Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from DOI:http://dx.doi.org/10.7554/eLife.03604.001 DNA molecules are made from four bases—often named ‘G’, ‘A’, ‘C’, and ‘T’—that are arranged along a backbone made of sugars and phosphate groups. Chemicals called nucleosides are essentially the same as these four building blocks of DNA (and other similar molecules) but without the phosphate groups.Proteins called nucleoside transporters are found in the membranes that surround cells and can pump nucleosides into the cell. These transporters also allow drugs that are made from modified nucleosides to enter cells; however, it was previously unclear how different transporters recognized and imported specific nucleosides.Like other proteins, nucleoside transporters are basically strings of amino acids that have folded into a specific three-dimensional shape. A protein's shape is often important for defining what that protein can do, as often other molecules must bind to proteins—much like a key fitting into a lock. Johnson et al. have now revealed the three-dimensional structure of one nucleoside transporter protein bound to different nucleosides and nucleoside-derived chemicals, including three anti-cancer drugs and one anti-viral drug. Some of these chemicals were shown to bind more strongly to the transporter protein than others, and examining the three-dimensional structures revealed that the different chemicals interacted with slightly different amino acids in the transporter protein.Johnson et al. then used this information to chemically modify an anticancer drug so that it is transported more easily into cells and is imported by only one of the subtypes of nucleoside transporters that are found in humans. This provides proof of principle that information about the structure and function of a transporter protein can help to redesign chemicals such that they can enter cells more efficiently, and to tailor them for transport by specific transporters. A similar approach may in the future allow researchers to design new nucleoside-derived drugs that are better at getting inside specific cells and, as such, provide effective treatments against cancers and viral infections.DOI:http://dx.doi.org/10.7554/eLife.03604.002 Nucleosides play critical roles in biology as precursors to nucleic acids and the energy currency of the cell and also serve as signaling molecules . FurtherIn addition to nucleosides, NTs are responsible for the transport of a wide range of nucleoside-derived anticancer  and antiviral  drugs . Both NTCNTs belong to the solute carrier (SLC) superfamily, constituting the family SLC28. The SLC superfamily, composed of 52 families, is responsible for the transport of ions, metabolites, neurotransmitters, and drugs in humans. Several SLC families are of particular clinical interest because of their roles in drug absorption, distribution, metabolism, and excretion (ADME) . The recVibrio cholerae (vcCNT) presents the first opportunity to examine specific nucleoside recognition by CNTs from a structural perspective (+ gradient for nucleoside transport like hCNTs and shares high sequence identity (36–39%) with hCNTs with particularly high sequence identity for the nucleoside-binding site . For these reasons, vcCNT has been identified as an optimal candidate for structure-based ligand discovery using computational methods  at 2.1 Å, which is higher resolution than the 2.4-Å wild-type structure. This mutant was originally designed to introduce binding sites for hydrophobic mercury compounds for heavy-atom phasing. The high-resolution mutant structure revealed another water in the binding site that bridges the 4-carbonyl of the uracil base with Glu 156 but was otherwise identical to the wild-type structure ; Table 1Ds) for a variety of nucleosides and nucleoside analogs using the fluorescent cytidine analog pyrrolo-cytidine , further suggesting that vcCNT is a good model system to study hCNTs  relative to uridine. To deduce the structural basis of the reduced binding affinity, we solved the crystal structure of vcCNT bound to zebularine . To examine the structural basis of the reduced affinity, we solved the crystal structures of vcCNT-7C8C bound to adenosine . However, when the large, weakly electronegative substituent iodine  or a methyl group  is added, we observe an increase in KD (∼60 μM). In short, we observed an increase in affinity with smaller, highly electronegative substituents but a decrease in affinity with larger, less electronegative substituents. What structural feature could account for the differing affinities of these compounds?To test the energetic contribution of substituents at the C5 position of the pyrimidine ring, we measured the Kouridine . The strouridine . FluorinThese differences may result from interactions of the nucleosides with Phe 366. In the structure of vcCNT-7C8C bound to uridine, Phe 366 appears to interact with both the uracil base and the ribose of the nucleoside. Phe 366 forms an offset π–π interaction with the aromatic pyrimidine ring and also forms CH–π interactions with the ribose . The addNotably, Phe 366 is universally conserved between members of the CNT family, and its functional importance has never been tested. To examine the role of Phe 366 in nucleoside recognition, we mutated this residue and performed isothermal titration calorimetry (ITC) experiments. We found that the requirement for Phe at this position is strict, as mutation of this residue to alanine or tyrosine or tryptophan results in significantly decreased binding affinity for uridine while not affecting the stability significantly . TherefoDs for deoxyuridines from which each of the ribose hydroxyls have been removed. Both 3′- and 5′-deoxyuridine yielded KD values greater than 2800 μM (D = 170 μM). Several anticancer nucleoside analog drugs contain modifications at the C2′ position of the ribose. Gemcitabine, for example, is a cytidine analog with two fluorine atoms bonded to C2′. The measured KD of gemcitabine for vcCNT (KD = 1370 μM) is ∼22-fold higher than that for cytidine (KD = 61 μM)  which has its 2′-hydroxyl flipped up above the ribose ring (D > 3000 μM). Interestingly, consistent with our observation with vcCNT, hCNTs show no significant binding and transport of cytarabine  are invariant between hCNTs and vcCNT. To probe these interactions, we measured K 2800 μM , while t= 61 μM) . To unde= 61 μM) . In the = 61 μM) . In orde= 61 μM) . Further= 61 μM) . In addi= 61 μM) . To furtose ring . Cytarabtarabine . Taken tD = 0.9 μM, Of all of the nucleosides and nucleoside analogs studied, the fluorescence probe pyrrolo-cytidine had the strongest binding affinity for vcCNT , their apparent affinities for hCNTs are low . We wond+-nucleoside symporters, one can measure the current generated by the Na+ transport that is coupled with nucleoside transport. We injected Xenopus oocytes with mRNA coding for each of the hCNTs and measured inward Na+ currents elicited by the addition of different nucleosides to the extracellular side. Addition of 200 μM gemcitabine to hCNT3-expressing oocytes induced Na+ currents  from hCNT3 and three amino acids  from hCNT1. Since the nucleoside-binding site of vcCNT is highly homologous to hCNTs, we generated models of the hCNT1 and hCNT3 nucleo-pockets by swapping out these residues in the vcCNT-pyrrolo-cytidine structure. The structural models of hCNT1 and hCNT3 suggest changes in the overall structure of the nucleo-pocket . In partOur structural and equilibrium-binding studies of vcCNT have allowed us to better understand the design principles of nucleoside recognition by CNTs. Two helical hairpins (HP1 and HP2) and two unwound helices (TM4 and TM7), related by twofold pseudo-symmetry, create a bowl-shaped nucleoside-binding site at the center of the transport domain of vcCNT . The intWith regard to binding energetics, our equilibrium-binding studies have shown that the ribose interactions are energetically important consistent with previous non-equilibrium studies with hCNTs . HoweverOur structural and equilibrium-binding studies highlighted the importance of Phe 366 in nucleoside recognition. The nucleoside-bound structures of vcCNT help to shed light upon the structural basis of the importance of this residue. Notably, Phe 366 is the only residue within the nucleoside-binding site that forms interactions with both portions of the nucleoside. Furthermore, all of the other binding-site residues on HP1, HP2, TM4b, and TM7b interact from the same side of the nucleoside as Phe 366, forming the shape of a bowl around the nucleoside with Phe 366 serving as its base. As a result, the nucleoside rests on the face of Phe 366, likely helping to orient the nucleoside so that it may form all of the other interactions within the binding site. Although π–π and CH–π interactions are generally weak, they can provide significant interaction energies depending on the circumstance . The impWhat is the molecular basis of having a bowl-shaped nucleoside-binding site? Several other nucleoside-binding proteins bind to their substrates by sandwiching the nucleobase between aromatic residues . The utiThis rigid-body conformational change also suggests that most of the interactions between transporter and nucleoside that are present in the inward-occluded structure are maintained in the outward-facing conformation, as observed with other sodium-coupled transporters . One excDespite the recent progress in the area of structural biology of transporters, most SLC transporter structures determined to date are not highly homologous to human transporters, and it is often the case that drug interactions with these non-human transporters are different from their human counterparts, rendering structural studies of drug–transporter interactions technically challenging and necessitating substantial engineering to mimic the behavior of the human transporters . AlthougUnderstanding the structural principles of nucleoside and nucleoside drug recognition by vcCNT not only allowed us to understand why a certain class of drugs  are not well recognized and transported by hCNTs, but also offered us an opportunity to modify an existing drug to improve its affinity for vcCNT. Furthermore, our electrophysiological studies show that it is not only transported more efficiently by hCNT1, but it is also selectively transported by hCNT1. Our structural models of hCNT1 and hCNT3 allowed us to hypothesize that the