Interventional ICU trials have followed up patients for variable duration. However, the optimal duration of follow-up for the determination of mortality endpoint in such trials is uncertain. We aimed to determine the most logical and practical mortality end-point in clinical trials of critically ill patients.We performed a retrospective analysis of prospectively collected data involving 369 patients with one of the three specific diagnoses (i) Sepsis (ii) Community acquired pneumonia (iii) Non operative trauma admitted to the Royal Perth Hospital ICU, a large teaching hospital in Western Australia (WA cohort). Their in-hospital and post discharge survival outcome was assessed by linkage to the WA Death Registry. A validation cohort involving 4609 patients admitted during same time period with identical diagnoses from 55 ICUs across Australia (CORE cohort) was used to compare the patient characteristics and in-hospital survival to look at the Australia-wide applicability of the long term survival data from the WA cohort.P = 0.19), community acquired pneumonia (P = 0.86), non-operative trauma (P = 0.47).The long term outcome data of the WA cohort indicate that mortality reached a plateau at 90 days after ICU admission particularly for sepsis and pneumonia. Mortality after hospital discharge before 90 days was not uncommon in these two groups. Severity of acute illness as measured by the total number of organ failures or acute physiology score was the main predictor of 90-day mortality. The adjusted in-hospital survival for the WA cohort was not significantly different from that of the CORE cohort in all three diagnostic groups; sepsis (A minimum of 90 days follow-up is necessary to fully capture the mortality effect of sepsis and community acquired pneumonia. A shorter period of follow-up time may be sufficient for non-operative trauma. Mortality is the most clinically relevant and commonly used primary outcome measure for phase III trials in intensive care. However, the optimal duration of follow-up for the determination of mortality in such trials is uncertain ,2. InterThe Australian and New Zealand Intensive Care Society (ANZICS) Centre for Outcome and Resource Evaluation (CORE) Adult Patient Database (APD) gathers information about the vast majority of admissions of critically ill patients from various intensive care units (ICUs) across Australia and New Zealand but currently does not follow up patients beyond hospital discharge . HoweverWe hypothesized that, if the characteristics and short-term outcomes of patients in the WA cohort and the various ICUs from Australia (as identified within the two databases) were comparable, then the follow-up data of the patients in WA cohort could be used to estimate the likely in-hospital and out-of-hospital long-term survival of critically ill patients in Australia.We conducted a retrospective analysis of prospectively collected data from two large, related databases. Access to the data was granted by the ANZICS CORE Management Committee in accordance with standing protocols. Data are collected primarily for ICU Outcome Peer Review under Quality Assurance Legislation of the Commonwealth of Australia . Such data are collected and transferred from hospitals to the database with government support and funding. Hospital data are submitted by or on behalf of the ICU Director and results are reported back to the Director. Each hospital allows subsequent data use as appropriate under the ANZICS CORE standing procedures and in compliance with the ANZICS CORE Terms of Reference  and waivThe study cohort consisted of all patients over 18 years of age who were admitted to ICU from emergency departments between 1 January, 2001 and 31 December, 2002 with one of three acute physiology and chronic health evaluation score (APACHE) II diagnoses : sepsis The data for the WA cohort were extracted from the Royal Perth Hospital ICU database. In this study, the survival outcome after hospital discharge of the WA cohort was assessed on 31 December 2003 by linkage to the WA death registry ,16. The The in-hospital and subsequent survival data of the WA cohort at different time points after ICU admission was used to assess whether a 'plateau' was observed. These data were then further analyzed to determine the incidence of death after hospital discharge and the quantum effect of various variables on survival at different time points. A formal landmark survival analysis was performed with the landmark time point chosen as ICU discharge. The variables assessed included age, gender, Charlson co-morbidity index , Acute pP value of 0.05 was considered to be statistically significant.Continuous data with a near normal distribution are presented as mean and standard deviation and data with a skewed distribution were expressed as median and interquartile range. Categorical variables and data with a skewed distribution are analysed by chi-squared and Mann-Whitney test, respectively. Kaplan-Meier survival analysis and log-rank test was used to compare the difference in hospital survival between the WA cohort and ANZICS APD cohort. Single variable and multivariable analyses were performed using logistic regression for binomial outcomes and reported using odds ratios ) and Cox proportional hazard regression for time to death with results reported using hazard ratios (95% CI). Survival analysis was performed with survival time measured from both ICU admission and ICU discharge. Multivariable models were constructed using both stepwise selection and backward elimination procedures before undergoing a final assessment for clinical and biological plausibility. Statistical analysis was performed using SAS version 9.1  and SPSS statistical software . A