Objective. To find a statistically significant separation point for the QuantiFERON Gold In-Tube (QFT) interferon gamma release assay that could define an optimal “retesting zone” for use in serially tested low-risk populations who have test “reversions” from initially positive to subsequently negative results. Method. Using receiver operating characteristic analysis (ROC) to analyze retrospective data collected from 3 major hospitals, we searched for predictors of reversion until statistically significant separation points were revealed. A confirmatory regression analysis was performed on an additional sample. Results. In 575 initially positive US healthcare workers (HCWs), 300 (52.2%) had reversions, while 275 (47.8%) had two sequential positive tests. The most statistically significant  separation point identified by the ROC for predicting reversion was the tuberculosis antigen minus-nil (TBag-nil) value at 1.11 International Units per milliliter (IU/mL). The second separation point was found at TBag-nil at 0.72 IU/mL . The model was validated by the regression analysis of 287 HCWs. Conclusion. Reversion likelihood increases as the TBag-nil approaches the manufacturer's cut-point of 0.35 IU/mL. The most statistically significant separation point between those who test repeatedly positive and those who revert is 1.11 IU/mL. Clinicians should retest low-risk individuals with initial QFT results < 1.11 IU/mL. We report the findings of a multisite study of United States healthcare workers (HCWs) that began as a quality control initiative in the Veterans Administration Palo Alto Health Care System (VAPAHCS) when QuantiFERON Gold In-Tube (QFT) serial screening tests were observed to be initially positive and were subsequently negative in those low-risk individuals. This seemingly spontaneous “reversion” has been reported around the world in the literature, and the variability that occurs mostly around the baseline is recognized –6. This study design was driven by the clinical experience: when an HCW presents with a positive QFT result, what can the clinician do to discern whether the next test is likely to remain positive or become negative? The foundation of the problem lies in the dichotomous nature of the results reported. Currently, a QuantiFERON tuberculosis antigen minus-nil (TBag-nil) ≥ 0.35 International Units per milliliter (IU/mL) is reported as “positive.” At that point the provider has a decision to make, one that is generally to investigate further with a chest radiograph, seek specialty consultation, and/or recommend medical treatment. Whereas positive tuberculin skin tests (TSTs) were often felt to be erroneous due to prior BCG vaccination, and compliance and treatment rates were low; studies are showing that positive interferon-gamma release assay (IGRA) results are more likely to lead to both the recommendation and the acceptance of chemotherapy –10. ChemIn response to the persistent concerns regarding reversions near the cut-point of 0.35 IU/mL, a 2010 Morbidity and Mortality Weekly Report published by the Centers for Disease Control and Prevention (CDC) recommended that quantitative QFT results should be reported. The CDC did not, however, provide guidance for either the interpretation or the use of these values . We inveData were obtained from a retrospective review of available clinical laboratory records from three different sites: (1) Veterans Administration Palo Alto Health Care System (VAPAHCS), California, (2) University of Illinois Chicago (UIC) Il, and the (3) Cleveland Clinic (CC), Ohio, where each HCW undergoes preemployment and annual QFT testing irrespective of previous results. All subjects are US HCWs who were serially tested by QFT Gold-in-Tube in their hospital's laboratory. All HCWs at least 18 years of age with available records were included. The study's date range was January 2009 through June 2011 at VAPAHCS, from August 2008 through June 2011 at UIC, and from October 2009 through December 2011 at the Cleveland Clinic. HCWs who tested consistently negative and those with only a single test result were excluded. Results reported without the QFT TBag-nil numerical value, as well as HCWs with negative-to-positive discordance/conversion at the conclusion of their testing series were removed from the dataset . To be included in the analysis, at least two QFT tests were required, one of which was a positive result that was followed by either a positive or a negative result. This reproduces the clinician's actionable decision point; that is, when a patient presents with a positive result, the action to test further, to refer, or to treat is initiated. Patients were only included once see .The VAPAHCS is a suburban teaching hospital located in Palo Alto, California. The county in which it resides, Santa Clara, has the 3rd highest tuberculosis (TB) rate in California  at 11.4%The University of Illinois, Chicago (UIC) is a public, urban academic teaching hospital. The HCW population is approximately 5,000. Their laboratory performed over 50,000 QFT-GIT tests by June 2011; 20,543 of these were on HCWs. Annual HCW TB screening is mandated and compliance is 99%, with most HCWs tested annually, but some who are on surveillance are tested every six months. UIC reports a HCW QFT negative rate of 89.5%, with 1.1% indeterminate and 9.4% positive at some point in their series. Illinois ranks 21st for tuberculosis cases in the nation , and ChiThe Cleveland Clinic Foundation  is an urban teaching hospital. The laboratory had performed over 10,000 QFT-GIT tests by June 2011. This includes patient and HCW testing. Cleveland Clinic hires approximately 2,500 HCWs annually. The HCW population is 98.5% negative with 0.5% indeterminate and 1% positive at some point in their series. Ohio ranks 35th in the nation for tuberculosis cases  with CleThe interferon gamma released was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol though with an 8-point standard curve for each microplate. The results were read at 450 nm by the Diamedix DS2 Automated ELISA System  at VAPAHCS, by the Diamedix DSX Automated ELISA System  at UIC, and by a BioTek ELx800 Absorbance Microplate Reader  at CC. All tests in this series met the nil, mitogen, and the equation criteria for test validity delineated in the manufacturer's package insert .In the absence of a gold standard against which to evaluate latent tuberculosis infection, the expected probability of two consecutive positive tests was employed as a proxy for corroboration of the test result in question, which is the implied presence of latent tuberculosis disease. In seeking what would best predict whether an individual was likely to be a “reverter”, the initial TBag-nil value in the series of two sequential tests was evaluated as a possible predictor variable. Note that the QFT result was considered positive if the TBag-nil was ≥0.35 IU/mL, so all TBag-nil values were at least 0.35 IU/mL in this analysis. We used a two-step data analytic approach. First, we employed a receiver operating characteristic analysis (ROC) , 21 on tn < 20) and/or when no further variables are selected because the P value associated with the Chi-square statistic is ≥0.01. If the association does not meet the criteria for the stopping rule, the sample is divided into two groups based on the optimal variable and identified separation point. The ROC analysis is then restarted, separately, for each of these two subgroups. The result is a decision tree identifying the HCW characteristics and associated separation points that best predict reversions, with P values, chi-square, and Kappa values calculated and reported. The ROC software developed by Drs. Yesavage and Kraemer is publicly available [Regarding the details of the ROC analysis, once the optimal variable and associated separation point are identified, the association with the success criterion is tested against a stopping rule. Stopping rules include a subgroup sample size too small for further analysis  or negative (reversion) result defined the two test results in the series that were analyzed see . The mean = 575) or the Confirmatory Group (n = 287). The Exploratory Group of tested HCWs had a 52.2% (300/575) reversion rate. The results of the ROC analysis performed on the Exploratory Group are presented as a decision tree shown in P < 0.001). Two groups of HCWs were identified: group 1: 75% reversions: 225/300 HCWs with TBag-nil <1.11 IU/mL; group 2: 27% reversions: 75/275 HCWs with Tbag-nil ≥1.11 IU/mL.The 862 HCWs who met inclusion criteria were randomly assigned to one of two groups: the Exploratory Group (P < 0.01): group a: 80% reversions: 163/204 HCWs with TBag-nil <0.72 IU/mL; group b: 65% reversions: 62/96 HCWs with TBag-nil ≥0.72 and <1.11 IU/mL.The ROC analysis further identified two subgroups of HCWs derived from group 1 above with a TBag-nil at 0.72 IU/mL :  group c: 43% reversions: 43/99 HCWs with TBag-nil ≥ 1.11 and <2.17 IU/mL; group d: 18% reversions: 32/176 HCWs with TBag-nil ≥ 2.17 IU/mL.Two subgroups of HCWs were also identified from group 2 above with TBag-nil at 2.17 IU/mL  using the same dependent measure (reversion) and predictor variable (TBag-nil) identified in the primary ROC analysis. The relationship remained statistically significant (P < 0.001). All three separation points at 0.72, 1.11, and 2.17 IU/mL (4 subgroups) identified in the ROC analysis also remained statistically significant for all subgroups by chi-square (P < 0.001). A logistic regression analysis was conducted in the Confirmatory Group  of reversions in this range, but this would include only 103/575 (18%) of the total population and 84/300 (28%) of the “reverters.” The sensitivity of this separation point would be only 0.28 and its Kappa 0.21. Thus with the ROC there are trade-offs in sensitivity versus specificity, depending upon the separation points selected. The 1.11 IU/mL measurement was chosen by the ROC analysis for this population because that value maximizes the percentage of “reverters” while optimizing sensitivity and specificity.P < 0.001). Our sample of HCWs had a 75% risk for reversion if their initial positive test fell within this range. While 0.35–1.11 IU/mL is therefore the optimal retesting zone identified here, 0.35–0.72 IU/mL  is another possible separation point also selected by the ROC and could be reasonably employed by providers based on the clinical situation much like the 5, 10, and 15 mm tuberculin skin test cut-off points that are used in different settings. Acceptance of TBag-nil values reported above as the delineators of a QFT retesting zone could lessen patient anxiety, decrease unnecessary radiographs, prevent unnecessary exposure investigations, and possibly spare patients from inappropriate medical treatment due to transiently “positive” QFT test results. We present a validated model on a sample of 862 US healthcare workers from three major US hospitals that could be used to define a QuantiFERON Gold In-Tube retesting zone between 0.35 and 1.11 IU/mL. The upper value was selected by a receiver operating characteristic analysis to maximize separation between HCWs who have two consecutive positive tests and those who have reversions (Limitations of the study include that while the current analyses incorporated over 850 positive HCW records, these data are from only three facilities. Furthermore, results in this study are weighted towards UIC, since their data comprise the majority of the sample group. While there could be selection bias among those HCWs who present for serial testing, it is not clear how that could influence these results. Finally, it should also be noted that prospective long-term followup would be required to provide thorough validation of the results. Future analyses using the same statistical methods could include additional data from other institutions in USA, Europe, or from countries with higher risk for HCWs. There is a possibility that there could be local variation based on biologic or regional laboratory differences that would be exposed when more data are analyzed.