subtype-specific differences in the structures of the nucleo-pocket in the inward-facing-occluded conformation give rise to the subtype-selectivity of the modified compound. The results of the mutational studies of the nucleo-pockets in hCNTs are consistent with this hypothesis. Although we do not know whether the modification of gemcitabine affects the outward conformation due to the lack of a structure of the outward-facing conformation, our studies suggest that destabilization of the inward-facing-occluded step would facilitate the release of the pyrrolo-gemcitabine. Conversely, stabilization of the outward-facing-occluded step would facilitate the capture of the substrate. Taken together, if one has knowledge of both the outward- and inward-facing conformations of the transporter, it might be possible, at least in principle, to modify a compound to bind to both conformations with differential affinities, which may improve its transportability and selectivity. Prior to our studies, the presence of the nucleo-pocket structure was unknown. Although our study with pyrrolo-gemcitabine serves merely as proof of concept, it is conceivable that the nucleo-pocket structure can be utilized in the design of nucleoside-derived drugs or prodrugs that can be specifically targeted only to cell types that express hCNT1 since expression levels of hCNT1 are closely related to the responsiveness of many different types of normal or cancer cells to chemotherapy treatment .Finally, our studies provide another valuable concept: even though two transporter subtypes may share substrate and drug specificity, as is the case with hCNT1 and hCNT3, it is still possible to use structural differences (with the help of modeling) between the two subtypes to design or modify a drug that can be selectively transported. This concept has broad applications to many SLC transporters that are involved in ADME since many families such as SLC21, SLC22, and SLC29 possess subtype-dependent drug specificities and/or tissue distributions .These results can also have an impact on basic scientific research. Since some transporter subtypes show significant changes in expression levels between normal and pathological conditions , and these changes in transporter expression are usually important for the pathological conditions to persist, a fluorescent compound that is subtype-specific  for a certain transporter family can be a valuable tool to study the role of transporter subtypes in human health and disease through live cell imaging . Taken t10-MBP fusion in C41 (DE3) cells, cells were lysed by homogenizer , protein was extracted from crude lysate using 30 mM dodecyl-maltoside, lysates were spun down to remove the insoluble fraction, and the supernatant was applied to a Co2+-affinity column for purification. The His10-MBP was cleaved by overnight digestion by PreScission Protease, and vcCNT was separated from His10-MBP by gel filtration using a Superdex 200 10/300 GL column in the presence of 5 mM decyl-maltoside. After gel filtration, protein was concentrated to ∼10 mg/ml and nucleoside was added to 1 mM (uridine), 2 mM , or 10 mM . Crystals were grown in the presence of 100 mM CaCl2, 37–42% PEG400, and 100 mM buffer: HEPES pH 7.5 (ribavirin), Tris–HCl pH 8.0–8.5 , or glycine pH 9.5 . Crystals were grown using the microbatch-under-oil technique. Crystals were harvested after 10–14 days, transferred to cryo solution containing 32.5% PEG400, and flash frozen in liquid nitrogen.Wild-type vcCNT and vcCNT-7C8C were expressed and purified as described  in the aX-ray data were collected at beamlines 22-ID-D and 24-ID-C at the Advanced Photon Source at Argonne National Laboratory. Data were processed using HKL-2000. The uridine, cytidine, zebularine, 5-fluorouridine, ribavirin, and pyrrolo-cytidine complex structures were refined using PHENIX with the original vcCNT structure (PDB ID 3TIJ) as the input model. The adenosine and gemcitabine complex structures were solved by molecular replacement with the original vcCNT structure as the search model using PHASER and refined using PHENIX.ex = 340 nm and λem = 467 nm was measured using a Cary Eclipse Fluorescence Spectrophotometer  with automated polarizers. Each titration was performed at least three times. The data for the three titrations were simultaneously fit to a single-site binding model based off of Morrison's quadratic equation using nonlinear least-squares analysis in GraphPad Prism to obtain a dissociation constant and standard error.To measure the binding affinity of vcCNT for fluorescent nucleoside analogs, individual 500-μl solutions were prepared containing varying concentrations of vcCNT in 5 mM DM and either 5 μM pyrrolo-cytidine or 2 μM pyrrolo-gemcitabine. The fluorescence anisotropy of each solution at λGraphPad Prism to obtain a dissociation constant and standard error.To measure the binding affinity of vcCNT for other nucleosides and nucleoside analogs, the nucleoside of interest was titrated 5 μl at a time into 500 μl of solution initially containing 5 μM of vcCNT in 5 mM DM and 1–2 μM pyrrolo-cytidine. The fluorescence anisotropy after each addition was measured. Each titration was performed at least three times. The data for the three titrations were simultaneously fit to a one-site competitive binding model based off of Wang's method  using noD and ΔHo as independent parameters.vcCNT mutants were prepared in the same manner as wild-type vcCNT. 15–30 mM of uridine was titrated 5 μl at a time into 25–40 μM of vcCNT solubilized in 5 mM DM using a MicroCal VP-ITC system . The total heat exchanged during each injection was fit to a single-site binding isotherm with K4OH and CH3OH completed the synthesis of the desired pyrrolo-gemcitabine in 75%.See Xenopus laevis oocytes were purchased from Ecocyte Bioscience . Individual oocytes were injected with 40 ng of mRNA using a 10-μl microdispenser  fitted with a tapered glass pipette tip and incubated at 17 °C for 4–5 days in ND96 buffer  with 0.1% penicillin and streptomycin before recording.The genes coding for the three hCNTs and vcCNT were cloned into a pGEM-HE vector. Plasmids were linearized using either SphI or NheI, and mRNA was transcribed using the mMESSAGE mMACHINE T7 Transcription Kit . Defolliculated The oocyte-recording chamber was gravity-perfused with ND96 buffer at a rate of 2 ml/min. Membrane currents were measured using an Oocyte Clamp . Individual oocytes were penetrated with two microelectrodes filled with 3 M KCl (0.5–1.0 MΩ). All electrophysiological experiments were conducted at room temperature. The OC-725C Oocyte Clamp was computer-interfaced via an Axon Digidata 1550 and controlled by Axoscope software . The current signals were filtered at 20 Hz and sampled at intervals of 20 ms. The signals were filtered at 0.5 Hz by use of pCLAMP 10.4 software for data presentation. Ooctyes were impaled with the electrode filled with 3 M KCl, and then membrane potentials were observed for 10 min. Cells were discarded if resting membrane potentials were unstable or more positive than −30 mV. Oocyte membrane potentials were clamped at −90 mV for holding potentials to measure transporter-generated currents. All data are shown as means ± SEM. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 2 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters” for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Volker Dötsch, Raimund Dutzler.The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The authors describe a thorough investigation of the interaction of vcCNT with different substrate analogues, including important antiviral and anticancer drugs. vcCNT is a close prokaryotic homologue of the SLC28 family of Na-coupled nucleoside transporters, that are responsible for the import of nucleoside analogues into cells. Its structure was previously determined at high resolution by the same group. The binding mode of ligand analogues to an inward-facing conformation of the protein was investigated by X-ray crystallography. The data is of high quality and of sufficient resolution to analyze interactions. Next to the crystallographic studies, the binding affinity to the detergent solubilized protein was quantified by a competition assay with a fluorescent ligand and by isothermal titration calorimetry. Taken together the data provides detailed insight into the structural and energetic determinants of the interaction.A few points, however, should be addressed before publication:1) To be able to judge the crystallographic data it would be helpful if the authors could provide stereo figures of the different complexes with superimposed 2Fo-Fc electron density in the supplemental materials.2) For the binding studies it is not clear whether the authors expect to measure binding to an inward-facing conformation, or whether they believe that the transporter may adopt different conformations in solution.3) 4) Is the change in subtype specificity from hCNT3 to hCNT1 important for the delivery of this class of compounds, or was this study designed as proof of concept?5) The results of the mutational analysis of F366 could potentially also be explained with misfolding of the protein. Has the integrity of the mutated transporter been investigated? 