolates against oxacillin (1 μg), trimethoprim-sulfamethoxazole (co-trimoxazole: 5.2-240 μg) and erythromycin 78 μg) was tested using disc diffusion method . Zone diameter breakpoint interpretation was done according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations as recommended by the manufacturer. The benzyl penicillin minimal inhibitory concentration (MIC) was measured using E-test strips . Clinical breakpoint interpretation of these MICs was done using the EUCAST clinical breakpoint tables for interpretation of MICs, using the recommendations for non-meningeal infections: MIC ≤ 0.06 mg/L: susceptible; > 2 mg/L: resistant; > 0.06 and ≤ 2 mg/L: intermediate category with dose specific breakpoints [ μg was tS. pneumoniae, according to the manufacturer’s instructions .A urine sample was collected from all patients for urinary pneumococcal antigen testing using Binax NOW S. pneumoniae. Probable pneumococcal pneumonia was defined as a respiratory syndrome in combination with a positive CXR and a positive ICT. In the absence of a CXR, or whenever a CXR was not positive, a respiratory syndrome with a positive sputum culture with S. pneumoniae AND a positive ICT was also defined as a probable pneumococcal pneumonia  pneumonia and given the lack of state-of-the-art blood culture capacity at the study site, the following definitions were used. Suspected pneumonia was defined as a respiratory syndrome combined with a positive CXR, as defined by showing evidence of any consolidation, and/or pleural effusion, and/or interstitial disease. Definite pneumococcal pneumonia was defined as a respiratory syndrome in combination with a positive CXR and a positive sputum culture with a Figure .Data were entered and analysed using the Epi Info package version 2002 .177 patients were screened. 97/177 (55%) patients were female and 150/177 (85%) patients were between 16–45 years. HIV status was known for 105/177 (59%) and in this group 84/105 (80%) patients were HIV positive. When asked about antibiotic use in the previous three months, 51/177 (29%) of patients was not able to answer this question because they were uninformed, physically unable to answer or because of a combination of the two. 48/177 (27%) of patients confirmed the use of antibiotics and another 78/177 (44%) denied its use. 30/177 (17%) of patients were receiving antibiotics at the time of study inclusion. Although the name ‘penicillin’ was often mentioned in this group (70%), it was not possible to verify this information in most cases. 13/177 (7%) of patients were found to be on TB treatment at study inclusion.53/170 (31%) patients had a positive urinary antigen test. In 124 patients a CXR was done. 86/124 (69%) patients had a positive CXR and were therefore qualified as having suspected pneumonia. 35 of these 86 (41%) patients turned out to have pneumococcal pneumonia, based on a positive culture and/or a positive urinary antigen test. An additional 6 patients had a positive culture and a positive urinary antigen test in the absence of a positive CXR, so overall, 41/177 (23%) patients with a respiratory syndrome fulfilled the criteria of either definite or probable pneumococcal pneumonia  have been expanded and re-set at ≤2 mg/L (susceptible) and ≥8 mg/L (resistant) in 2008 [After a critical appraisal of microbiological, pharmacokinetic and pharmacodynamic data, as well as clinical studies on the use of penicillin in non-meningeal pneumococcal infection, current penicillin MIC breakpoints for treatment of non-meningeal  in 2008 . In contAs for adult benzyl penicillin treatment in Mozambique, the national formulary recommends the use of ‘1 million International Units (IU) or more, 4–6 times daily’ for non-meningeal infections . MICs asPneumococcal susceptibility rates appear to vary across Africa, but a comparison of data is complicated by the use of different antimicrobial susceptibility testing methods and interpretive criteria. In a recent systematic review on community acquired bloodstream infections in Africa, 9.7% (range 0-36%) of pneumococcal isolates was resistant to ampicillin, 40% to co-trimoxazole and 2% to erythromycin . South AOur results have to be interpreted with caution as the pneumococcal culture sample size was small and a positive sputum culture may represent colonization rather than infection, although we believe that this risk is low as we selected patients on the basis of a clinical syndrome in combination with radiologic and microbiological criteria.In conclusion, the proportion of pneumonia attributable to pneumococcus was high in adults in an urban referral hospital in Mozambique. All pneumococcal strains turned out to be penicillin susceptible as based on benzyl penicillin MIC results and there seems to be no need to abandon penicillin as the main treatment for pneumococcal pneumonia. At the same time, given the presence of strains with MICs > 0.5 mg/L, current standard penicillin dosing for pneumonia may be insufficient. For Mozambique, as well as for other sub-Saharan African countries, we would like to emphasize the need for the use of a standard dosage that would be effective against strains with penicillin MICs within the locally observed range. More extensive, continued monitoring is needed to guide prudent empiric antibiotic choices.CLSI: Clinical and laboratory standards institute; CTX: Co-trimoxazole; CXR: Chest x-ray; EUCAST: European committee on antimicrobial susceptibility testing; HCB: Hospital Central da Beira; IU: International units; MIC: Minimal inhibitory concentration; RS: Respiratory syndrome; ICT: Immunochromatographic test; ND: Not done.The authors declare that they have no competing interests.JCB, CS and JMP conceived of the study and designed it. JCB, SB, GM, ESG, EC, CM and JMP designed the study protocol. SB, GM and JCB carried out the study participant recruitment and screening. SB performed all microbiological analyses, under supervision of EC, JCB and CS. RS reviewed all CXRs. JCB and SB analysed the data and JCB, CS and JMP carried out the interpretation of the data. JCB drafted the manuscript. CS, RS and JMP critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.JCB and JMP are internists and infectious diseases specialists who have been collaborating extensively with the Faculty of Health Sciences of the Catholic University of Mozambique (UCM) in Beira and the Ministry of Health of the Republic of Mozambique (MISAU) since 2006 for the improvement and expansion of graduate and post-graduate internal medicine training, including infectious diseases.