N-methyl-D-aspartate receptors (NMDAR), such as ketamine, elicits behavioral effects, and alterations in cortical interneurons similar to those observed in Sz. However, the chronic effects of ketamine on neocortical GBO are unknown. Thus, here we examine the effects of chronic (five daily i.p. injections) application of ketamine (5 and 30 mg/kg) and the more specific NMDAR antagonist, MK-801 , on neocortical GBO ex vivo. Oscillations were generated by focal application of the glutamate receptor agonist, kainate (KA), in coronal brain slices containing the prelimbic cortex. This region constitutes the rodent analog of the human dorsolateral prefrontal cortex, a brain region strongly implicated in Sz-pathophysiology. Here we report the novel finding that chronic ketamine elicits a reduction in the peak oscillatory frequency of KA-elicited oscillations (from 47 to 40 Hz at 30 mg/kg). Moreover, the power of GBO in the 40–50 Hz band was reduced. These findings are reminiscent of both the reduced resonance frequency and power of cortical oscillations observed in Sz clinical studies. Surprisingly, MK-801 had no significant effect, suggesting care is needed when equating Sz-like behavioral effects elicited by different NMDAR antagonists to alterations in GBO activity. We conclude that chronic ketamine in the mouse mimics GBO abnormalities observed in Sz patients. Use of this ex vivo slice model may be useful in testing therapeutic compounds which rescue these GBO abnormalities.Abnormalities in EEG gamma band oscillations  serve as a prominent biomarker of schizophrenia (Sz), associated with positive, negative, and cognitive symptoms. Chronic, subanesthetic administration of antagonists of  Mounting evidence suggests that the symptoms of neuropsychiatric disorders, such as schizophrenia (Sz), arise from a failure of the brain to properly integrate activity across local and distributed neuronal circuitry –3. NeuroN-methyl-d-aspartate receptor (NMDAR) antagonist, ketamine, have provided the most reliable and widely used means to mimic Sz-like symptoms, and currently represents the “gold-standard” for modeling this disorder in both humans, and animals , the rodent analog of the human dorsolateral prefrontal cortex  heterozygous GAD67-GFP “knock-in” mice (Swiss Webster background), which express GFP under control of the promoter for GAD67  of eitheChronic drug administration was conducted similar to the manner described in Behrens et al. . Mice we2PO4, 2 MgSO4, 25.6 NaHCO3, and 10 glucose saturated with 95% O2/5% CO2. 450 μm slices were cut between +2.96 and +1.54 mm with respect to bregma  using aAs described in McNally et al. , extraceKainate-elicited oscillations were characterized using both time-frequency, and power spectral density (PSD) analysis. Grand average time-frequency plots Figure  were genbetween groups of animals, i.e., drug treated vs. saline controls [note: this band is narrower than that used in our previous study . The frequency at which the peak power was observed was defined as the peak frequency. Total GBO power was determined by integration of averaged PSD between 40 and 50 Hz, which represents roughly the average peak frequency of the oscillatory response observed under control (no drug treatment) conditions ±5 Hz. This narrow frequency band was chosen to reduce variability of the PSD measures, when compared us study  where wet-test for statistical comparison of individual epochs of oscillatory response. All statistical analysis was performed using SPSS 10 (SPSS Inc.). Differences were considered to be significant at p < 0.05. Averaged values reported in this manuscript are expressed as mean ± SEM.Overall analysis of chronic drug effects were initially tested using repeated measures ANOVA. Further analyses of the results were performed using Student’s n = 9) or control animals which received saline injections (n = 7). Visual inspection of a grand average of KA-elicited oscillations recorded from animals receiving chronic ketamine (30 mg/kg) injections revealed a clear reduction in the KA-elicited oscillatory response when compared to saline treated controls  decrease in the peak frequency  of elicited oscillations in slices from chronic ketamine treated mice .This data was initially analyzed using the method employed in our earlier acute NMDAR antagonist study , which eF1,13 = 52.77; p < 0.01), and total GBO power , across the full 30 s of KA-elicited activity. No effect was observed on peak power . Further statistical analysis, comparing each epoch individually, showed that the significant decrease in peak frequency observed above was maintained throughout all periods of analyzed activity. While total GBO power was reduced in chronic ketamine treated animals throughout the entire 30 s of analyzed activity, this reduction was only statistically significant in the second , third , and fourth  epochs. No significant change in peak power was seen during these periods.Visual inspection of the grand averages suggested that oscillatory activity elicited in 30 mg/kg chronic ketamine treated slices decayed faster than in saline treated animals. Thus, to determine if chronic ketamine treatment impaired the ability of the PrL slice to maintain the KA-elicited GBO, we additionally analyzed the first 30 s of elicited oscillatory activity subdivided into 5 s epochs Figure ; Table 1n = 5), using the same dosing regimen described above conversely appeared to lead to a mild potentiation of the power of the KA-elicited oscillatory response . This decrease was of a similar magnitude as observed with the higher ketamine dose when compared across all epochs of analyzed activity at 5 mg/kg, however, it was only significant in epochs 5 and 6 , and reached a trend level of significance in epochs 1, 2, 3, and 4 . 5 mg/kg ketamine did not lead to a decrease in the GBO power of KA-elicited oscillations  and a trend level increase in the third  and fifth  epochs, however.Chronic treatment with a lower concentration of ketamine  and saline control (n = 7) animals. As shown in Figure F1,11 = 3.83; p = 0.08), peak frequency , or peak power . Comparing across individual epochs . Additionally, no significant (p > 0.05) effect was observed on peak power, or peak frequency across the entire range of analyzed KA-elicited activity  and then 2 mg/kg  had no effect on in vitro PrL oscillatory activity compared to saline controls (data not shown).To ensure that the observed lack of effect was not caused by the dose of MK-801 being too low, the above experiments were repeated using first 0.5 mg/kg (ex vivo. There were three main findings: (1) Administration of chronic subanesthetic ketamine at 30 and 5 mg/kg reduced the peak frequency of the elicited oscillations; (2) Chronic ketamine at 30 mg/kg also reduced the power of cortical GBO within the 40–50 Hz band; (3) Somewhat surprisingly, the effect of ketamine was not mimicked by a more selective NMDAR antagonist, MK-801. In the following sections we discuss these three findings in more detail.The results reported in this study represent the first investigation of the effects of chronic (5 days) application of NMDAR antagonists, ketamine, and MK-801, on gamma band oscillations (GBO) in the adult mouse neocortex, Several studies have suggested that the neocortical circuitry has an intrinsic resonance frequency , 30. IntWhile our previous findings with acute ketamine  are not evoked GBO in a variety of sensory or cognitive paradigms  recently coauthored a review article and have collaborated on funded and unfunded NIH research grants. However, Dr Kocsis was not involved in the design, execution, analysis or writing of the work presented in this manuscript.


Serbian soybean genotype Ana was gamma irradiated at doses of 1, 2, 4, and 10 kGy in order to evaluate the influence of gamma irradiation on isoflavone  contents and hydroxyl radical scavenging effect (HRSE). The increase in genistin and daidzin contents as well as antioxidant activities was observed especially at doses of 4 and 10 kGy. Results were also compared with our previous results relating to total phenol content (TPC), DPPH radical scavenger capacity (DPPH RSC), and ferric reducing antioxidant power (FRAP). Our results indicated that doses up to 10 kGy improve the antioxidant activities of soybean and also nutritional quality with respect to isoflavone content. All results were analyzed by multivariate techniques . Significant positive correlations were observed between genistin, daidzin, DPPH RSC, and HRSE. Legumes play an important role in the traditional diets of many regions throughout the world. Soybean and its processed products have been acclaimed as health foods due to their high content of protein and essential amino acids, omega-3 fatty acids, fat-soluble vitamins, polysaccharides, and insoluble fibers . Besidesp-coumaric acid. A form of flavonoid in soybean seeds, isoflavones, has been found to have important secondary compounds with many chemical actions. Isoflavones are categorized chemically by their functional groups. There are four subgroups: aglycones, glycosides, malonyl-glucosides, and acetyl-glucosides [Soybean seeds contain many phenolic compounds such as 5-O-caffeoylquinic acid, caffeic acid, ferulic acid, and ucosides . Isoflavucosides . DaidzeiGamma irradiation has long been known as a method for food preservation . IonizinGlycine max (L.) Merr.) of yellow coated genotype Ana were obtained from the Institute of Field and Vegetable Crops in Novi Sad. Soybean seeds were irradiated with the following doses of gamma radiation: 1, 2, 4, and 10 kGy. For that purpose the 60Co was used. The dose rate was 228 Gy/min. Seed irradiation was performed in the Laboratory for Radiation Chemistry and Physics “Gama” at the Institute of Nuclear Sciences in Vinča, Belgrade. Irradiated and nonirradiated soybean seeds (100 seeds) were ground in a mill and reduced to a fine powder. Description of the determination of total phenol and tannin contents, DPPH radical scavenging capacity, and FRAP was described in our previous work [Soybean seeds (ous work .•) formation was studied by electron spin resonance (ESR) using a spin trapping method [h0 and hx are heights of the second peak in ESR spectra of the control and sample, respectively. The influence of the soybean ethyl-acetate extract on hydroxyl radical  0.1% glacial acetic acid in ACN. Following injection of 20 μL of sample, solvent B was increased from 15% to 35% over 50 min and then held at 35% for 10 min. The solvent flow rate was 1 mL/min. A Waters 991 series photodiode array detector monitored from 200 to 350 nm. The minimum detectable concentrations for daidzein and genistein were 185 and 100 ng/mL, respectively. UVspectra were recorded and area responses were integrated by Waters PDA software.A linear HPLC gradient was composed of (A) 0.1% glacial acetic acid in Ht-test for independent observations. Differences were considered significant at P < 0.05. The antioxidant test results were investigated with multivariate analysis. The correlation matrix was calculated, giving the correlation coefficients between each pair of variables, that is, the analytical parameters tested. Each term of the matrix is a number ranging from −1 to +1; the + or − sign indicates a positive or negative interdependence between variables (direction), and the absolute value indicates the strength of the interdependence. Correlations between different parameters were considered significant at r > 0.95 (P < 0.05). Autoscaling transformation of data for antioxidant markers  was done using STATISTICA.All procedures (extraction and measurements) were obtained from triplicate measurements. Statistical comparisons between samples were performed with Duncan r = 0.8987).Data for total phenol and tannin contents in soybean were obtained from Štajner et al. . All datr = 0.9664) and isoflavone glycosides (r = 0.8785). Total isoflavone content was positively correlated with both glycosides, genistin (r = 0.9831) and daidzin (r = 0.9496). High negative correlations were found between isoflavone glycosides and their aglycones.Under the influence of gamma irradiation genistein content decreased from 21.19 (control) to 18.18 mg/kg (under dose of 10 kGy), daidzein content also decreased from 34.57 (control) to 29.00 mg/kg (10 kGy), genistin content increased from 368.2 (control) to 425.0 (10 kGy) mg/kg, and daidzin content increased from 242.1 (control) to 283.0 mg/kg (10 kGy). Total isoflavone content (TIC) gradually increased from 666.1 (control) to 755.2 mg/kg (10 kGy) . SignifiThe trend in total phenol content changes under the gamma irradiation in tested genotype Ana is consistent with the results obtained by Dixit et al.  where, aDixit et al.  investigr = 0.9182). Daidzin was significantly positively correlated with both parameters, DPPH RSC (r = 0.9581) and HRSE (r = 0.9873). Significant negative correlations were found between isoflavone aglycones (genistein and daidzein) and DPPH RSC and HRSE. Line plot of multiple variables after autoscaling transformation of positively correlated parameters  was presented by Data for DPPH RSC, FRAP, and HRSE are presented in •OH radical scavenging capacities. The structural heterogeneity of flavonoids in different genotypes of soybean, with multiple mechanisms of action, the diverse methods used to evaluate their antioxidant activity, and different experimental conditions of irradiation may all lead to different responses of soybean under irradiation. Our result that Ferric reducing antioxidant power significantly increased at low level dose of 1 kGy was in accordance with results of Dixit et al.  and it cPresent results showed that gamma irradiation of soybean with doses up to 10 kGy enhanced HRSE and accumulation of genistin and daizin. Significant positive correlation between genistin, daidzin, DPPH-RSC, and HRSE was found and negative correlation between isoflavone glycosides and aglycones was found. Thus, besides antimicrobial activity, radiation induces antioxidant activity and therefore increases nutritional quality.