1) To be able to judge the crystallographic data it would be helpful if the authors could provide stereo figures of the different complexes with superimposed 2Fo-Fc electron density in the supplemental materials.We have now provided supplemental stereo figures of the nucleoside-binding site of vcCNT in complex with each of the nucleoside analogs with 2Fo-Fc electron density maps superimposed.2) For the binding studies it is not clear whether the authors expect to measure binding to an inward-facing conformation, or whether they believe that the transporter may adopt different conformations in solution.Ph). On the basis of our vcCNT structure and structural and biophysical studies of related transporters, we previously hypothesized that transport is achieved by a rigid-body motion of the transport domain that maintains similar structures for both the inward- and outward-occluded conformations, and thus the interactions with the nucleoside are similar in both conformations. Our hypothesis is in line with recent studies done by the Boudker group that show that GltPh samples both conformations with equal probability and the affinities of both conformations are similar in detergent micelles. Thus we assume that most interactions that we showed to be important using nucleoside analogs have effects in both conformations. We think that one exception is the interaction between the methylpyrrole ring of pyrrolo-cytidine with the nucleopocket, which must affect the inward-occluded conformation preferentially because of the involvement of part of the static scaffold domain (TM6). Consistent with our assumption, several mutational studies done on human CNTs show that most of the residues that we believe are important in the inward-facing conformation are also important for the binding and transport of nucleosides. Further structural and biophysical studies of the outward-occluded conformation and associated conformational dynamics will help test our hypothesis.vcCNT, although it is a novel fold, adopts a common overall architecture of a static scaffold domain and a mobile transport domain, which is shared by several sodium-coupled transporters show bars of the average of the integrated charge from 10-12 independent experiments. It is not clear how the authors have accounted for differences in the transport currents due to different expression levels. For this figure, it would also be necessary to show the corresponding traces from non-injected oocytes as control.+ transport within the same oocyte, we collected data from oocytes displaying high levels of currents, which could be the reason for the small deviations in the total charge transported from different oocytes. The control water-injected oocytes showed no response to either gemcitabine or pyrrolo-gemcitabine. We have now included the corresponding current traces from the control water-injected oocytes in To accurately measure differences in the compound-dependent Na4) Is the change in subtype specificity from hCNT3 to hCNT1 important for the delivery of this class of compounds, or was this study designed as proof of concept?Our study was solely designed as proof of concept. We just want to point out that at least in principle this type of specificity switch could be utilized to target cancer cells that express higher levels of hCNT1 while having little effect on those expressing higher levels of hCNT3. There are multiple studies that have shown that several gemcitabine-resistant pancreatic cancer cells reduce their hCNT1 surface expression levels, suggesting that hCNT1 is the main gemcitabine transporter for these cancer cell types. Conversely, there is a clinical study showing that expression levels of hCNT3 are important for the survival of pancreatic cancer patients with a specific type of gemcitabine treatment, suggesting that an hCNT3-specific gemcitabine derivative could also be effective in this specific case.We have modified a paragraph in the Discussion section to clarify that our studies with pyrrolo-gemcitabine are designed as proof of concept: “Although our study with pyrrolo-gemcitabine serves merely as proof of concept, it is conceivable that the nucleo-pocket structure can be utilized in the design of nucleoside-derived drugs or prodrugs that can be specifically targeted only to cell types that express hCNT1 since expression levels of hCNT1 are closely related to the responsiveness of many different types of normal or cancer cells to chemotherapy treatment.”5) The results of the mutational analysis of F366 could potentially also be explained with misfolding of the protein. Has the integrity of the mutated transporter been investigated?The F366 mutants  behave similarly to the wild-type transporter in terms of their biochemical stability and elution volumes in size-exclusion chromatography. Because we demonstrated in our initial structure paper that wild-type vcCNT exists as a trimer both in detergent micelles and in the membrane, the similar elution volumes of the mutants strongly suggest that the mutants adopt trimer stoichiometry as well. Furthermore, we did not observe any aggregation of the mutants during our ITC experiments. We have now included gel filtration traces of the mutants as well as wild-type vcCNT in


Empathy is a multidimensional construct referring to the capacity to understand and share the emotional and affective states of another person. Cerebral γ-aminobutyric acid (GABA)-ergic levels are associated with a variety of neurological and psychiatric disorders. However, the role of the GABA system in different dimensions of empathy has not been investigated.Thirty-two right-handed healthy volunteers took part in this study. We used proton magnetic resonance spectroscopy to determine GABA concentrations in the anterior insula (AI) and the anterior cingulate cortex (ACC) and to examine the relationship between the GABA concentrations and the subcomponents of empathy evaluated by the Interpersonal Reactivity Index (IRI).Pearson correlation analyses (two-tailed) showed that AI GABA was significantly associated with the empathy concern score  and the personal distress score  but not significantly associated with other empathy subscales. No significant correlation was found between ACC GABA and empathy subscores.Left AI GABA was positively correlated with the emotional aspects of empathy. These preliminary findings call into question whether AI GABA alterations might predict empathy dysfunction in major psychiatric disorders such as autism and schizophrenia, which have been described as deficits in emotional empathic abilities. Empathy is a multidimensional construct and usually refers to the capacity to understand and share the emotional and affective states of another person. Empathy is believed to be important in social behaviors 1H-MRS) to measure the absolute concentrations of cerebral glutamate in the ACC, left dorsolateral prefrontal (PFC) and left hippocampus and found that dorsolateral prefrontal cortex (DLPFC) glutamate levels showed a significant negative correlation with the “perspective taking” score. GABA, the main inhibitory transmitter in the brain, has been shown in a number of studies to be correlated with various disorders such as autism As the primary neurotransmitters in the human brain, glutamate (Glu) and γ-aminobutyric acid (GABA)-ergic mechanisms are important for a variety of neurological and psychiatric disorders, including Parkinson's disease, and Alzheimer's dementia 1H-MRS to detect GABA, the AI and the ACC were chosen as regions of interest due to their involvement in empathy. Specifically, in the present study, 1H-MRS was used to measure the concentrations of cerebral GABA in the AI and the ACC at rest and to examine the relationship between the GABA concentrations and the subcomponents of empathy evaluated by Interpersonal Reactivity Index (IRI) As mentioned above, the AI and the ACC have been identified as key regions involved in empathy for pain. However, to our knowledge, the role of the GABA system for different dimensions of empathy has not been investigated thus far, and the relationship between the GABA system and empathy remains unclear. In this study of the regional brain using SD = 1.8, range = 22–30 years) were recruited from the East China Normal University. None of the subjects had a history of neurological or psychological disorders. All participants gave written informed consent before scanning. The study was approved by the Ethics Committee of the East China Normal University.Thirty-two right-handed volunteers . Single voxel edited 1H-MR Spectra were acquired at rest from two volumes of interest (VOI) in each subject. One VOI of 20×18×30 mm3 = 10.8 ml was placed in the ACC (3 = 12.5 ml) was located in the left AI  using a twelve-channel, phased array, receive-only head matrix coil for signal detection. Before MRS acquisition, a three-dimensional high-resolution T the ACC , while a left AI . A singlAll spectra were analyzed using jMRUI software The signal-to-noise ratio (SNR) was calculated using the difference spectrum following phase adjustment such that the NAA peak was upright with a phase of 0 degrees for each subject The subjects' empathic abilities were evaluated using a multidimensional questionnaire, the Interpersonal Reactivity Index (IRI), which has been widely used to assess empathy ability Participants were asked to complete the IRI independently after MRS data acquisition. Statistical calculations were carried out using SPSS 16.0  for Windows.SD = 1.9, range = 22–30) in AI group and 27 subjects  in ACC group.All 32 subjects underwent MRS measurements as scheduled. Nine subjects whose spectra were acquired in the AI and five subjects whose spectra were acquired in the ACC were excluded due to non-valid  MRS data. The MRS data were analyzed for 23 subjects  in the AI of 23 subjects and in the ACC of 27 subjects, as well as mean IRI subscores, are presented in 1H-MRS study reporting associations between the subcomponents of empathy and regional brain GABA+ concentrations in the AI and ACC. The main findings in this preliminary study were that there were significant positive correlations of the AI GABA+ concentrations with the two emotional empathy scales . No associations were found with regard to the other subscales of the IRI. No significant relationship was found between the ACC GABA+ concentrations and empathy subscales.To our knowledge, this is the first The GABA system is the major inhibitory system in the human brain Empathy is a complex form of psychological inference in which observation, memory, knowledge, and reasoning are combined to yield insight into the thoughts and feelings of others A considerable neuroimaging studies have convincingly demonstrated that anterior insula cortex and anterior cingulate cortex are both activated during empathic pain processing. However, Gu et al Studies have suggested that women may be more empathetic than men In this study, we sampled MR spectra only in the ACC and the AI. An fMRI study 1H-MRS GABA in the left AI was positively correlated with the emotional facets of empathy subscales of the IRI. This may be consistent with well-replicated evidence of insula activity in the functional magnetic imaging studies of empathy for pain. Our findings call into question whether AI GABA alterations might predict dysfunction of empathy in major psychiatric disorders such as autism and schizophrenia, which have been suggested to involve deficits in emotional empathic abilities.In summary, File S1Table S1, GABA quantification in ACC and IRI subscores. Table S2, GABA quantification in AI and IRI subscores.(DOCX)Click here for additional data file.