two-sided When considering patients with pneumonia or sepsis, 28-day mortality only effectively captured 67% and 70% of deaths that occurred within six months of ICU admission. By considering 90-day mortality, the proportion of deaths captured increased to 89% and 93%, respectively Figures  and 2. TSingle-variable analysis showed the APACHE score to be the most consistent predictor of mortality but not a statistically significant predictor of time to death after ICU discharge for either pneumonia or sepsis Table . GCS wasMultivariable analysis showed that markers of acute illness, such as the number of organ failure and APACHE score, were the strongest predictors of mortality for sepsis, community acquired pneumonia and non-operative trauma Table . AlthougWhen the two cohorts were compared patients from the WA cohort were slightly younger, had less co-morbidity, and a longer length of ICU and hospital stay across all three diagnostic subgroups Table . HoweverUsing the WA data, we found that the mortality of sepsis and community acquired pneumonia reached a plateau by 90 days and that mortality after hospital discharge was common. We further found that at 90 days after ICU admission the severity of acute illness on ICU admission was still the most important predictor of mortality.We compared the characteristics, severity of illness and in-hospital outcomes of 55 ICUs across Australia (CORE cohort) with those of a cohort of patients with identical diagnoses from a university teaching hospital in Western Australia (WA cohort) for whom long-term outcome was available. We found that the APACHE II-predicted mortality, hospital mortality, and in-hospital survival curves were similar between the WA and CORE cohorts.This study uses very high quality prospectively collected data (ANZICS CORE APD) that is representative of the ICU patient population in Australia and provides a valid comparator with which to evaluate how general the WA data is ,12,20. WMany interventional ICU trials have used different durations of follow up with which to assess mortality but the most appropriate duration of follow up is uncertain -7. Our rEpidemiological data shows that severity of illness and organ failure that requires intervention can have a mortality effect long after hospital discharge -23 It isThis study has several strengths. It formally addresses the important issue of what might be an appropriate duration of follow-up for the assessment of mortality as an outcome. It used high-quality databases for this assessment and confirmed the biological and clinical appropriateness of 90-day follow up by showing that 90 days after ICU admission, the degree of illness severity at ICU admission remained an important predictor of outcome. However, our study also has limitations. Although the WA cohort was comparable with a wider Australian ICU sample in severity of illness and hospital survival, it is still possible that the survival pattern of the two cohorts could be different and we failed to detect such a difference. This seems unlikely given the striking similarity in illness severity, short-term outcome similarities, and the general uniformity of the urban Australian population. It further seems unlikely given that the observations are internally consistent for three separate conditions. However, our results may not be generally applicable to ICU patients in other countries because hospital and healthcare systems vary. Thus, similar studies in other countries are now desirable.The sample size of the WA cohort in this study was relatively small and the results, therefore, have wide confidence intervals. We acknowledge that our study may not have enough power to truly assess the importance of the selected predictors of mortality. Accordingly, studies involving larger samples may also be desirable to confirm these findings. In addition, we only examined three specific subgroups of critically ill patients. The survival pattern during the first 180 days after the onset of other critical illness may be different in other diagnostic groups . HoweverA minimum follow-up time of 90 days without censoring at hospital discharge is necessary to fully capture the mortality effect of community acquired pneumonia and sepsis. For non-operative trauma, a shorter follow-up time appears to be sufficient. This information is important in providing an evidence-based approach in designing and interpreting randomized controlled trials involving these conditions.• Hospital or 28-day mortality is not an adequate follow up end-point for interventional trials in ICU that involve sepsis and community acquired pneumonia.• Mortality after hospital discharge is significant up to 90 days when it appears to reach a plateau.• Severity of illness is the main determinant of mortality at 90 days and, as such, any interventions that aim to attenuate physiological derangement from sepsis or community acquired pneumonia are likely to have a significant effect on mortality up to 90 days.ANZICS: Australian and New Zealand Intensive Care Society; APACHE: Acute Physiology and Chronic Health Evaluation; APD: Adult Patient Database; CI: confidence interval; CORE: Centre for Outcome and Resource Evaluation; GCS: Glasgow Coma Score; ICU: intensive care unit.The authors declare that they have no competing interests.GT designed the study, collected the data, performed the statistical analysis and drafted the manuscript. KMH performed data analysis and helped to draft the manuscript. CG, RB, GH, and SW participated in its design and analysis of the study, and coordinated the drafting of the manuscript. MB performed additional statistical analysis and responded to reviewers. All authors read and approved the final manuscript.