GATA4 gene polymorphism with coronary artery disease (CAD) and its metabolic risk factors, including dyslipidaemic disorders, obesity, type 2 diabetes and hypertension, following a preliminary study linking early onset of CAD in heterozygous familial hypercholesterolaemia to chromosome 8, which harbours the GATA4 gene.The study was designed to evaluate the association of GATA4 gene in 250 individuals to identify variants of interest and then investigated the association of 12 single-nucleotide polymorphisms (SNPs) with the disease traits using Taqman chemistry in 4,278 angiographed Saudi individuals. Of the studied SNPs, rs804280 (1.14 (1.03 to 1.27); p = 0.009) was associated with CAD , hypercholesterolaemia  (1.61 (1.03–2.52); p = 0.037) and elevated low-density lipoprotein-cholesterol (hLDLC)  (1.87 (1.10–3.15); p = 0.020). Additionally, rs3729855_T ) and rs17153743 (AG + GG) (2.30 (1.30–4.26); p = 0.005) were implicated in hypertension , following adjustments for confounders. Furthermore, haplotypes CCCGTGCC  and GACCCGTG  constructed from the SNPs were associated with CAD and ACCCACGC  with myocardial infarction, while hypercholesterolaemia  and hLDLC  shared the AACCCATGT, and AACCCATGTC was associated with hLDLC . A 10-mer GACCCGCGCC  was associated with obesity , and the GACACACCC  was implicated in type 2 diabetes mellitus 2,378 vs 1,900).We first sequenced the whole Our study implicates GATA4 in CAD and its metabolic risk traits. The finding also points to the possible involvement of yet undefined entities related to GATA4 transcription activity or gene regulatory pathways in events leading to these cardiovascular disorders. GATA4 gene, as a plausible candidate for CAD, HFH and harbouring of low high-density lipoprotein levels. This led to the notion that GATA4 presents a potential candidate for CAD onset, especially in dyslipidaemic conditions. Given the lack of information on the role of this gene in the etiology of atherosclerosis, our study sought to comprehensively investigate the likelihood of GATA4 polymorphism predisposing individuals to acquiring the cardiovascular metabolic risk traits, particularly dyslipidaemia, obesity, type 2 diabetes mellitus and hypertension, as a potential trigger for the disease onset related to these disorders. To this effect, we first sequenced the gene in the two HFH families and an additional 250 individuals from the Saudi general population to identify potentially informative variants, and then performed a population-based association study for selected variants with CAD and its risk traits in a larger cohort of angiographed Saudi individuals.The GATA binding proteins constitute a family of cell-restricted zinc-finger transcription factors (TFs), which recognize the GATA motif present in the promoters of many genes. This family, comprising six developmental/cell-type specific transcription factors, GATA1-6, is critical to the development of diverse tissues  and actsThe initial scanning of the HFH yielded several peaks in different genomic regions including Chromosome (Chr) 8, among others, giving a logarithm of the odds (LOD) score of 1.8 that isolated at least three of the affected siblings with the early onset of CAD in both families. One of the potential culprits at this locus is GATA4, which we elected to pursue further for its role in dyslipidaemia-related onset of CAD. Subsequent sequencing of the genes in all members of the two HFH families and an additional 250 individuals led to the selection of 12 single-nucleotide polymorphisms (SNPs), rs2740434_G > A , rs17153743_A > G (0.02), rs13264774_C > T (0.14), rs56298569_G > C (0.44), rs804280 A > C (0.04), rs3729855 C > T (0.18), rs3729856_A > G (0.45), rs1062219 C > T (0.45), rs12825_C > G (0.09), rs804291_C > T (0.32), rs11785481 C > T (0.12), rs3203358 C > G (0.20) for further case-control studies. Selection of the SNPs was based partly on the prevalence in our general population and partly on currently available information of their role in the disease. Furthermore, these SNPs reside in the later portion of the gene , which encodes the C-terminal of the protein and harbours gene regulatory motifs, both of which are thought to be important in the transcriptional activity of the GATA4  in our study population, using a homogenous set of individuals with dilated cardiomyopathy (n = 272) as controls. The results pointed to a significant causative association for the rs3729856 A > G (p.S377G)  = 1.76 (1.19–2.61); p = 0.005), the rs3729856_GG (5.09 (2.14–12.06); p < 0.000001), and rs11785481_TT (6.23 (1.93–20.17); p < 0.002) with congenital heart disease. Interestingly, in the multivariate analyses for congenital heart disease versus the rest of the studied population , only the rs11785481_T (1.78 (1.06–3.00); p = 0.030) retained its significant association (Table p = 0.009) with CAD (Table p = 0.036) lost this property, following the adjustment for confounders. Interestingly, the rs804291_TT was associated with both hypercholesterolaemia (hChol) (1.61 (1.03–2.52) and elevated low-density lipoprotein-cholesterol (hLDLC) (1.87 (1.10–3.15); p = 0.020). In contrast, the rs3203358_G was protective against acquiring both CAD (0.88 (0.78–0.99); p = 0.042) and hChol (0.86 (0.75–0.98); p = 0.026) (Table p = 0.005) were implicated in hypertension, while the former appeared to be protective against type 2 diabetes mellitus (0.68 (0.53–0.88); p = 0.003)  was weakly associated with CAD, its 8-mer derivative CCCGTGCC  was associated with the disease. This relationship became stronger with the shortening of these haplotype sequences, culminating in the 4-mer ACCC  displaying the most significant relationship with disease  which also culminated in the 4-mer GACC  showing the most significant association with the disease. Put together, these trends indicate that the SNPs rs17153743 (2), rs13264774 (3), rs56298569 (4) and rs804280 (5) constituted the primary core for this association with CAD. Myocardial infarction was equipotently linked to the 8-mer ACCCACGC  and its 7-mer derivative ACCCACG , thereby sharing with CAD, the same three causative nucleotides of the four SNPs comprising the core of the relationships. Notable relationships also included the 10-mer AACACATCCC  and the 4-mer ATAC  showing the most significant protective property against acquiring MI.Since several SNPs were associated with the different cardiovascular disease traits, we were interested in testing the likelihood of these relationships being delineable at the haplotype level. We employed the most frequent 10-mer GACACACCCG (frequency = 0.145) as the baseline for the analyses. While the 9-mer haplotype, ACCCGTGCC  and elevated hLDLC levels  shared the common 9-mer AACCCATGT, while the 10-mer AACCCATGTC was associated with hLDLC , but only weakly so with hChol  , while low high-density lipoprotein was associated with the CCCAC .Since the outgoing notion was that GATA4 may mediate dyslipidaemia-mediated onset of CAD, it was interesting to investigate whether haplotyping might reveal any link with lipidaemic disorders. Interestingly, hChol  was associated with obesity, while a 9-mer GACACACCC  was implicated in type 2 diabetes mellitus  and its involvement in other cardiovascular disease pathways, such as those leading to atherosclerosis. Hence, the primary question raised in our study was whether a link existed between the impact of GATA4 on CAD manifestation and the presence of metabolic disorders, as our linkage study in HFH seemed to suggest.In the present study, we first screened two families with HFH in which the primary probands presented with severe phenotypes of early onset CAD. We identified several potential loci of which Chr 8 appealed as the most attractive choice for detailed investigation on the genetic basis for dyslipidaemia-related onset of the disease. This locus harbours the ulations ,26,30. TTo begin with, our results pointed to an association of the rs804291 and rs11785481 with both hChol and hLDLC, which appeared to follow primarily an autosomal recessive mode of inheritance. A closer analysis of the data also indicated that while the rs11785481 was not directly related to CAD per se, the probability of an individual acquiring CAD increased greatly in hypercholesterolaemic individuals. We also noted that two other SNPs that were not directly associated with hChol became so in the presence of MI, similarly indicative of a possible interaction of the dyslipidaemic disease traits with changes in GATA4 as a possible link to CAD/MI manifestation in these individuals. Moreover, the analyses for the other metabolic risk factors revealed the association for two variants with hypertension, further linking risk traits for metabolic syndrome to GATA4 polymorphism. Altogether, these observations furnish support for the notion of a contribution of some interactions of metabolic risk traits with GATA4 to the disease pathways leading to atherosclerosis, an assertion which requires further investigation.GATA4 variants relate to alterations in lipid levels and CAD/MI in this study lead to important questions regarding the possible mechanisms or pathways linking them with one another. Based on our results, it appears that such mechanisms may involve events associated with the harbouring of lHDLC, for example, being linked to pathways directly influencing circulating lipid levels and possibly leading to acquiring CAD in dyslipidaemic individuals, as indicated by the inverse relationship of some of these traits with the different GATA4 variants. Accordingly, the processes appear to be separable from those leading to cardiac malformations. Hence, the novel finding implicating the GATA4 in dyslipidaemia seems to point to yet undefined entities, possibly involving the regulation of the GATA4 functional state, as playing an important role in the disease process. In this regard, perhaps the most revealing observation of the present study is the linking of non-coding variants of the gene to disease. Of