Sepsis is a leading cause of death in intensive care units and is increasing in incidence. Current trials of novel therapeutic approaches for sepsis focus on 28-day mortality as the primary outcome measure, but excess mortality may extend well beyond this time period.We used relative survival analysis to examine excess mortality in a cohort of 1,028 patients admitted to a tertiary referral hospital with sepsis during 2007–2008, over the first 5 years of follow up. Expected survival was estimated using the Ederer II method, using Australian life tables as the reference population. Cumulative and interval specific relative survival were estimated by age group, sex, sepsis severity and Indigenous status.Patients were followed for a median of 4.5 years (range 0–5.2). Of the 1028 patients, the mean age was 46.9 years, 52% were male, 228 (22.2%) had severe sepsis and 218 (21%) died during the follow up period. Mortality based on cumulative relative survival exceeded that of the reference population for the first 2 years post admission in the whole cohort and for the first 3 years in the subgroup with severe sepsis. Independent predictors of mortality over the whole follow up period were male sex, Indigenous Australian ethnicity, older age, higher Charlson Comorbidity Index, and sepsis-related organ dysfunction at presentation.The mortality rate of patients hospitalised with sepsis exceeds that of the general population until 2 years post admission. Efforts to improve outcomes from sepsis should examine longer term outcomes than the traditional primary endpoints of 28-day and 90-day mortality. Severe sepsis is the most common cause of death in intensive care units Most existing studies investigating longer-term outcomes of sepsis are difficult to extrapolate for several reasons. The majority of sepsis outcome studies include only patients admitted to an intensive care unit (ICU), but only 50–70% of patients hospitalised with severe sepsis ever enter an ICU Relative survival analysis is a statistical technique commonly used in oncology We aimed to describe the long term outcomes of a prospectively recruited cohort of patients with sepsis, including those admitted to ICU and non-ICU wards. Our primary aim was to estimate the duration of the excess mortality risk in patients with sepsis over the first 5 years of follow up, using relative survival analysis.The patients included in this cohort have been previously described in detail Where patients were admitted with more than one episode of sepsis over the 12-month course of the original study, only the first episode was included in the current analysis. Furthermore, all patients who were resident outside the Northern Territory in the year of initial admission (n = 62) were excluded from the current analysis, because their vital status was difficult to determine accurately in the longer term.This study was approved by the Human Research Ethics Committee of the Menzies School of Health Research and Northern Territory Department of Health, who waived the requirement for individual informed consent.Severe sepsis was defined as sepsis plus at least one attributable organ dysfunction within the previous 24 hours, as per the definitions used in the PROWESS study Information about all deaths that occur in the Northern Territory is provided to the Northern Territory Department of Health by the Registrar of Births, Deaths and Marriages and recorded in the public hospitals' client administration system. We accessed this deaths information to determine vital status for all patients included in this study at 12 months, 3 years and 5 years after the commencement of the study.Patients were included in the survival model until death from any cause, censoring due to loss to follow up, or the end of the follow-up period (30/06/2012). Mortality of sepsis patients was measured as both overall survival and relative survival. Overall survival  is the proportion of sepsis patients still alive at a certain point in time after their sepsis episode; deaths include those unrelated to the sepsis episode, so overall survival does not measure excess mortality related specifically to the sepsis episode. Overall survival analysis was calculated using the Kaplan-Meier method.By comparing deaths among the sepsis patients with the expected number of deaths based on general population mortality rates, relative survival is an estimate of excess mortality in the sepsis patients. To estimate relative survival, the background mortality rate for the general population was derived from life tables. Expected survival was estimated using the Ederer II method 2 test. Crude survival rates were derived using Kaplan-Meier estimates. Interval-specific relative survival ratio (RSR) and cumulative RSR were derived using the Stata user-defined program “strs” All analyses were conducted using Stata/SE 12 . Continuous variables were compared using t-test for normally distributed data and Mann-Whitney U test for non-normal data. Categorical variables were compared using χA generalized linear model with Poisson error structure was used to model the excess mortality based on collapsed data, in which the outcome was the observed number of deaths and was assumed to be Poisson distributed. The predictors of excess mortality were chosen using forward selection, resulting in the final model with the following covariates: follow-up time since diagnosis, gender, Indigenous status, age group, Charlson index group, severe sepsis and bacteraemic.Patients were followed for a median of 4.5 years (range 0–5.2 years), giving a cumulative time at risk of 3,997 patient-years. Of the 1,028 patients with sepsis, 218 had died by the end of the follow-up period. Severe sepsis was present in 228 patients 22% - Table 1.One-year and five-year crude survival for all sepsis patients  were 87.5% (85.3%–89.3%), and 77.6%(74.5%–80.4%) respectively. The corresponding figures for the severe sepsis subgroup were 74.1% (67.9%–79.3%) and 66.2% (59.7%–72.0%).Kaplan-Meier estimates by age group are shown in Interval-specific relative survival 3 lead tAcross all subgroups except males greater than 64 years old, Indigenous patients had lower five-year relative survival than non-Indigenous patients Table 4,In multivariable regression analysis, Table 5 In this cohort of patients hospitalised with sepsis, and including approximately 4,000 patient years of follow up, the excess mortality persisted for 2 years overall and for 3 years in the subgroup with severe sepsis.Although there is increasing interest in longer term outcomes following sepsis Although we have demonstrated excess mortality in the sepsis cohort compared to the general population, we cannot determine the mechanism of mortality, as we did not have access to cause of death data. Acute sepsis-related organ failure generally resolves by the time of hospital discharge in those who survive. However, systemic inflammatory activation persists for weeks-months afterwards Our finding that excess mortality persists for at least 2–3 years supports calls to extend the traditional end-points of interventional sepsis studies beyond the most commonly used end-point 28-day mortality Our study has some limitations. We studied a cohort of patients from tropical Australia of whom a substantial proportion were Indigenous Australians, and with a relatively young mean age, and thus the applicability of our findings to other populations is unclear. However, the fact that the post-sepsis mortality excess has been shown to persist for well beyond 90 days in several other cohorts support our findings. We used life tables from 2006 (the most recent available robust data), but the period under study was 2007–2012. If the mortality rate in the background population had decreased over this time, our models may have overestimated the excess mortality in the sepsis population. However, given the short time between these two periods, this is unlikely to have been significant. We did not have access to data on cause of death, nor on comorbidities in the reference population. However, a strength of our study is that, unlike the majority of sepsis studies investigating longer term outcomes post-sepsis In conclusion, the risk of mortality from an acute episode of sepsis extends well beyond hospital discharge. Relative survival analysis is a useful tool for examining the excess mortality in patients with sepsis. Future efforts to improve outcomes from sepsis should focus on endpoints as long as 3 years post discharge, and such endpoints should no longer be limited to the first 90 days following hospital admission.


The correct methodology of thrombelastography might be influenced by elapsing time. In our study we investigated kaolin activated citrated samples together with non-activated citrated samples in relation to the elapsed times of 0, 15 and 30 minutes to compare both methods and to find out if there is an impact of time on results of thrombelastography.Blood samples obtained from 10 healthy volunteers were analyzed after 0, 15 and 30 minutes from sampling with kaolin activation and without activation. Then the results were analysed and compared between the non-activated and the kaolin-activated method.All blood samples became more hypercoagulable with the time elapsing, both in non-activated and kaolin-activated samples and differences between both groups were found statistically and clinically significant after only 0 minutes.The non-activated citrated method seems to be reliable and suitable for thrombelastography in non-emergency cases  when we have time to wait 15–30 minutes to get results. In urgent situations a rapid thrombelastography test should be preferred. Although the kaolin-activated method can also be used, results must be interpreted with caution. Thrombelastography (TEG) has been used as a laboratory and bed-side method since 1948 -7. BecauWe decided to compare the results of kaolin activated citrated samples with results of non-activated sample in relation with elapsed time of 0, 15 and 30 minutes. We wanted to find out if time impacts the activation of citrated samples. The time period of 15 to 30 minutes would be very practical for transporting blood samples to lab. It is very important to elaborate the TEG method, which could be used in non-emergency situations  especially in patients with intensive care unit (ICU) coagulopathy represented by prolonged prothrombin time (PT) because TEG as a global coagulation test may lead to reduced FFP transfusion administered preventively (before surgery) just on the base of prolonged PT and thus minimize related adverse side effects of FFP .This study was approved by the Ethics Committee for Multi-Centric Clinical Trials of the University Hospital Motol  and written informed consent was obtained from all subjects.2, TEG Hemostasis System, Haemoscope Corp., USA) and the sample was started. Meanwhile (after 2–3 minutes from sampling) 340 μl of blood was pipetted from the citrated tube to the other cuvette containing 20 μl of calcium and the non-activated sample was initiated. After 15 minutes the second 2 ml citrated tube was used and the process was repeated. After 30 minutes the third 2 ml citrated tube was used and the process was repeated again. Three TEG machines were used simultaneously in this study .In the first step of study we investigated the role of time and its influence on the stability of blood samples after 0, 15 and 30 minutes in both non-activated citrated and kaolin activated citrated samples. Blood was obtained from 10 healthy volunteers  from the cubital vein using a 10 ml syringe and needle and blood was squirted into 3 citrated tubes with a volume of 2 ml . Immediately after obtaining blood (and gentle mixing the blood sample 5–6 times) - after 0 minutes, 1 ml of blood was poured from the first 2 ml citrated tube to the kaolin vial and after 3–4 minutes 340 μl of this blood was pipetted to the cuvette containing 20 μl of calcium  was used to compare the effect of time on sample stability. Then in post hoc analysis the Tukey test was used. In the second step to compare non-activated with kaolin-activated, a paired t-test was used (p < 0.05 was considered significant).In all measurements, both non-activated and kaolin activated, the time elapsed from blood sampling played a role and the samples became more hypercoagulable. The most significant time effect (p < 0.05) occurred within the first 15 minutes (between 0 and 15 minutes) both for non-activated and kaolin activated samples. After 15 minutes (between 15 and 30 minutes) time does not seem to play a significant role and the results do not differ significantly (p > 0.05) in either group Figures . InteresIn the second step we compared the results of non-activated samples with the results of kaolin-activated samples of all time points 0, 15, 30 minutes. Significant differences (p < 0.05) between non-activated and kaolin-activated samples were found mostly in 0 group for parameters R, K and alfa angle  could produce an apparent heparin reversal effect . This seA second limitation that has to be mentioned is that the observed time effect found in healthy volunteers will need to be confirmed in ICU patients before assuming that the time effect would lead to equivalent results in this population.The relatively small sample size of the study (10 volunteers) considering the relative large interindividual variability of TEG data is another limitation of the study. However differences were statistically significant.In conclusion, the time activation of the blood sample plays an important role in thrombelastography both in the kaolin activated and the non-activated method citrated method. Non-activated citrated TEG method used between 15–30 minutes from sampling seems to be reliable and suitable for analysing blood coagulation in haematology lab. This may be especially appropriate when TEG is used for whole blood coagulation assessment in patients with ICU coagulopathy (prolonged PT) before planned surgical intervention when FFP is administered preventively. This method of using TEG can help in decreasing often unnecessarily administered FFP.Authors declare that the experiments comply with the current laws of Czech republic performed.