particular interest was the finding of causative haplotypes for CAD/MI as well as the metabolic risk traits encompassing variants in the 3′-UTR of the gene. Thereby, isolating the two coding SNPs from those residing in the 3′-UTR did not alter the significance level for the latter, possibly pointing to the changes at this chromosomal locus rather than the individual variants as the underlying genetic basis for these manifestations. Notably, the significance levels for the haplotype associations were higher than those of individual constituent variants, pointing to the importance of these genomic sequences in revealing the impact of a gene on disease that would otherwise remain uncovered through associations with changes at individual loci. Implications are that these events may be due to changes other than those directly pertaining to a functional motif of the GATA4 protein. Furthermore, the close proximity of these variants may also suggest the presence of sequences encoding some other yet unidentified entities as the potential culprits, especially considering the fact that several 3-mer haplotypes at this locus were shared by some of the traits. Thus, it is plausible to postulate that alteration at this genomic locus, and not necessarily in the GATA4 gene function per se, may offer an explanation for the observed link to alterations in the metabolic risk traits and CAD manifestation.The various ways in which the GATA4 gene may influence disease pathways. For example, both germline and somatic mutations have recently been described in this region that were predicted to affect RNA folding as cause for congenital heart disease t test asCAD: coronary artery disease; CHD: congenital heart disease; CBP: cAMP-response element-binding (CREB) protein; FH: family history of CAD; GATA4: GATA binding protein 4; hChol: hypercholesterolaemia; HFH: familial hypercholesterolaemia; hLDLC: high low-density lipoprotein-cholesterol; hTG: hypertriglyceridaemia; HTN: hypertension; lHDLC: low high-density lipoprotein-cholesterol level; MI: myocardial infarction; T2DM: type 2 diabetes mellitus; TFs: transcription factorsThe authors declare that they have no competing interests.NPM was involved in running the Affymetrix assays, designing probes, screening for gene mutations as well as participating in the write-up of the manuscript. SMW performed Affymetrix genotyping analysis. AIT performed the statistical analysis. SH ran the Affymetrix assays. MN and DG were responsible for the overall running of the Taqman assays. NAT contributed to statistical analysis. EA was responsible for procuring patient material and data. NM was responsible for the clinical patient data and material acquisition. BFM contributed to the write-up of the manuscript. ND is the Principal Investigator, with the overall responsibility for the project and preparation of the manuscript. All authors read and approved the final manuscript.GATA4 Suppl data: analysis for GATA4 interaction with age, sex and other confounders for the analyses of the different disease traits.Click here for fileGATA4 Haplo Suppl data: GATA4 haplotyping for the cardiovascular disease traits.Click here for file


Achyranthes aspera (family Amaranthaceae) is known for its anticancer properties. We have systematically validated the in vitro and in vivo anticancer properties of this plant. However, we do not know its mode of action. Global gene expression analyses may help decipher its mode of action. In the absence of identified active molecules, we believe this is the best approach to discover the mode of action of natural products with known medicinal properties. We exposed human pancreatic cancer cell line MiaPaCa-2 (CRL-1420) to 34 μg/mL of LE for 24, 48, and 72 hours. Gene expression analyses were performed using whole human genome microarrays . In our analyses, 82 (54/28) genes passed the quality control parameter, set at FDR ≤ 0.01 and FC of ≥±2. LE predominantly affected pathways of immune response, metabolism, development, gene expression regulation, cell adhesion, cystic fibrosis transmembrane conductance regulation (CFTR), and chemotaxis ). Disease biomarker enrichment analysis identified LE regulated genes involved in Vasculitis—inflammation of blood vessels. Arthritis and pancreatitis are two of many etiologies for vasculitis. The outcome of disease network analysis supports the medicinal use of A. aspera, viz, to stop bleeding, as a cure for pancreatic cancer, as an antiarthritic medication, and so forth. Large sections of the world population depend on medicinal plants  for their health care needs. The indigenous population use crude preparations of natural products in the form of decoction, paste, or as food supplement. The prevailing belief is that crude mixtures contain many compounds. Several of these compounds might be inactive by themselves but are essential for the effectiveness of the “active molecule” in the crude preparations. In other words, synergism negates the additive effect of individual molecules . Global Modern scientific standards emphasize thorough understanding of the molecular mechanism of action of single agent drugs. However, this approach is limited to single molecules, which have been chemically characterized. Even these drugs modulate the activities of disparate metabolic/signaling pathways . These rAnalytical methods like microarray, pathway focused PCR arrays, RNA seq, and cGH array give a snapshot of large scale changes at genome level. These methodologies generate large data sets. The development of data mining software has enabled discovery of molecular markers from the mass of data possible. Thus, the ability to analyze gene expression changes at genome level had revolutionized the molecular investigations that until now were difficult. For example, understanding global gene expression changes that accompany certain diseases like cancer has enabled the development of personalized medicine. Data analysis of microarray results also facilitated deciphering molecular pathways that are affected by both existing as well as newly discovered drugs .Achyranthes aspera (Family Amaranthaceae) is a medicinal plant in Ayurvedic pharmacopoeia. It is an annual shrub found in India, Southeast Asia, America, and Sub-Saharan Africa. It is classified as a weed [A. aspera leaves (LE) has antiproliferative activity against a variety of human cancer cell lines cultured in vitro. Our results also showed pancreatic cancer cells to be the most sensitive to LE. These exciting outcomes indicate the presence of active anticancer molecule(s) in A. aspera. Unpublished, anecdotal observations suggest that A. aspera benefited pancreatic cancer patients. Animal studies carried out with A. aspera extract confirm LE to be nontoxic to mice. Most importantly, these studies have shown that LE inhibited human tumor growth in mice [A. aspera on global gene expression pattern in human pancreatic cancer cell line MIA PaCa-2. Using MetaCore software, we narrowed down the pathways affected in MIA PaCa-2 cells treated with LE for three different time points.s a weed . Traditis a weed . Earlier in mice . In prel in mice , 8. ThesAchyranthes aspera leaves, and the preparation of whole leaf extract [A. aspera leaf powder was serially extracted in 100% hexane followed by 100% acetone over night at room temperature. The acetone extract was concentrated in a rotary evaporator. The residue was dissolved in 100% methanol and called leaf extract (LE).We have recently published the details about the collection, authentication of  extract , 8. Brie2 atmosphere. It was seeded in a six-well plate at an initial density of 5 × 105 in 2 mL medium. Overnight cultures were exposed to 34 μg/mL of LE for 24, 48, and 72 hours, respectively. Two percent methanol exposed cells served as a control. Control and experimental groups were set up in duplicate.The human pancreatic cancer cell line MIA PaCa-2 (CRL-1420) was purchased from ATCC (USA) and maintained in our laboratory in RPMI 1640 medium supplemented with 10% FBS  at 37°C in a humidified 5% COTotal RNA was prepared from control and LE treated MiaPaCa-2 cells using TriReagent . At the end of the treatment, cells were directly lysed in TriReagent and total RNA isolated. From this point, the rest of the gene expression related procedures were performed at the microarray and gene expression Core facility of the University of Miami. The quality of the extracted RNA was evaluated using BioAnalyzer 2100 . RNA samples with RNA integrity number (RIN) ≥9 have been used for the microarray experiments [Target RNA was amplified using Amino Allyl Message Amp kit . Amplified RNA from the control group was labeled with Cy-3 and the test group with Cy-5 dye . Duplicate microarray hybridization was set up using biological replicates that were labeled by dye-swap; that is, control RNA was labeled with Cy-5 and test RNA with Cy-3. The dye labeled cDNA samples were hybridized to the whole human genome microarray . We followed the experimental instructions provided by the kit supplier for RNA amplification, cDNA preparation, dye labeling, and array hybridization.