Despite Internet-based interventions that incorporate pedometers with appropriate goal-setting processes and other theoretically-based behavior change strategies being proposed as a means of increasing walking behavior, few have incorporated all of these key features or assessed maintenance of behavior change.The objective of our study was to investigate the effect of a 12-week pedometer step goal walking program individually tailored to baseline step counts, combined with an interactive support website for step counts, health parameters and motivation over 12 and 24 weeks.Low active participants (mean [SD] 46.2 [11.2] years) were randomly assigned to the Stepwise (SW) intervention group (n=49) or a comparison (CP) group (n=48). SW received a pedometer, step goal walking program and access to the SW website . CP received a pedometer and locally available physical activity information. Step counts, BMI, resting heart rate, blood pressure and glucose, cholesterol and triglyceride levels, psychological well-being, perceived health, self-efficacy and self-determined motivation were measured at baseline, 12 and 24 weeks.P<.001) and 24 . Group step counts were significantly different at week 24 with SW taking 8939  more steps compared to CP. Compared to baseline, both groups had improved triglyceride levels  at week 12, decreased diastolic blood pressure  at weeks 12 and 24 , improved positive  and negative affect  at week 12, and perceived health at week 12  and 24 . Total cholesterol increased at week 12  and week 24 . Repeated measures ANOVA found motivation for walking improved from baseline with higher task self-efficacy  and autonomous motivation  at weeks 12 and 24 and decreased controlled motivation  at week 24.Linear mixed model analysis found that both groups’ step counts increased from baseline to week 12 (β = 11,002, CI 5739-16,266, Both groups had similar improvements in step counts and physical and psychological health after 12 weeks but only the SW group successfully maintained the increased step-counts 24 weeks post-intervention. This suggests the step-goal based walking program combined with Internet-based behavior change tools were important for sustained behavior change. Regular moderate-intensity physical activity amounting to at least 150 minutes per week is one of the best lifestyle choices an individual can make to improve health, sustain good health and prevent ill health . DespiteIncorporating a pedometer into a walking intervention with a nonclinical population of adults is associated with significant increases in physical activity of 2000-2500 steps per day -15 and tPedometer-based interventions of varied length have been conducted using Internet technology -29,34. TTo overcome the limitations of previous research, we created the StepWise intervention which combines an individually-tailored step goal pedometer walking program with an interactive support website. The website allows the participants to enter their step counts, graphically see their goal achievement, obtain automated and individualized motivational messages and plan their walking activities. This pilot study consisted of a 12-week intervention with a 12-week follow-up to investigate whether the StepWise intervention would increase step counts, improve health parameters and motivation for walking compared with a comparison group in a community sample of apparently healthy adults.This pilot randomized trial compared a StepWise intervention group (SW) to a comparison group (CP) over a 12-week intervention and a 24-week follow up. The intervention was fully Web-based but the study also involved face-to-face components, specifically to collect outcome data and for the intervention and comparison procedures to be explained to participants. The trial is reported in accordance with CONSORT-EHEALTH guidelines. The research was approved by the University Human Ethics Committee in accordance with all applicable regulations .A targeted recruitment strategy was employed  to attraAll measurements took place in a room at the University in the morning. Participants arrived in a fasted state having done minimal physical activity that morning. The same measurements were taken at baseline, 12 weeks, and 24 weeks.Physical activity was assessed by step counts. Participants were given a Yamax PW-610 pedometer, individually calibrated consistent with manufacturers’ guidelines and asked to wear the pedometer during all waking hours for the next 7 days and to remove it only when sleeping, bathing or during water-based activities. The screen of the pedometer was covered so participants could not see their step counts. At baseline they were encouraged to continue with the same amount of physical activity as they had been doing the previous week. One week later, the researcher removed the screen cover and step counts were recorded. The pedometer was returned to the participant to use in the study.2), seated resting heart rate (measured using a Polar PE3000 heart rate monitor) and blood pressure . A trained phlebotomist then drew a 6 ml blood sample from the participant’s arm by venipuncture and the sample was analyzed for glucose, total cholesterol, HDL cholesterol and tryglycerides. After collection the venous blood was centrifuged and the heparinized plasma was analyzed for Glucose, Total Cholesterol, HDL cholesterol and Triglycerides using a Cobas C111 analyzer (Roche Diagnostics). Staff taking these measurements and doing the analysis were blinded to group allocation. Participants completed the Positive and Negative Affect Schedule [Participants were measured for height and weight in order to calculate Body Mass Index  whether they had used the SW website (yes/no), (2) how often , and (3) to write down what aspects of the website they had found most and least useful.One week after baseline testing, participants attended a second face-to-face session where they were randomized into either SW or CP groups. To ensure equal representation in the groups, randomization was stratified by gender  and age (<45years or >45years) creating 4 distinct stratification groups. Group assignment was placed inside sealed envelopes and the envelopes shuffled to produce an unpredictable sequence of group assignment. When each participant arrived, the next envelope in the pile representing that individual  was opened to reveal their group assignment. As far as we are aware, participants could not tell whether they were in the SW or CP group because all study information stated participants would be given access to a pedometer walking program and a supportive physical activity website.The SW intervention consisted of 2 components: (1) an individualized pedometer-based walking program with weekly step goals, and (2) a website (created by the University Web development team) where individuals entered their step counts, received goal feedback, their next weekly goal  and tailored motivational feedback, and created a physical activity plan. At the second face-to-face session, the intervention components were discussed with each participant and the participant left with an information sheet summarizing the discussion. The same researcher met with each participant and the information discussed was standardized across all participants to ensure accuracy and consistency in the delivery of the intervention.The walking program was structured around each participant’s baseline step counts and designed so that physical activity increased gradually ,32-33. BWeek 1: Walk an extra 1500 steps  on at least 3 days of the weekWeek 2: Walk an extra 1500 steps  on at least 3 days of the weekWeek 3: Walk an extra 1500 steps  on at least 5 days of the weekWeek 4: Walk an extra 1500 steps  on at least 5 days of the weekWeek 5: Walk an extra 3000 steps  on at least 3 days of the weekWeek 6: Walk an extra 3000 steps  on at least 3 days of the weekWeek 7: Walk an extra 3000 steps  on at least 5 days of the weekWeek 8: Walk an extra 3000 steps  on at least 5 days of the weekWeeks 9-12: Maintain walking levels using the week 7 goalEach participant’s baseline step counts and reported barriers to physical activity   were entAt the end of the 12-week intervention, participants met with the researcher and were encouraged to maintain their new level of physical activity over the following 12 weeks. They kept the pedometer and were told they could continue to access and use the StepWise website to receive step goals. To support maintenance of behavior change, there was a discussion of strategies relating to relapse prevention. This involved encouraging the participant to identify factors that could interfere with their being able to continue their new walking behavior and thinking of strategies they could put in place to overcome these factors so they could remain active. In the 12-week follow-up period, participants were sent 2 emails reinforcing the messages that had been discussed.Comparison group (CP) participants were given a pedometer and informed of the public health guidelines for physical activity  and how the guidelines translated into pedometer steps. Participants were shown the MoveMe website  a nonintAt the end of the 12 weeks, participants met with the researcher and were encouraged to maintain their new level of physical activity over the following 12 weeks, or to continue to try and meet the public health guidelines for physical activity and to continue to use the MoveMe website. Participants kept their pedometer to use during this period if they wished. In this follow-up period, participants were sent 2 emails reinforcing the messages that had been discussed.G-Power analysis  was usedt-tests were used to analyze demographic variables and baseline data. The step counts and health variables were analyzed by linear mixed effect models using the R statistical package [2(η2).To ensure the groups were comparable, independent  package . Group . After baseline testing, 97 participants  were eligible to be randomized, and 42 females and 7 males with a mean age of 47.1 years (SD 11.3) were allocated to the SW group while 40 females and 8 males with a mean age of 45.3 (SD 11.1) were allocated to the CP group see . ParticiP<.001) more steps at week 12 and 6,810  more at week 24 (see P=.04) more steps at week 24 than CP (see P=.055). CP step counts did not change significantly (P=.15).There were no group differences in step counts between baseline and 12 weeks. All participants increased their step counts per week from baseline taking 11,000  and decreased diastolic blood pressure  at week 12 and week 24 . Less positive was an increase in total cholesterol from baseline to week 12  and 24  without a concomitant increase in HDL Cholesterol. There were positive effects for psychological health with increased positive affect and decreased negative affect at week 12 and participants perceived they were in better health at week 12 and 24 compared to baseline  and autonomous motivation  increased over time while controlled motivation decreased . Compared to baseline, participants felt more confident walking at week 12  and 24  had higher autonomous motivation at week 12  and week 24  and had lower controlled motivation at week 24 . There were no differences between the groups.A time main effect showed that task self-efficacy  stated they had used it at least once a week, with 15% (5/33) once every 2 weeks. The website usage statistics that were downloaded also confirmed this and showed that 82% of participants (27/33) logged into the website weekly, 1 participant logged in 8 out of 12 weeks, 4 logged in 9 out of the 12 weeks and 1 logged in 10 out of 12 weeks. Encouragingly, all participants used the step log function and therefore automatically received the goal accomplishment and tailored motivation messages and 45% (15/33) regularly used the activity plan function. Together, these results show the participants were engaged with the intervention and used the intervention tools provided on the website. At 24 weeks, self-report data and the website usage statistics showed that 48% of participants (16/33) were still accessing the website with 69% (11/16) accessing it (unprompted by email) at least once every 2 weeks.Participants reported three website features they found most useful: (1) The step goals and pedometer because they provided a weekly challenge, (2) the self-monitoring tools of entering their step count each week and the graph to have a visual representation of the goal and to see whether or not it had been achieved, and (3) the feedback from the motivational messages that provided encouragement and positive reinforcement.We developed an intervention containing an individually tailored step goal pedometer walking program, combined with an evidence-based interactive website. In this pilot study we examined whether it would increase walking behavior, improve health and motivation over the short (12-week) and medium-term (24-week) in a community sample of apparently healthy adults. Results showed there were no differences in step counts between the SW and CP groups at week 12, all participants, irrespective of group, increased their step counts from baseline to week 12 and 24. However, importantly, SW participants had significantly higher step counts at week 24 compared to CP participants, suggesting that the intervention successfully helped individuals maintain their new levels of physical activity. In conjunction with the increase in walking, triglyceride levels and diastolic blood pressure improved as well as positive and negative affect  and perceived health in participants of both groups. Surprisingly, total cholesterol increased across the study without a significant change in HDL cholesterol. Motivationally, all participants gained greater confidence for walking and their motivation became more autonomous, irrespective of their group assignment. These results are generalizable to a community sample of relatively healthy volunteers wanting to increase their walking behavior.Participants increased their step counts by 21-24% above baseline at week 12. These results are smaller, but still comparable with, other pedometer interventions with similar samples that have shown increases of around 27% -15. The Consequently, despite the lack of group differences at 12 weeks, providing participants with a step goal walking program and access to a website containing the evidence-based behavior change tools to self-monitor their behavior, get feedback and motivational support, and plan their activities, seemed to encourage individuals to maintain the proposed increased physical activity at 24 weeks ,11-12. Twant to be physically active rather than feeling they have to be physically active and the changes are related to increased and sustained physical activity behavior [The study was successful in changing motivation for physical activity in both groups. Participants felt more confident in their ability to walk for longer periods of time. Increased confidence has been demonstrated in a number of walking studies and is a strong predictor of physical activity behavior . The perbehavior . InteresBoth groups received a pedometer and physical activity device, therefore the study did not have a no treatment control group. Consequently, there is still the possibility that some other factor other than the elements contained in the intervention and comparison group caused the change in physical activity behavior. Step counts were only recorded for analysis at baseline, 12 weeks and 24 weeks. It may be that participants simply increased their walking behavior in the measurement week and these values do not actually reflect the activity they achieved during the intervention. However, for someone to have such a large increase in activity in one week is unlikely. The walking program automatically increased weekly step count goals, so consequently if something happened to disrupt (decrease) normal walking patterns  then the individual would be expected to meet a new, harder goal on resumption of the program. Ideally, the person would have been prompted by the program to go back to the walking goal from the week prior to the disruption. However, our StepWise software was not able to do this. This should be taken into account in future development of the intervention. Additionally, the intensity at which participants walked was not measured. Although we endeavored to emphasize that participants walked at moderate intensity, we cannot be sure they adhered to this. It was hoped that the study would have 80% power to detect differences in step counts (the primary outcome), however, after drop-outs we did not achieve our target sample size of 43 per group. Our large drop-out rate is a limitation; we were able to elicit reasons for why 9 SW and 3 CP participants decided not to continue with the study but we were not able to ascertain the reasons why 5 SW and 6 CP did not show to the testing sessions. As a result, we cannot be sure whether there was some underlying factor that caused these individuals to drop out and therefore bias the results. Furthermore, it is likely the study was underpowered to detect differences in the biochemical health measures taken. Finally, in hindsight, it would have proved useful to also ask participants if there were any other features they would have liked the website to have to encourage and support their physical activity. This would have been advantageous for future development of the StepWise website.There were no differences in step counts between the SW and CP groups following the 12-week intervention; all participants increased their walking behavior and had improved physical and psychological health. However, only the StepWise group successfully maintained their increased walking behavior 3 months post-intervention and achieved step counts that met the physical activity guidelines for health. Sustained behavior change is supported by a step-goal-based walking program with behavior change tools that allow a person to self-monitor behavior, get feedback and motivational support, and make activity and coping plans.


Childhood vaccination rates in Manitoba populations with low socioeconomic status (SES) fall significantly below the provincial average. This study examined the impact of a pay-for-performance (P4P) program called the Physician Integrated Network (PIN) on health inequity in childhood vaccination rates.The study used administrative data housed at the Manitoba Centre for Health Policy. We included all children born in Manitoba between 2003 and 2010 who were patients at PIN clinics receiving P4P funding matched with controls at non-participating clinics. We examined the rate of completion of the childhood primary vaccination series by age 2 across income quintiles (Q1–Q5). We estimated the distribution of income using the Gini coefficient, and calculated concentration indices for vaccination to determine whether the P4P program altered SES-related differences in vaccination completion. We compared these measures between study cohorts before and after implementation of the P4P program, and over the course of the P4P program in each cohort.The PIN cohort included 6,185 children. Rates of vaccination completion at baseline were between 0.53 (Q1) and 0.69 (Q5). Inequality in income distribution was present at baseline and at study end in PIN and control cohorts. SES-related inequity in vaccination completion worsened in non-PIN clinics , but remained constant in P4P-funded clinics .The P4P program had a limited impact on vaccination rates and did not address health inequity. Inequity in health outcomes is a global challenge that creates economic losses and health care burdens, such as loss of productivity and tax payments, higher welfare payments and health care costs –7. InequThe PIN program provided funding to clinics in Manitoba for meeting quality of care targets on selected clinical process indicators, including meeting childhood vaccination targets. The provincially recommended childhood vaccination program in Manitoba follows a vaccination schedule that stipulates a primary series be administered by age 2 . The vacAlthough the PIN program aimed to improve the overall quality of primary care and did not specifically target low-SES Manitobans, there is concern that P4P programs operating within the current pattern of health inequity can widen the socioeconomic gap. It has long been recognized that inequities left unaddressed have damaging effects on health outcomes in vulnerable populations . TherefoThe PATHS Data Resource  is a de-Income quintiles were constructed using public-use census data for each dissemination area  in Manitoba based on the postal code of the child at age 2. The dissemination areas were sorted by average income and divided into quintiles of equal population size. About 1 % of individuals in Manitoba were excluded from the income quintiles, because their postal code did not link with a dissemination area, their dissemination area had a suppressed average household income, or they lived in a dissemination area where 90 % or more of the population was institutionalized .The PIN program was implemented in two phases: Phase 1 was launched in 2007 with the participation of four primary care clinics, and eight additional clinics were recruited for Phase 2 in 2008. The twelve clinics were distributed across Manitoba regional health authorities (RHAs) and included 170 physicians with over 180,000 patients assigned to their care during the period of study . P4P funThe development of the study cohorts is described in Fig. We determined the rate of vaccination completion by income quintile in each cohort by counting the number of vaccine doses children had received by the age of 2 and comparing these to the Manitoba Immunization Guidelines . To measChanges in concentration indices for vaccination could be influenced by corresponding changes in underlying income inequality . We therWe also estimated the Kakwani Progressivity Index (KPI). The KPI is defined as the difference between the concentration index  and the Gini coefficient (income distribution), and ranges from -2 to 1 . For ourFigure Figure Table At the end of the 5-year PIN program, Gini coefficients in the PIN clinics and non-PIN clinics remained significantly higher than 0, signifying that a significant difference in income distribution between the two groups persisted. Concentration indices for the PIN clinics and the non-PIN clinics remained significantly higher than 0, indicating that health inequity in vaccination continued in both groups after the PIN intervention. The mKPI showed that health inequity was less than expected when accounting for the underlying income inequality in these groups.In Table The findings of this study suggest that the PIN program helped to maintain the pre-intervention level of inequity in childhood vaccination rates by the age of 2. Although income distribution remained unequal in the both the PIN and non-PIN groups throughout the course of the P4P intervention, inequities in vaccination across income quintiles worsened in non-PIN clinics while remaining stable in PIN clinics. We expected to see a decrease in health inequity in the PIN clinics, but this was offset by the health inequity increase in the non-PIN clinics.In the last decade, P4P programs have become a popular method of encouraging improved primary health care, although it is still uncertain how they impact patient care and disease management , 42. WitTo our knowledge, our study is the first to examine whether a P4P intervention can effectively alleviate SES-related health inequities in vaccination. Our findings suggest that in the absence of a P4P funding initiative, inequity in vaccination completion increased in the general population. Although the PIN program mitigated the increasing negative effect of low SES on vaccination during the study period, the program did not address the pre-existing health inequity. The social determinants of health, including SES, have a direct effect on the health of individuals and populations, and disparities in health affect the health status of the overall population , 47. AltStrengths of our study include the ability to link administrative data across several databases, allowing us to capture virtually all of the eligible population for our study cohort, including rural and urban populations. Although our findings are specific to the primary care renewal initiatives implemented in the province of Manitoba, the inclusion of different geographic regions lends generalizability to the results for the entire population of the province. The analysis was limited by the use of area-level income data. However, studies have shown that area-level measurements, such as those collected from dissemination areas, provide a good approximation of individual-level SES . AlthougOverall, our study suggests that a P4P incentive program was effective at maintaining SES-related health equity in vaccination completion, but did not improve it. Given how few studies address equity in the delivery of primary care, future research should take into consideration not only whether primary care renewal initiatives have an impact on health, but also the role of social determinants in driving inequities in health and health care use.