μm resolution using a GenePix 4000B scanner . The scanned images were analyzed with GenePix Pro 6.1, the software package provided by Molecular Devices, USA. Quality control of the images was performed with Acuity 4.0  software package. The following quality criteria have been used: (1) at least 90% of the pixels in the spot had intensity higher than background plus two standard deviations, (2) less than 2% saturated pixels in the spot, (3) signal to noise ratio (defined as ratio of the background subtracted mean pixel intensity to standard deviation of background) was 3 or above for each channel, (4) the spot diameter was between 45 and 70 μm, (5) the regression coefficient of ratios of pixel intensity was 0.6 or above, and (6) feature has opposite change direction at dye-swap. Only features that passed all quality control criteria in all microarrays were analyzed further. To identify significantly expressed genes, the R package “limma” was used. “Within array” normalization was carried out with Lowess method, and “between arrays” normalization with “quantile” algorithm in the “limma” package. Features that did not pass the quality control criteria in all arrays were given weight of 0 and were excluded from the analyses. Differential expression and false discovery rate (FDR) were assessed using a linear model and empirical Bayes moderated F statistics. Genes that passed FDR ≤ 0.01 in all time points with a fold change of 2 or more in at least one time point and in the same direction in all time points were uploaded into MetaCore tool  for the identification of pathways affected by LE. The same data sets were uploaded to TM4, microarray data management, and analysis software to generate the heat map [The microarrays were scanned at 5 β-actin. The mean normalized expression (MNE) was calculated using the method developed by Muller et al. [The microarray gene expression data were verified by quantitative RT-PCR. Premade validated RT-PCR primers  used forr et al. . The folThe whole genome microarray  featured approximately 41,000, 60 nucleotide long oligonucleotide probes. These probes represent all known human transcripts. The microarray data were deposited in NCBI's “Gene Expression Omnibus” database repository; accession number is GSE44290 .Seven genes differentially regulated in microarray were selected for validation using quantitative real time PCR. The direction of fold change in gene expression observed in microarray analyses matched with the real time PCR data .http://dx.doi.org/10.1155/2013/471739). By this screening, we found that 1178 genes were common for all the three time points irrespective of the direction of expression change viz up or down regulation. Details of these common genes are listed in the Supplementary File S-1. In this data table, the same gene may be upregulated at one time point and downregulated at other time points. These 1,178 genes represented expression data that passed initial QC for all the three time points irrespective of direction of gene expression change triggered by LE treatment. Except for the QC described above, no preset fold change (FC) or false discovery rate (FDR) parameters were applied to these data sets. The lists of genes thus identified to be common for all three time points are detailed in “Spot test” sheet in the Supplementary File S-1.17,135 features including replicated spots passed technical QC see “” in all From this data set, we selected genes that passed FDR ≤ 0.01 at each of the three treatment time points. This analyses yielded 971 (637/334), 806 (325/481), and 3,075  genes that were unique to 24 h, 48 h, and 72 h time points, respectively (Supplementary File S-1). The numbers in parenthesis indicate the number of underexpressed and over expressed genes, respectively. A detailed gene list corresponding to the three time points is listed in sheets 24hFDR001, 48hFDR001, and 72hFDR001, respectively (Supplementary File S-1). The genes selected by this method were compared for commonly regulated genes between the different treatment time points. This analysis found 223, 191, and 2,009 genes to be unique to 24, 48, and 72 h, respectively. 38 genes were established to be common between 24 and 48 h, 489 between 24 and 72 hours, and 356 genes between 72 and 48 h. 221 genes were common for all the three time points. Though the genes may be common for the indicated parameters, the direction of regulation may not be the same for these genes at the compared time points. In this list, only the gene names were common. Other than FDR ≤ 0.01 the FC was not factored in this selection. Therefore, the same gene may have a positive and negative fold change at different time points . This data is graphically represented in Next, we limited the data analyses to genes that showed similar expression trend either up or down regulated over the three different experimental time points. This analysis yielded the following results. From across three different time points, 941 516/425) out of 1,178 genes showed similar expression pattern . From this list, 362 (248/114) genes showed FC ≥ ±2 . This list was further screened for genes with FDR ≤ 0.01 and thus we picked 89 (58/31) genes . Of the 89 genes, 82 had known valid accession IDs. Thus, from 41,000 probe sets on Human Genome microarray, we successfully selected 82 (54/28) genes that showed similar trend across all the three time points with FDR 0.01. In this list, all the genes in at least one of the three points qualified FDR ≤ 0.01 and FC ≥ ±2  to determine the possible signaling pathways affected by LE. This enrichment analysis identified 98 pathways to be regulated by LE. We grouped these 98 pathways that have similar effect. The top ten pathway maps of the 98 identified as above are given in P value was 1.216 × 10−5 , immune response_IL-27 signaling (P ≤ 1.712E − 03), immune response_HSP60 and HSP70/TLR signaling (P ≤ 8.466E − 03), translation_IL-2 regulation of translation (P ≤ 5.066E − 02), CFTR folding and maturation (norm and CF) (P ≤ 3.572E − 02), wtCFTR and delta F508 traffic/Late endosome and lysosome (norm and CF) (P ≤ 3.823E − 02), protein folding_membrane trafficking and signal transduction of G-alpha (i) heterotrimeric G-protein (P ≤ 4.819E − 02), DNA damage_role of SUMO in p53 regulation (P ≤ 4.322E − 02), DNA damage_NHEJ mechanisms of DSBs repair (P ≤ 4.819E − 02), transport_RAB3 regulation pathway (P ≤ 3.572E − 02), and cell cycle_role of Nek in cell cycle regulation (P ≤ 3.037E − 03). The rest of the 86 pathways possibly affected by LE had a P value ≥0.05.Besides the above, additional 11 pathways were significantly affected by LE. They are immune response_MIF-mediated glucocorticoid regulation . As reported in the literature, A. aspera is used as a cure for pancreatic cancer [in vivo [The expression of 82 genes that were identified to be influenced by LE was subjected to disease biomarker enrichment analysis in MetaCore tool. This investigation picked up a large number of diseases from this data set. The top 10 diseases along with the corresponding c cancer , as an ac cancer , and to [in vivo . Therefo[in vivo .In complementary and alternate medicine (CAM), unrefined (crude) preparations of natural products are used for the treatment of various illnesses. It is believed that compounds present in a crude preparation modulate the effect of the active molecule . In thisA. aspera. It is noteworthy that LE modulates the expression of genes involved in embryonic development, cell adhesion, transcription factors that regulate cell growth, and so forth. These effects support its' use as abortifacient [A. aspera, a medicinal plant used in Ayurvedic medicine, inhibited the growth of cancer cells in vitro and orthotopic pancreatic tumor in vivo. Many parts of this plant are used as a cure for a wide array of ailments that include fertility control, fetal expulsion, wound healing, and cancer therapy. Since LE is a crude extract we expected a large number of genes would be affected in the treated cells. However, stringent data filtering determined just 89 genes were affected by LE. Of these, seven were probes used for quality control. Thus, effectively, we have narrowed down the number of genes affected by LE to 82. The pathway data analyses demonstrated predominantly immune response is affected by LE, followed by metabolism, development, transcription, and so forth. The effects on developmental, gene expression regulation, and cell adhesion pathways imply that LE directly affects cancer growth and metastasis.We are interested in the anticancer properties of ifacient . Hithertin vitro [Mutations in cystic fibrosis gene are identified as a risk factor for chronic pancreatitis and pancreatic ductal adenocarcinoma , 23. Molin vitro  and the Our results demonstrate that it is possible to pin point possible mechanism of action of crude extract using carefully planned experiments. We applied several assumptions in our data analyses. For instance, we eliminated expressions that did not follow the same pattern. There are instances where a group of genes may be up regulated in the short term and down regulated over long term exposure to LE or vice versa. Though less likely, another set of genes may be up and down regulated in a cyclical manner. By considering unidirectional regulation, we would have eliminated such unique changes effected by LE. Ambiguities in gene expression analysis may be reduced and the robustness of expression data may be increased by using several experimental replicates, multiple different cell lines, and different drug concentrations. Additionally, gene expression analyses may be performed in tumor samples obtained from animal model that had been treated with LE.Anoectochilus formosanus and a single compound drug plumbagin [The identification of regulation of 82 genes from a large data set encourages us to think that natural products may target a limited set of genes. There are evidences to support such observations. For instance, almost identical results were obtained in MCF-7 breast cancer cells that were treated with a crude extract of lumbagin . HoweverIn conclusion, we have demonstrated that LE affected the expression of only a few genes. Using similar methodology, it is possible to get reasonably good overview of the changes induced by crude preparation of natural products with medicinal properties.The gene expression data is deposited with the NCBI's “Gene Expression Omnibus” database repository under the accession number GSE44290.Supplementary data Table S-1 and supplementary Figure SF-1 are available online athttp://dx.doi.org/10.1155/2013/471739. The data referred in sections 3.1, 3.2 & 3.3 are included in the supplementary data Table S-1. The legends for the objects in Figure 4 are given in the supplementary Figure SF-1. Click here for additional data file.Click here for additional data file.