Subarachnoid hemorrhage (SAH) is a neurological disease with a high mortality rate. Several serious complications frequently arise after successful surgery for this condition. Cerebral vasospasm, one such complication, occurs in 50 to 70% of SAH patients. These patients suffer neurological symptoms known as delayed ischemic neurological deficit (DIND); however, the effect of treatment of vasospasm is limited. The major pathogenesis of cerebral vasospasm is the reduction of nitric oxide (NO) and activation of vasoconstrictors. Acupuncture is known to increase the production and activity of vascular endothelial cell-derived NO and improve endothelium-dependent vasodilatation. A preliminary retrospective case study to investigate the ability of acupuncture to prevent the occurrence of cerebral vasospasm has been conducted. However, no randomized, controlled clinical trials have been carried out to evaluate the efficacy of acupuncture for cerebral vasospasm.This trial will be a single-center, randomized, placebo-controlled, parallel group, patient-assessor-blinded clinical trial. A total of 80 patients with SAH will be randomized into two groups: a study group given acupuncture, electroacupuncture, and intradermal acupuncture, and a control group given mock transcutaneous electrical nerve stimulation and sham intradermal acupuncture. Intervention will start within 96 h after SAH, and a total of 12 sessions will be performed during a 2-week period. The primary outcome measure will be the occurrence of DIND, and the secondary outcomes will be vasospasm as measured by cerebral angiography, transcranial Doppler, clinical symptoms, vasospasm-related infarcts, NO and endothelin-1 plasma levels, mortality, and modified Rankin Scale scores.This trial will examine the efficacy and safety of acupuncture for cerebral vasospasm after SAH. The placebo effect will be excluded and the mechanism of action of the treatments will be evaluated through blood testing.NCT02275949, Registration date: 26 October 2014.ClinicalTrials.gov  Subarachnoid hemorrhage (SAH) can occur spontaneously or as a result of trauma . The mosTreatment of SAH involves early ligation of the aneurysm or insertion of a coil; however, complications such as cerebral vasospasm, rebleeding, or hydrocephalus frequently occur after successful surgery -4,7,8. SSpontaneous vasoconstriction and vasodilatation are adjusted by the physiological regulation of vascular endothelial cells . If a ceAcupuncture is an effective and safe treatment method that has been used for the treatment of acute stroke including SAH for centuries . In receBased on previous reports, a clinical study was conducted in hypertensive patients with damaged arterial endothelial cell function, which confirmed that acupuncture at ST36-PC6 improved endothelium-dependent vasodilatation . After tThis study will be a single-center, randomized, placebo-controlled, parallel group, patient-assessor-blinded clinical trial. The study will be conducted at Kyung Hee University Hospital in Gangdong, Seoul, Korea.www.clinicaltrials.gov (NCT02275949) and will be reported in compliance with the CONSORT statement (www.consort-statement.org).The trial will be carried out in accordance with the Declaration of Helsinki and the Korean Good Clinical Practice Guidelines and has been approved by the ethical committee of the Kyung Hee University Hospital at Gangdong (KHNMC-OH-IRB 2014-001-002). It has been registered on A total of 80 participants will be recruited for this trial. Patients with SAH admitted to our hospital are potential candidates for the study. Eligible participants will be randomized to the study group or the control group after written informed consent is obtained. Concealed allocation will be achieved by randomization and allocation by an assigned researcher without patient contact. Randomization will be done using a computer-generated allocation list by an assigned researcher not involved in the intervention or assessment. Stratified block randomization will be carried out according to sex and the Hunt and Hess Scale (HHS) . The phyThe participants and the outcome assessors will be blinded to the type of intervention used. The practitioner will not provide any clues about the allocation information to the participants during the study. Blinding will be maintained until the study is completed.Participants conforming to all the following conditions will be included: (1) SAH verified by computed tomography (CT) and cerebral angiography; (2) aneurysm treated by endovascular coiling or surgical clipping; (3) age > 18 years; (4) HHS 1 to 4; (5) acupuncture treatment can start within 96 h after SAH; and (6) participation is voluntary and the informed consent form is signed.Participants with any of the following conditions will be excluded: 1) traumatic or infectious SAH; 2) HHS 5; 3) transcranial Doppler (TCD) cannot be performed; 4) heart, liver, or renal failure; 5) presence of cardiac pacemaker; or 6) have experience of electro-acupuncture.Intervention will start within 96 h of SAH, after ruptured aneurysms have been secured by endovascular coiling or surgical clipping. Interventions will be applied once a day, 6 days a week for 2 weeks  in addition to standard treatments such as prophylactic hypertensive, hypervolemic, and hemodilution (triple-H) therapy and nimodipine. Sterile disposable stainless steel acupuncture needles  will be used. All interventions will be performed by one Korean Medicine doctor with over 3 years of working experience who completed a college education of 6 years. This doctor will be trained in the study protocol before the start of the trial.de qi (a subjective experience in which patients feel a radiating sensation considered indicative of effective needling) is achieved.Acupuncture, electroacupuncture, and intradermal acupuncture will be performed at every session. Acupuncture needles  will be inserted bilaterally at the acupuncture points Zusanli (ST36), Neiguan (PC6), Gongsun (SP4), and Xiangu (ST43). After insertion to a depth of approximately 1.5 cm, the needles will be manually stimulated until An electric stimulator  will be connected to the handle of each needle in the ST36 and PC6 positions and a current of 5 Hz applied. The current intensity will be increased until light muscle contraction is evident and reaches approximately 70% of bearable intensity. The needles will be left in place for 20 min and then removed. The practitioner will be able to regulate the intensity in response to requests from the subjects.After the needles are removed, intradermal acupuncture needles with tape  will be inserted bilaterally in the ST36, PC6, SP4, and ST43 positions and maintained until the next session.Mock transcutaneous electrical nerve stimulation and sham intradermal acupuncture will be carried out by the same Korean Medicine doctor. After an electrical insulator  is attached bilaterally in the ST36 and PC6 positions, the same electric stimulator will be connected to the same points as used in the study group, but without application of current. The subjects in the control group will see the same lights and hear the same sounds coming from the machine as the study group. After 20 min, the electrical insulator will be removed and sham intradermal acupuncture will be performed at the same points. 3 M micropore medical tape (1 cm × 1 cm) will be placed on the skin. After intradermal acupuncture needles are placed over the tape but not penetrating the skin, micropore medical tape of the same size will be placed over the needles. This will remain in place until the next session.The evaluation of each assessment scale will be performed by an investigator blinded to the assigned group. A baseline assessment will include Fisher grade (FG), HHS, modified Rankin Scale (mRS), NO and endothelin-1 in plasma . The occAll participants will receive conventional therapy in the same way. This is defined as general neurosurgical management carried out in SAH patients to stabilize the vital signs, control pain, and prevent vasospasm through fluid or medication therapy. Any use of medication will be recorded on the case report forms every day.Participants conforming to any of following conditions will be removed from the study: (1) more than three sessions  are not performed; (2) withdrawal of patient consent; (3) severe adverse events making the trial unsustainable; (4) worsening condition making it difficult to participate in the trial; or (5) impractical to continue with the trial, as judged by the principal investigator.The occurrence of DIND will be compared between the study group and the control group. DIND is defined as an unaccountable new focal neurological deficit lasting ≥2 h. Any occurrence of DIND will be recorded by the physician and verified by the investigator every day.Incidence of angiographic vasospasmAngiographic vasospasm is defined as focal or generalized reduction of cerebral arterial caliber on conventional cerebral angiogram or CT angiography confirmed by a neuroradiologist and a neurosurgeon.Incidence of TCD vasospasmMCA) of >200 cm/s and a Lindegaard ratio of >3  [TCD vasospasm is defined as a peak systolic middle cerebral artery velocity (PCA artery) -30.Incidence of vasospasm-related infarct on CT or MRIA vasospasm-related infarct is defined as a cerebral infarction in the region of angiographic vasospasm or TCD vasospasm as shown on CT or MRI.Changes in plasma NO and endothelin-1www.rndsystems.com).A blood sample (3 mL) will be taken from the brachial vein, centrifuged at 3,000 rpm for 15 min, and kept in a freezer at -70°C before analysis. The concentration of NO will be analyzed using a Total Nitric Oxide and Nitrate/Nitrate Parameter Assay Kit  and the level of endothelin-1 will be measured using an Endothelin-1 Quantikine ELISA Kit . All samples will be discarded after analysis Modified Rankin Scale (mRS) scoreFunctional status is assessed with mRS score at the end of treatment and 14 days after treatment (or at discharge).Secondary outcome measures are as follow:Any adverse events or abnormalities will be recorded in the case report forms irrespective of the intervention used. Severity will be evaluated as mild, moderate, or severe, and the relation of any events to the intervention will be evaluated as not related, possibly related, or related.If any serious adverse events occur, the intervention will be stopped immediately and appropriate action will be taken. This will be reported promptly to the institutional review board, according to the protocol.c - Pt) is therefore 28.9%. To detect this difference with a two-sided 5% significance level and a power of 95%, 39 participants per intervention arm have to be randomized, allowing for a 20% dropout rate [DIND, the primary outcome of the trial, was used to calculate the sample size. According to our pilot trial carried out in 2012, the incidence rate of DIND was 10% in the study group and 38.9% in the control group . The difout rate .\document-test or Mann- Whitney U test will be used for continuous outcomes, and a χ2-test or Fisher’s exact test will be used for noncontinuous outcomes. To control for possible confounding factors , multiple regression analysis will be performed for the comparison of two groups. Confidence limits of 95% will be calculated and P values of < 0.05 will be considered statistically significant.According to the principle of intention-to-treat, all randomized participants will be analyzed using SPSS version 18.0  software. All data will be represented as mean ± standard deviation (SD) or number (%). For comparisons of two groups, an independent samples In this randomized controlled trial, we aim to observe the effect of acupuncture treatment on the prevention of vasospasm after SAH. In addition to the occurrence of DIND and vasospasm, plasma levels of NO and endothelin-1 will be evaluated to identify the mechanism of action of the treatments used. Stratified block randomization will be used according to sex and severity (HHS). For the placebo control, we will use an electric stimulator without electrical flow and sham intradermal acupuncture designed not to penetrate the skin. With use of the same electrical stimulator and intradermal acupuncture method as the treatment group, better blinding of the control group is expected. Participants will be treated alone in a treatment room in order to avoid any communication with other participants.In summary, the effect of acupuncture treatment on the prevention of vasospasm after SAH will be assessed in this trial. The placebo effect should be excluded and the trial design should allow for evaluation of the efficacy and safety of acupuncture for SAH patients.Recruitment started in September 2014 and will be completed by the end of May 2016.