Background. Oxidative stress plays a role in the pathogenesis of many chronic inflammatory lung diseases. Exhaled breath condensate (EBC) collection is a noninvasive method to investigate pulmonary oxidative stress biomarkers such as malondialdehyde (MDA). Subjects and Methods. We measured MDA levels in EBC in a large number of patients (N = 194) with respiratory diseases: asthma (N = 64), bronchiectasis , chronic obstructive pulmonary disease , idiopathic pulmonary fibrosis . Fourteen healthy nonsmoking subjects were included as controls. Results. Excluding IPF subjects, MDA levels were significantly higher in all disease groups than in control group. MDA was significantly higher in COPD than asthmatic and BE subjects. Among asthmatics, corticosteroids-treated subjects had lower MDA levels than untreated subjects. COPD subjects showed an inverse correlation between MDA concentrations and FEV1% . Conclusions. EBC-MDA is increased in subjects with chronic airway disorders, particularly in COPD, and it is related to FEV1 reduction. Oxidative stress plays an important role in the pathogenesis of many chronic inflammatory lung disorders, particularly in COPD and asthma, where it is an important consequence of irritant-induced damage of bronchial epithelial cells and represents an amplifying mechanism through the recruitment of inflammatory cells in the airways , 2.Among the many biological targets of oxidative stress, membrane lipids are the most commonly involved class of biomolecules. Lipid peroxidation yields a number of secondary products able to boost oxidative damage . In addi2O2, adenosine, isoprostanes, leukotrienes and cytokines, and an increasing number of studies report the use of EBC in the investigation of airway lining fluid composition in several respiratory diseases [Exhaled breath condensate (EBC), obtained by cooling exhaled air, has recently been proven as a good biological tool to monitor inflammatory and oxidative stress of lower respiratory tract . Severaldiseases .Malondialdehyde (MDA) has been widely studied as a product of polyunsaturated fatty acid peroxidation. High MDA levels have been observed in several biological fluids from patients with different airway diseases including asthma –11, COPDIn this study, we measured and compared EBC levels of MDA in a large group of subjects with different pulmonary diseases in order to explore the possible usefulness of this measurement in the assessment of different lung diseases. N = 64), bronchiectasis , chronic obstructive pulmonary disease  and idiopathic pulmonary fibrosis  were included in the study. Fourteen healthy nonsmoking subjects were included as controls.Exhaled breath condensate was collected from 194 subjects affected by different airway diseases attending to our pulmonary unit. Patients with asthma  and the results were expressed as percentage of CECA  predicted values .All patients were examined in a stable phase of the disease, with no acute exacerbation in the previous month. Informed consent for participation to the study was obtained from all patients. EBC was collected cooling exhaled air with a specifically designed condenser . Subjects breathed tidally for 15 min through a two-way non-rebreathing valve in order to prevent inspiratory and respiratory air mixing and trap saliva , 11. 2PO4 0.3 M, pH 3.5) in a water bath at 95°C for 60 minutes, cooled in ice for 5 minutes, and then allowed to recover at room temperature for 40 minutes before analysis. Malondialdehyde (MDA) concentrations were measured in EBC samples according to the method described by Lärstad et al. . Samplesλ fluorescence detector, Waters, Milan, Italy), equipped with a Polaris C18 column, 150 × 4.6 mm ID, particle size 5 μm . Excitation and emission wavelengths were of 532 and 553 nm, respectively. The mobile phase was composed by acetonitrile: 20 mM potassium phosphate buffer pH 6.8 , the flow rate was 1.3 mL/min. The method had a limit of detection of 4.1 nM and a recovery of 96%. Intra- and interassay reproducibility were 1.9 and 8.7%. Ri) was 0.854. Analyses were performed using a high-performance liquid cromatography (HPLC) connected to a fluorescence detector (Binary HPLC pump 1525 and 2475 multi N = 52), COPD (N = 41) and bronchiectasis (N = 15). Sputum induction and analysis were carried out as previously described [1 lower than 35%, isotonic saline (IS: NaCl 0.9% w/v) was used as recommended in [1 fell by 20% or more from baseline values. Before and every 5 min after the start of nebulization, FEV1 was measured, and patients were then asked to rinse their mouth and throat carefully to discard saliva and to try to cough sputum into a container. Sputum was performed in 108 subjects with asthma  in a shaking bath at 37°C for 15 min, then centrifuged to separate cells from supernatant. The cell pellet was resuspended in PBS for viability and total cell count, and aliquots were used to prepare slides for differential cell counts. At least 300 inflammatory cells were counted. Macrophage, lymphocyte, neutrophil, and eosinophil values were expressed as percent of total inflammatory cells. Slides with cell viability <50% and an amount of squamous cells such that 300 inflammatory cells could not be counted were considered inadequate and discarded. t-test and Mann-Whitney test for normally and nonnormally distributed variables, respectively. Differences among three or more groups were tested using ANOVA and Kruskal-Wallis test for normally and nonnormally distributed variables, respectively. Correlations between functional and biological indices were evaluated using Spearman's rank correlation test. A P value lower than  .05 was considered as significant.Functional data were expressed as mean and standard deviation and biological data as median and range. Differences between two groups were tested using unpaired Ri values over 0.70 are usually interpreted as satisfactory) and the Bland-Altman plot was used to graphically represent the variability between the two measurements [Intraclass correlation coefficient was calculated to assess MDA interassay reproducibility . COPD patients had significantly higher levels of MDA than asthmatic and BE subjects (37.0 (8–126) versus 26.3 (6–91) nM, P < .05, and 19.2 (6–54) nM, P < .05, resp.). Within the COPD group, subjects with severe COPD showed higher levels of MDA than patients with moderate COPD (39.0 (8–126) versus 29.1 (9–81) nM, resp., P < .05).Except for IPF patients, who showed MDA levels similar to those of healthy controls (15.2 (4–79) versus 15.6 (4–25) nM, resp.), all the disease groups showed higher levels of MDA than healthy controls . Among a1 was observed both in the whole group of patients  and in the COPD subgroup  (P = .52). Considering each disease group separately, we found that among COPD patients, MDA levels in current smokers were not different from ex- and nonsmokers (P = .11),   whereas among asthmatics, current smokers had higher MDA levels than ex- and nonsmokers (38.5 (19–91) versus 25.5 (6–72) nM, P < .05).An inverse correlation between MDA concentrations and FEV: −0.24) . No diffP < .05) than patients with low sputum neutrophils, even when COPD patients only were considered  sputum nsidered . No diffnsidered . 1 and neutrophilic inflammation, particularly in COPD patients. We found that subjects with chronic airway disorders have increased levels of MDA in EBC, thus confirming that oxidative stress plays a role in the pathophysiology of lung diseases. We also found that MDA concentrations in EBC are related to FEVMDA is a marker of oxidative stress that can be measured, using different and sometimes complex analytical procedures , 11, in In our experience, MDA measurement in EBC was reproducible and accurate: interassay reproducibility was good and not different from what reported by other authors using different, more complex methods . We checOur method differentiates normal subjects from patients with different airway diseases despite some overlapping of single measurements. Furthermore, MDA was sensitive to changes induced by therapeutic intervention, as shown by the lower levels of MDA in asthmatics treated with ICS in comparison with untreated asthmatics. This issue is controversial in literature: while some studies reported a reduction in MDA levels after corticosteroid treatment , other s1. These results are in agreement with other reports [COPD subjects showed the highest values of MDA in EBC. This observation was expected, considering that oxidative stress plays a relevant role in smoke-induced airway damage and chronic inflammation. This was confirmed by the higher levels of MDA in patients with higher percentage of sputum neutrophils, and by the inverse correlation between EBC-MDA and FEV reports , suggest reports –33. No d reports .In asthmatic patients, EBC-MDA levels were lower than those observed in COPD patients, but higher than normal subjects. There was no difference according to asthma severity, but ICS treatment was associated with lower values of MDA. While some authors demonstrated a significant decrease in blood MDA during the recovery from asthma exacerbation treated with oral corticosteroids , other aIn bronchiectasis, we found EBC-MDA levels slightly higher than normal subjects, but lower than COPD patients. This result may be unexpected, considering that sputum neutrophilia is a prominent feature of this kind of patients. We found no papers evaluating EBC-MDA levels in bronchiectasis, and only one paper reports increased blood MDA levels in children with bronchiectasis . IncreasIn IPF, no difference in EBC-MDA levels was observed in comparison with healthy subjects. In the literature, few data are reported showing EBC increase of hydrogen peroxide, 8-isoprostane and nitrates in comparison with healthy subjects , 39. TheIn conclusion, we demonstrated that MDA may be accurately measured in EBC of most patients with chronic pulmonary diseases and that it is partially related to the severity of airway obstruction and neutrophilic inflammation. This was particularly evident in COPD patients, where oxidative stress seems to play a major role in the pathophysiology of the disease. Thus, MDA may be a promising marker of oxidative stress in asthma and COPD. Its usefulness in the management of these diseases, as well as for other inflammatory markers, needs to be explored with well-designed, specific studies.


Rapid diagnostic tests (RDTs) are the current complement to microscopy for ensuring prompt malaria treatment. We determined the performance of three candidate RDTs  for rapid diagnosis of malaria in the Central African Republic.Blood samples from consecutive febrile patients who attended for laboratory analysis of malaria at the three main health centres of Bangui were screened by microscopy and the RDTs. Two reference standards were used to assess the performance of the RDTs: microscopy and, a combination of microscopy plus nested PCR for slides reported as negative, on the assumption that negative results by microscopy were due to sub-patent parasitaemia.We analysed 436 samples. Using the combined reference standard of microscopy + PCR, the sensitivity of Paracheck™-Pf was 85.7% , that of SD Bioline Ag-Pf was 85.4% , and that of SD Bioline Ag-Pf/pan was 88.2% . The tests performed less well in cases of low parasitaemia; however, the sensitivity was > 95% at > 500 parasites/μl.Overall, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan performed slightly better than Paracheck™-Pf. Use of RDTs with reinforced microscopy practice and laboratory quality assurance should improve malaria treatment in the Central African Republic. In countries in sub-Saharan Africa, malaria management is frequently based on clinical criteria, leading to overuse of antimalarial agents ,2. AfterPfHRP-2 gene [The World Health Organization (WHO) recommends parasitological confirmation for all patients suspected of having malaria before starting treatment. This recommendation includes high-quality microscopy or, when that is not available, use of easy-to-use rapid diagnostic tests (RDTs) . Panel sP-2 gene -21, the P-2 gene , cross-rP-2 gene ,24 and tP-2 gene . Local dP-2 gene ,26-28.PfHRP-2 and −3 found wide variation in PfHRP-2 sequences in samples from the Central African Republic. The authors speculated that this might reduce the sensitivity of RDTs for detecting malaria in people with very low parasite density [A recent multi-centre study assessing global sequence variation in  density . In this density . The Min density . The objPlasmodium falciparum, and malaria transmission occurs throughout the year, with peaks at the beginning and the end of the rainy season, although no data are available on the intensity of transmission. Malaria accounts for more than 40% of morbidity in the country .This cross-sectional study was conducted in August 2011 in Bangui, capital of the Central African Republic, in the Hospital de l’Amitié, the Complexe Pediatrique and the Institut Pasteur of Bangui. Bangui is located beside the Oubangui River, north of the Democratic Republic of the Congo . The climate is tropical, and rainfall peaks between April and November. The average temperature varies from 19°C to 32°C. The main malaria parasite is The Hospital de l’Amitié and the Complexe Pédiatrique are tertiary referral public health centres equipped with laboratories where thin and thick smears are analysed. The Institut Pasteur of Bangui is a private centre  for biomedical research, in which laboratory diagnosis for malaria is performed for patients referred by clinicians at health centres where this test is not available.P. falciparum-specific histidine-rich protein type 2 (PfHRP2), while Bioline Malaria Ag Pf/Pan contains antibodies targeting both PfHRP2 and lactate dehydrogenase specific to P. falciparum and other Plasmodium species . Blood samples from people attending each study centre for laboratory analysis were tested for the presence or absence of malaria parasites by microscopy and the RDTs. Samples from patients with negative microscopy (regardless of RDT result) were tested P. falciparum by nested PCR, on the assumption that false-positive results with RDTs are due to sub-patent parasitaemia [We analysed the performance of three RDTs: Paracheck™-Pf, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf and /pan . SD Bioline Malaria Ag Pf and Paracheck™-Pf contain antibodies against sitaemia .Consecutive patients of all ages presenting at each study site were approached by the study technician for recruitment. Each patient’s medical card was checked, and those with a request for smear analysis were considered eligible if they were febrile (axillary temperature ≥ 37.5°C) or had a history of fever in the previous 24 h. They were included in the