As people infected with the human immunodeficiency virus (HIV) in Sub-Saharan Africa live longer due to availability of antiretroviral treatment (ART), so is the rise of associated infections with their burdens on patients. But reliable data on the prevalence of co-infection with hepatitis B (HBV) or C (HCV) still remains sparse and many individuals with HIV do not know their co-infection status. This study attempted to estimate the seroprevalence and identify risk factors associated with hepatitis B and/or C co-infections in HIV-infected individuals from five Regions of Cameroon by screening 531 HIV infected subjects for the presence of HBV surface antigen (HBsAg) and antibodies to HCV (HCV-Ab). A Screening and a confirmatory Enzyme linked immunosorbent assay were used to detect presence of markers of infection. CD4 count levels were also examined. The results indicate that of the 531 participants, 68% were females and 32% males. Mean CD4 count was ~400 cells/μl. Seroprevalence rates for HBsAg and HCV-Ab were 23.7%, and 7.2%, respectively. Associations assessed using logistic regression revealed that HBsAg but not HCV-Ab positivity was linked to age, lower CD4 count and residing in an urban rather than in a rural setting. This high prevalence of co-infection with HBV raises the urgent need to systematically screen all newly diagnosed HIV cases for co-infection in Cameroon and other regions of sub-Saharan Africa where HIV accounts for the majority of the global infection, so as to improve management strategies for HBV infection and ART implementation. People infected with the human immunodeficiency virus (HIV) are at a greater risk of co-infection with either hepatitis B (HBV) and/or hepatitis C virus (HCV) compared to the general population . Up to 3Although the mechanisms by which the hepatitis virus interacts with HIV to influence disease progression are not well understood, it has been reported that HIV/HBV co-infection facilitates HBV replication and reactivation leading to higher HBV DNA levels and a reduced spontaneous clearance of the virus –8. On thIn most developing countries, including Cameroon, HCV and/or HBV testing and monitoring in HIV patients is not routine. As a result, the challenge of implementing directives from the World Health Organization (WHO) that ART be commenced in HIV co-infected patients irrespective of CD4 count results remains daunting. Therefore many HIV/hepatitis virus infected patients do not benefit from programs aimed at treating HIV patients since only the patients’ CD4 count level is taken into consideration for the purpose of initiating ART .Worldwide an estimated 350 million people are chronically infected with HBV while 185 million are chronic carriers of HCV . On the Ethical approval for the current study was obtained from the National Ethical Review Board in Cameroon and the Institutional Review Board at the New York University School of Medicine. Patients’ records were anonymized and de-identified prior to analysis.All participants were initially enrolled as part of a large cohort study on HIV-1 genetic diversity in Cameroon –20. All Testing for the hepatitis B surface antigen (HBsAg) was performed using the HBsAg enzyme linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions. All non-reactive samples were recorded as negative. All reactive samples were tested a second time using the HBsAg confirmatory test according to the manufacturer’s instructions. All samples reactive to both assays were recorded as positive while those that were non-reactive in the second test after being reactive in the first test were considered non concordant and were subjected to a sensitivity analysis before being included as either positive or negative .All samples reactive by ELISA for HBsAg were further tested for the presence of hepatitis “e” antigen (HBeAg) and IgM antibody to hepatitis B core antigen (HBc IgM) using ELISA kits .The presence of hepatitis antibodies is a strong marker of infection with HCV. HCV-Ab was detected using the Anti-HCV ELISA V 4.0 , a fourth generation enzyme immunoassay kit, which uses recombinant HCV antigens . HCV-Ab reactive samples were confirmed with the same ELISA kit using the algorithm shown in CD4 count of participants was determined by Guava Easy CD4 system that uses combination of monoclonal antibody and volumetric system to count cell numbers with high precision .Participants positive for HBsAg or anti-HCV following testing were considered infected. Continuous variables including age and CD4 counts were compared in males and females using the Student’s t-test. Categories were compared using the chi squared test for trend. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the association between chronic infection with HBV or HCV and each of the defined risk factors including age, sex, CD4 count, residence, region, and number of sexual factors. We did not analyze by year of enrollment as participants in different regions were enrolled during different years. Each of the risk factors was entered into the regression model as a main effect and adjusted for the other co-factors including being infected with either HBV or HCV. P trend was estimated by treating categorized markers as ordinal variables. In a sensitivity analysis (not presented), inclusion of subjects with non-concordant results following testing with the second ELISA assay as non-reactive had no substantial effect on the results. Two-sided P values of <0.05 were considered statistically significant. All statistical analyses were performed using Stata .The total number of people initially screened for hepatitis viral infections were 531. Of these, 168 or 32% were positive for hepatitis B surface antigen (HBsAg+) while HCV antibodies (HCV-Ab) were detected in 61 or 11.5% . Nine orAge specific prevalence for HBV was 10% for individuals 40 years old or younger, 8% for those of ages between 41–50 and 3% in subjects older than 50 years. Likewise HCV age specific prevalence was lowest (2%) in participants in the highest age group (>50 years) and highest (4%) in those with age 31–40 years .Analysis by region indicates that the Center Region had the highest HBV prevalence (18%) among the 181 HIV infected subjects studied . This isTo determine the independent risk factors for co-infection with HBV or HCV in this HIV infected population, we performed an unconditional logistic regression with HBsAg positivity or HCV Ab positivity as outcomes adjusting for age, sex, CD4 count, residence, regions and number of sexual partners. Our results show that although age is not statistically associated with being chronically infected with HBV (HBsAg+), individuals older than 30 years were less likely to be infected compared to those with a younger age. These results also show that men had a higher chance of being infected with HBV than women,  .We studied the effect of a lowering of CD4 level on co-infection in this HIV infected population by stratifying our study subjects into those with a CD4 count of below 250 cells/μl and those with CD4 counts ≥250 cells/μl. Our results indicate that subjects with a CD4 count superior to 250 cells/μl were less likely to be co-infected when compared to individuals with a lower CD4 count,  for HBV and  for HCV .We examined the influence of residence  on the chance of being co- infected with HBV or HCV. Our results show that participants living in urban cities were more likely to be chronically infected with HBV than those residing in rural villages, , We tested the hypothesis that participants with multiple sexual partners were more likely to be co-infected with either HBV or HCV compared to those with only one sexual partner. In support of our hypothesis, we observed a 28% increased chance of being infected with HBV in people with multiple sexual partners compared to those with only one, , Although we collected data from five political regions, for the purpose of this analysis and to maintain statistical power, we pooled data from the five political regions to make up two regions based on their geographical location, Western and Eastern regions. Compared to the western regions, participants from Eastern regions were statistically significantly at higher risk of being chronically infected with HBV, , To better control for geographical location within the country, we performed separate analysis to determine which risk factors influence hepatitis virus co-infection in HIV infected people living in each of the two geographical settings, Western and Eastern parts of the country. The results are presented in Tables A and B in Hundreds of thousands of people in Cameroon are infected with HIV with an estimated prevalence of 4.3% in calendar year 2011  but littThe high frequency of co-infection with HBV in this population has important consequences for the emergence of liver-related mortality  and seriThe frequency of anti-HCV in this study (7.2%) is much lower than that reported previously in Cameroon  but muchWe examined the influence of several factors including, age, sex, CD4 cell count, residence, geographical region and number of sexual partners on the risk of co-infection with HBV or HCV  and 3. OAccepted values for CD4 cell counts in healthy individuals range from 500–1200 cells/μl . HIV infWe found a significantly elevated risk of co-infection in residents of urban cities compared to those residing in rural villages. Although we did not examine the direct influence of socioeconomic status (SES) on risk of co-infection in this study, it has previously been reported that higher SES is associated with a more mobile lifestyle and a higher risk of infection with HIV . TherefoAlthough the proportion of women with chronic HBV or HCV infections (63%) is much higher than that for men , in adjuThere are some limitations of this study that warrant further research. The analysis has been performed with samples from HIV-1 infected persons that were studied for HIV-1 prevalence and genetic diversity; thus, the samples were a convenience sampling set and were not collected randomly to represent the general population. A larger sample size representing the general HIV-1 infected population and/or a more complete data set will benefit more statistical power for some risk factors. The analysis performed here were all based on ELISA screening and no molecular tests were performed. We acknowledge that the use of additional HBV and HCV confirmatory tests as well as molecular tests for HBV DNA will also benefit the study. We note that in the absence of molecular tests for HBV DNA, it was not possible to take into account occult HBV infections in those with HBsAg negative results thereby possibly reporting a lower HBV prevalence than would otherwise have been the case. We also note that we did not perform test for HCV RNA and were therefore unable to identify active HCV infections among those who were anti-HCV positive. About 25% of HCV infections clear spontaneously resulting in a positive anti-HCV test but a negative HCV RNA PCR test . Thus, wS1 FileTable A. Risk factors associated with infection with Hepatitis B or C in patients infected with HIV in the Western Regions of Cameroon, Table B. Risk factors associated with infection with hepatitis B or C in patients infected with HIV in Eastern regions of Cameroon.(PDF)Click here for additional data file.