study if they gave written consent; for patients aged ≤ 18 years, informed consent was provided by a parent or guardian.This project was reviewed and approved by the ethical committee of the University of Bangui. The Central African Republic Health Ministry also gave approval for this study.For an estimated annual malaria rate in Bangui of 40% , an expected sensitivity of Bioline RDTs of 95%  and a seIn each study centre, the laboratory team consisted of three technicians with university training, all of whom had been re-trained for diagnosing malaria in the laboratory by standard operational procedures . The tecP. falciparum. Parasite density was determined as the number of parasites per 200 leukocytes on the assumption of an average leukocyte count of 8000/μl of blood. A result was considered negative if no parasites were detected per 200 leukocytes. Each slide was read independently by two study technicians. In the case of a discrepant qualitative result (negative or positive), a third reading was done by the designated technician at the Institut Pasteur. All the laboratory technicians were blinded to the RDT results. The results of both microscopy and the RDTs were reported to the clinicians, who were advised to treat the patient for malaria if the results of these two analyses were discrepant.Finger-prick blood samples were obtained for slides and for testing with the RDTs. Blood smears were air-dried, stained with 4% Giemsa and analysed under a light microscope (× 100 oil immersion) to detect asexual forms of The RDTs were performed by the third technician at the study centre following the manufacturer’s instructions.For patients with negative microscopy, three drops of blood were collected on a piece of filter paper (Whatman®). The blood spots were air-dried and stored at 4°C in individual sterile plastic bags for PCR analysis.Plasmodium DNA [Parasite DNA extraction and PCR assays were performed at the Institut Pasteur. The blood-impregnated filter paper piece was washed with distilled water and placed directly in a PCR tube containing the PCR reaction components. Genomic DNA was determined in an assay based on nested PCR for dium DNA .Data were entered onto Excel spreadsheets and analysed with MedCalc®software .P) of 0.05.The first step of our analysis was to determine the performance of the RDTs according to the falciparum density in samples found positive by microscopy. Density was categorized as ≤ 100, 101–200, 201–500, 501–1000, 1001–5000, 5001–50 000 and > 50 000 parasites/μl. RDT results were compared (paired proportions) with the McNemar test, at a level of significance . According to the PCR results, the results of the RDTs were designated as TP2 if PCR was positive in FP1, FP2 if PCR was negative in FP1, FN2 if PCR was positive in TN1 and TN2 if PCR was negative in TN1. Combination of the results of the RDTs for samples positive by microscopy and of PCR for samples negative by microscopy gave the values TP = TP1 + TP2, TN = TN2, FP = FP2 and FN = FN1 + FN2, which were used to calculate the performance of the RDTs.The second step was to estimate the performance of the RDTs against the combined results of microscopy and nested PCR, expressed as true-positive (TP), true-negative (TN), false-positive (FP) or false negative (FN). The formulas used to calculate performance were TP/TP + FN for sensitivity, TN/TN + FP for specificity, TP/TP + FP for positive predictive value (PPV) and TN/TN + FN for negative predictive value (NPV). The results were interpreted with 95% confidence intervals (CIs). At baseline, the results of the comparison between the RDTs and microscopy were expressed as TPP. falciparum. In 234/235 (99.6%) cases of infection, P. falciparum was the only parasite species identified. One infected individual had a P. ovale mono-infection  and was hence excluded from the study, which was confined to detection of falciparum malaria.A total of 437 patients were recruited for this study between 8 and 22 August 2011. Microscopic analysis showed that 53.8% (235/437) of the blood slides were positive for The RDTs gave positive results in 52.3% (228/436) of cases with Paracheck™-Pf, 57.8% (252/436) with SD Bioline malaria Ag-Pf and 58.0% (253/436) with SD Bioline malaria Ag-Pf/pan.Comparison of the results of the RDTs with those of microscopy showed concordant positive results in 85.9% (201/234) of cases with Paracheck™-Pf, 92.3% (216/234) with SD Bioline malaria Ag-Pf and 92.3% (216/234) with SD Bioline malaria Ag-Pf/pan. Of the samples that were negative by microscopy, 13.4% (27/202) were found to be positive with Paracheck™-Pf, 17.8% (36/202) with SD Bioline malaria Ag-Pf and 18.8% (38/202) with SD Bioline malaria Ag-Pf/pan.p = 0.04)  by microscopy and 7 false-negative results (7/202 or 4.2%) by both microscopy and the RDTs. Overall, the combined results of microscopy and PCR showed a sensitivity of 85.7% for Paracheck™-Pf, 85.4% for SD Bioline malaria Ag-Pf and 88.2% for SD Bioline Ag-Pf/Pan. The specificity was 86.0% for Paracheck™-Pf, 86.3% for SD Bioline malaria Ag-Pf and 80.4% for SD Bioline malaria Ag-Pf/pan.The overall sensitivities, specificities, NPV and PPV for the three RDTs, evaluated against microscopy and microscopy and PCR are detailed in Table P. falciparum samples be at least 75% at 200 parasites/μl, although discrepancies between these scores and clinical sensitivity are limitations of these tests [The sensitivity and specificity of the tested RDTs found in this study appear to fall within the ranges quoted in other studies, which reported a sensitivity of Paracheck™-Pf of 82.9–100% and a specificity of 56.0–96.4% -42 and ase tests ,47.HRP2 during a malaria episode with low parasitaemia might not be detected by RDTs [In this study, some blood samples that were negative by microscopic examination gave negative results in all three RDTs, while some gave positive results in all RDTs. Factors that affect the sensitivity and specificity of RDTs, such as low parasitaemia, are particular challenges for malaria diagnosis with these new devices. We found that a parasitaemia between 40 and 400 parasites/μl in blood samples with positive results by microscopy gave negative results in all three RDTs. This finding corroborates those of other studies -52, whic by RDTs  because  by RDTs .HRP 2 gene may reduce the sensitivity of these RDTs [PfHRP2 sequences in samples from the Central African Republic might reduced the sensitivity of RDTs for detecting malaria at very low parasite density [HRP 2 in humans [Furthermore, deletions or mutations in the ese RDTs -21. It h density . Other f density  and the n humans , might ePfHRP2 from previous malaria infections [Cases incorrectly identified as positive by the RDTs might be due to the persistence of fections , cross-rfections ,24 or otfections .This study has some limitations. First, PCR was performed only on samples found negative by microscopy. It might be considered incorrect to use a different “gold standard” for slides found to be positive and those found to be negative by microscopy, by using PCR only for slides found negative by microscopy. However, the risk for false-positive microscopy results was considered low, as the slides were read independently by two experienced technicians. The fact that the microscopists counted only 200 negative leukocytes before declaring a slide as negative might have decreased the sensitivity of microscopic detection in this study. PCR is a useful gold standard because it is highly sensitive, can detect cases with low parasitaemia that are missed by other techniques and is easily reproducible . NeverthPlasmodium, even if they are less prevalent in the Central African Republic. Moreover, introduction of these tests will indisputably help to identify malaria cases in this area, where microscopy is still of poor quality. Microscopy should nevertheless complement RDTs for determining parasite density, and therefore reinforcement of microscopy skills by ongoing training of technicians and quality assurance of both RDTs and microscopy should be considered priorities in national malaria programmes. The results of this study also indicate that RDTs should be evaluated in each new setting before they are deployed, in view of possible variations in performance in different populations.This study showed that the performance of SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan was better than that of Paracheck™-Pf. SD Bioline malaria Ag-Pf/pan is the most useful test because it detects other species of The authors declare that they have no competing interests.DD and JMM conceived the study, with substantial contributions from AM and NM. DD, JI, JCG and GT participated in field data collection and laboratory analysis. AM, DD and SB interpreted the data and drafted the manuscript. All the authors have read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/109/prepub


With growing evidence on the role of inflammation in cancer biology, the presence of a systemic inflammatory response has been postulated as having prognostic significance in a wide range of cancer types. The derived neutrophil to lymphocyte ratio (dNLR), which represents an easily determinable potential prognostic marker in daily practise and clinical trials, has never been externally validated in pancreatic cancer (PC) patients.Data from 474 consecutive PC patients, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the prognostic relevance of dNLR, univariate and multivariate Cox regression models were applied.p = 0.041) was identified.We calculated by ROC analysis a cut-off value of 2.3 for the dNLR to be ideal to discriminate between patients’ survival in the whole cohort. Kaplan-Meier curve reveals a dNLR≥2.3 as a factor for decreased CSS in PC patients . An independent significant association between high dNLR≥2.3 and poor clinical outcome in multivariate analysis (HR = 1.24, CI95% = 1.01–1.51, In the present study we confirmed elevated pre-treatment dNLR as an independent prognostic factor for clinical outcome in PC patients. Our data encourage independent replication in other series and settings of this easily available parameter as well as stratified analysis according to tumor resectability. Pancreatic cancer (PC) is the ninth most common cancer but ranks forth as a cause of cancer-related mortality worldwide Tumor inflammation and immunology have recently been identified as enabling cancer characteristics, and increasing evidence supports their involvement in cancer progression and metastases This retrospective study included data from 474 consecutive patients with histological confirmed pancreatic adenocarcinoma, who were treated at the Division of Clinical Oncology, Medical University of Graz between 2004 and 2012. Patients where PC diagnosis was made by cytology or assumed by radiological assessment without proven histology from biopsy or surgical resection samples were not included in this study. Also other rare histological subtypes such as azinus cell carcinoma or neuroendocrine carcinoma were not included as they are associated with different prognosis p<0.05 was considered statistically significant.Cancer-specific survival was defined as the time (in months) from date of surgery or date of histological proven diagnosis to cancer-related death. First, the pre-published cut off value of 2 was applied for the continuous dNLR. Nevertheless, we seek an ideal cut-off value for the continuous dNLR by applying receiver operating curve analysis (ROC) analysis as previously reported Of the 474 patients with PC, there were 256 (54%) male and 218 (46%) female patients diagnosed with PC. The mean age at diagnosis was 64.5±10.4 years. Median survival was 7 months (range 0–79 months) and 408 (85.7%) patients died by their most recent follow-up visit. The mean dNLR was 2.77±4.19 and the median dNLR was 2.17. The AJCC tumor stage was defined as stage I in 5 patients, stage IIa in 18 patients, stage IIb in 85 patients, stage III in 33 patients and stage IV in 333 patients. Three hundred forty four patients underwent chemotherapy with gemcitabine. The patients who did not receive chemotherapy were either in a poor Karnofsky index, had contraindication with regard to co-morbidities or reject the recommended chemotherapeutic treatment. In 135 (28.5%) patients, a surgical resection of the tumor has been performed see also .Primarily, we validated the pre-published value of 2 as the cut-off for the continuous dNLR. To investigate whether a high dNLR was associated with clinical outcome in PC patients, univariate and multivariate analyses were performed. Univariate analysis identified older age , high tumor stage , high tumor grade , no chemotherapeutic treatment , low Karnofsky index , surgical resection (p<0.001) and increased dNLR ratio  as prognosticators of poor outcome for patients’ CSS, whereas gender and level of CA19-9 were not statistically significantly associated with CSS .Furthermore, high dNLR was significantly correlated with high tumor stage, lower rate of surgical resection, higher CA19-9 levels and the lack of administration of chemotherapy (p<0.05), whereas no association with gender, age and tumor grade could be found (data not shown). To determine the independent prognostic value of the dNLR for CSS, a multivariate analysis using a Cox proportional hazard model was performed. In the multivariate analysis that included age, gender, CA19-9 levels, tumor stage, tumor grade, administration of chemotherapy, Karnofsky index, surgical resection and dNLR, we identified tumor stage (p<0.001), tumor grade (p<0.001) and administration of chemotherapy (p<0.001) as independent prognostic factors for CSS, whereas the other parameters including the dNLR ratio (cut off at 2) were not significantly associated with CCS . Therefore, applying the criteria mentioned above, we determined by using ROC analysis a cut-off value of 2.3 for the dNLR to be best to discriminate between patients’ survival in the whole cohort. This cut-off value prompted us to re-evaluate the dNLR as a universally useful prognostic biomarker in our study cohort. In the present study, we externally validated for the first time the dNLR in patients with PC and found a significant association between an elevated pre-treatment dNLR and poor clinical outcome. A high dNLR reflects an increased neutrophil and/or a decreased leucocyte ratio. It is generally accepted that inflammatory processes in the tumor microenvironment play a crucial role in promoting proliferation, invasion and metastasis of malignant cells In conclusion, our study provides evidence that pre-treatment dNLR can be considered as a promising independent prognostic parameter in PC patients. Further independent prospective trials are warranted to confirm these results.


Aeromonas hydrophila (AH) is an aquatic bacterium. We present a case of fifty-five-year-old gentleman with chronic kidney disease (CKD) due to crescentic IgA nephropathy who presented to us with fever. He was recently pulsed with methyl prednisolone followed by oral prednisolone and discharged on maintenance dialysis through a double lumen dialysis catheter. Blood culture from peripheral vein and double lumen dialysis catheter grew AH. We speculate low immunity due to steroids and uremia along with touch contamination of dialysis catheter by the patient or dialysis nurse could have led to this rare infection. Dialysis catheter related infection by AH is rare. We present our case here and take the opportunity to give a brief review of AH infections in CKD patients. AH is Gram-negative, rod-shaped facultative anaerobe. It can exist in aquatic environment, fish, food, birds, pets, and natural soil. AH can cause gastrointestinal and Nongastrointestinal infections. Nongastrointestinal infections include hemolytic syndrome and kidney disease, cellulitis, wound and soft-tissue infection, meningitis, bacteremia and septicemia, ocular infections, pneumonia and respiratory tract infections, and urinary tract infection in neonates, osteomyelitis, peritonitis, and acute cholecystitis –4. AH can cause infection in immunocompromised host. Ko and his colleagues found AH sepsis in liver cirrhosis 54%) and malignancy (21%) 4% and ma diabetes  hemoglobin: 9 g/dL,  white cell counts: 4,700 u/L, 3. platelet count: 171,000/mmUrine DR showed yellow color with clear appearance, pH of 5, specific gravity of 1.010, 4 red blood cells per high-power field, 1 WBCs per high-power field, urine protein of 1.0 g/L, and 2 RBCs cast and urine protein to creatinine ratio: 3.9,   BUN: 109 mg/dL,  creatinine: 4.8 mg/dL,  electrolytes: serum sodium 141 mmol/L, potassium 5.0 mmol/L, chloride 116 mmol/L, and bicarbonate 14.5 mmol/L,  ANA: negative,  C-ANCA: negative,   P-ANCA: negative,  hepatitis B surface antigen: nonreactive,  hepatitis C antibody: nonreactive,  PT/APTT/INR: 10.5/30.2/1.Because of symptomatic uremia, a right internal jugular nontunneled catheter was inserted on 28 of July 2012 for hemodialysis. In view of his normal size kidneys, a biopsy was done on 1 August 2012 to look for reversible causes. The biopsy showed 20 glomeruli in which 14 were globally sclerosed or severely collapsed and condensed. Several of them were associated with fibrocellular crescents with disruption of bowman's capsule and 4 had cellular crescents. Immunofluorescence showed IgA positivity in the mesangium and along the capillary walls. He was pulsed with methyl prednisolone 500 mg once a day for 3 days followed by oral prednisolone 25 mg twice a day. Cyclophosphamide or mycophenolate mofetil was not added in view of 14/20 sclerosed glomeruli and fibrocellular crescents. As there was no sign of renal recovery, the patient was discharged on three times a week dialysis through right internal jugular catheter with followup in nephrology outpatients clinic.A fifty-five-year-old Afghanistan gentleman with a history of hyperthyroidism (5 years) and ischemic cardiomyopathy (2 years) presented to Aga Khan University Hospital in July 2012. He was found to have kidney failure 2 months previously and was initiated on hemodialysis due to uremic encephalopathy. He was admitted to our institution for further work-up investigations for his kidney failure. His laboratory investigations were as follows:3. There was no evidence of hemolysis as his reticulocyte count was 2% and LDH was 250 IU/L. His iron study showed trasferrin saturation of 12%. Chest X-ray showed bilateral hilar congestion and minimal bilateral pleural effusion. Blood cultures were sent both from peripheral vein and dialysis catheter. In view of one episode of hemoptysis, bronchoscopy was done which was normal. Bronchoalveolar lavage showed no growth of acid fast bacilli and fungal smear was negative. Blood cultures from peripheral veins showed AH which was sensitive to piperacillin/tazobactam, imipenem, ceftriaxone, cefixime, ciprofloxacin, and gentamicin. Blood culture from dialysis catheter also grew the same organism with the same sensitivities. Dialysis solution culture did not grow anything. His dialysis catheter was removed after hemodialysis and 1 pint of pack cells was transfused. The patient was also started on erythropoietin 4000 unit subcutaneously three times a week. Intravenous iron was not given in view of sepsis. He was treated with piperacillin-tazobactam 2.25 gm 8 hourly for a total of 14 days. He responded to antibiotics and became afebrile after 48 hours. A second right internal jugular nontunneled catheter was inserted after 48 hours of antibiotics. He was discharged on 16 August 2012 with a new right internal jugular temporary catheter with the advice of three times a week hemodialysis. A plan for arteriovenous fistula after two weeks of antibiotics was also made. His repeat blood cultures on day 5 were negative. His anemia responded to erythropoietin and pack red cell transfusion. After completion of course of antibiotics, he was put on intravenous iron sucrose 100 milligram each dialysis for a total of 10 doses. Subsequent laboratory results on day 5 showed hemoglobin of 10 gm, hematocrit of 30%, MCV 82 fl, WBC 5000 u/L, and platelet count of 170000/mm3.He was readmitted on 8 August 2012 approximately one week after discharge, with a two-day history of high grade fever, mild productive cough, frothy sputum, and hemoptysis. On physical examination, he was febrile with a temperature of 39°C, a heart rate of 100 beats per minute, a respiratory rate of 24 breaths per minute, blood pressure of 139/68 mmHg, and oxygen saturation of 99% at room air. He was pale and nonedematous. He had bilateral basal crepitations on chest examination. The rest of the physical exam was unremarkable. His laboratory workup showed BUN 74 mg/dL, serum creatinine, 4.8 mg/dL, serum sodium 135 mmol/L, potassium 4.7 mmol/L, chloride 109 mmol/L, and bicarbonate 19.1 mmol/L. His CBC showed hemoglobin of 7.1 g/dL, hematocrit of 25%, MCV 82 fl, WBC 3000 u/L, and platelet count of 70000/mm Uremia is associated with alternation in primary host defense system. Neutrophils exhibit impaired chemotaxis, oxidative metabolism, phagocytic activity, degranulation, intracellular killing, and dysregulated programmed cell death. Factors contributing to neutrophil dysfunction include malnutrition, trace element deficiencies, iron overload, impaired glucose metabolism, hyperparathyroidism, dialysis, and uremic retention solutes. These immunologic abnormalities are complicated by the use of immunosuppressive drugs to treat and control underlying diseases and exacerbated by nutritional deficiencies, the dialysis procedure, and the disruption of cutaneous and mucosal barriers to infection . There are various case reports of AH infections in CKD patients and presentations tend to vary with dialysis modalities. Systemic infections appear to be more common in patients undergoing hemodialysis (HD). On the other hand, peritoneal dialysis (PD) patients generally present with peritonitis. Our case is unique because of the rare AH involvement of hemodialysis catheters. Although there are multiple case reports of peritoneal dialysis catheter, our case is the first that reported hemodialysis catheter related bacteremia. We speculate that there may be some skin carriage of these organisms which led to dissemination of bacteria through the open skin access of the large bore catheters. Alternatively, touch contamination of dialysis catheter by the patient or dialysis nurse after contact with contaminated water supply or dialysis solution could have resulted in this infection. Steroid exposure and the intrinsic low immunologic threshold of dialysis patients may have contributed to the pathogenesis and virulence of the disease. Catheter removal and early initiation of antimicrobials resulted in successful treatment in our case. The different spectrum of disease presentations in HD and PD patients are summarized in  Out of 22 cases reported, 14 patients were cured, 7 patients died, and outcome of 1 patient could not be determined. Overall mortality was 33.33%. The infection appeared to be more serious in HD patients as reflected by the worse mortality  as compared to peritoneal dialysis . Uremia, malnutrition, phagocytic dysfunction, and concurrent immunosuppression were identified as potential risk factors for development of AH sepsis. Contamination of dialysis solution and touch contamination may be the potential sources of infections. Prevention, early detection, and treatment are of paramount importance as the mortality is very high (33.33%). In conclusion, AH infections do occur in chronic kidney disease. Judging from our literature review, systemic infections occur more frequently in HD patients while localized peritonitis is more common in PD patients. Chronic kidney disease, concurrent immunosuppression, lifestyle factors, and endemicity of AH can predispose to sepsis. Early dialysis catheter removal and initiation of antibiotics can result in successful treatment of dialysis catheter related sepsis by AH. Our case highlighted an interesting and unreported complication of AH infections, which seemingly can produce a wide spectrum of presentation in CKD patients.