A large amount of experimental data generated by modern high-throughput technologies is available through various public repositories. Our knowledge about molecular interaction networks, functional biological pathways and transcriptional regulatory modules is rapidly expanding, and is being organized in lists of functionally related genes. Jointly, these two sources of information hold a tremendous potential for gaining new insights into functioning of living systems.Genomics Portals platform integrates access to an extensive knowledge base and a large database of human, mouse, and rat genomics data with basic analytical visualization tools. It provides the context for analyzing and interpreting new experimental data and the tool for effective mining of a large number of publicly available genomics datasets stored in the back-end databases. The uniqueness of this platform lies in the volume and the diversity of genomics data that can be accessed and analyzed , and the integration with an extensive knowledge base that can be used in such analysis.http://GenomicsPortals.org.The integrated access to primary genomics data, functional knowledge and analytical tools makes Genomics Portals platform a unique tool for interpreting results of new genomics experiments and for mining the vast amount of data stored in the Genomics Portals backend databases. Genomics Portals can be accessed and used freely at  A large amount of experimental data generated by modern high-throughput technologies is available through public repositories such as GEO  and ArraInadequacies of the major data repositories to serve as access points to genomics data have resulted in numerous fragmented projects providing access to data from a single dataset ,7, a setOn the other end of the spectrum, several prominent efforts are directed towards constructing lists of functionally related gene lists ,4, but tThe power of integrative analyses utilizing genomics data and functional knowledge has been demonstrated in the analysis of individual datasets  and systThe access to gene expression regulatory data such ChIP-chip and ChIP-seq transcription factor binding and epigenomics data is even more difficult and fewer resources are available. Most of the datasets are still deposited to the main genomics repositories. However, the only meaningful way to access this data is through UCSC and ENSEMBL Genome Browsers ,22. BothGenomics Portals platform was designed around the three conceptual problems faced daily by biomedical scientists:Characterizing experimentally derived gene lists in the context of relevant publicly accessible genomics data . By simply pasting the experimentally derived gene list  into the query box and then selecting the relevant datasets, one is able to download the primary data, perform basic analysis and generate publication-quality graphics depicting the expression patterns of the genes queried. The whole process can take less than one minute.1. Functional analysis of newly generated data. By depositing newly generated data into Genomics Portals databases, one can leverage extensive knowledge base , or browse our collection of analytical results to construct meaningful gene lists for querying their own data.2. Integrative mining of public genomics data. Researchers can simply use the knowledge base and their own imagination to construct query gene lists and select genomics datasets to mine.3. constructing a list of genes by either using the predefined lists, or pasting a gene list of interest; querying one of the databases with genome-scale data; and producing analysis summaries. Based on the analysis results, one can further refine their gene query list and repeat the procedure on a different dataset.The conceptual structure of Genomics Portals is depicted in Figure Genomics Portals knowledge base consists of >20,000 lists of functionally related genes, Entrez GeneRIFs, and BioGRID gene-gene interaction data. One can construct a query gene list by pasting their own list, by manipulating the predefined gene lists, by an open text search of GeneRIFs, and by searching for interaction partners of a gene or a group of genes. One can also elicit a gene list by browsing pre-computed clustering results for different datasets.Numerous efforts are underway to systematically collect and organize the functional information about genes. Our first goal is to assemble lists of functionally related genes from such collections. Following are sets of gene lists we gathered using publicly available resources: Gene Ontologies , KEGG paFor a most of gene expression datasets we also performed unsupervised cluster analysis followed by in-depth functional annotation of the clustering structure. The cluster analysis was performed using the Bayesian model-based procedures ,30 as weThe vast majority of the genomics data deposited in our databases is public data downloaded from the major repositories , or produced by the computational analyses of genomics data . A small portion of the database is private data belonging to our collaborators which is accessible after a log-in. No registration or login is required for accessing the public data. In general, "genomics data" refers to genome-scale vector of measurements produced by various experimental assays  or computationally constructed scores . Genomics datasets are organized thematically into different data portals. Different portals can contain datasets related to different diseases (e.g. Breast Cancer and Prostate Cancer), specific types of genomics data (e.g. Epigenomics and Transcription Factors), or different biological processes (e.g. Development). The same dataset can be assigned to different portals.Gene expression data in our databases consists of the complete collection of human, mouse and rat GEO DataSets , and close to 100 manually curated GEO series of particular interest. The majority of the manually curated datasets are not available as GDS DataSets. For some of the manually curated datasets we have re-processed the raw outputs (e.g. CEL and GPR files).Most of these datasets have been manually curated and re-processed. The data is summarized as the average measurement intensities within non-overlapping 50 bp windows for the -4 kb to +1 kb regulatory region around each RefSeq sequence for the given genome build. This representation provides for a straightforward manipulation and graphical representation of such data for gene lists. The definition of "measurement intensities" depends on the primary data that was downloaded from the repository and it can range from typical log2-scaled fluorescence intensities for hybridization-based experiments to sequence read counts and various approaches to identifying and quantifying "peaks" for high-throughput sequencing technologies.Most of this type of data is derived from computational assessments of DNA features within the genomic regulatory regions such the existence of transcription factor (TF) binding motifs and CpG islands. Computationally predicted transcription factor binding scores are provided as whole gene (i.e. Refseq) scores that assess the overall likelihood of a transcription factor binding within the gene's promoter and high resolution datasets that provide locations of putative TF binding sites at the same resolution used for ChIP-chip and ChIP-seq data. All scores were calculated using in-house developed scoring algorithms and TRANSFAC transcription factor binding motif definitions .Rserver infrastructure. The basic manipulations consist of sub-setting the data, selecting grouping variables for the samples in the datasets, identifying differentially expressed genes, cluster analysis, assessing enrichment of differentially expressed genes within the query list, etc. The results of such analyses are provided as static annotated heatmaps, or can be browsed using our FTreeView browser. Analyses of results can also be incorporated within the images of KEGG pathways.In designing Genomics Portals we sought to strike a balance between the key limiting factors such as the complexity of query interfaces and the computational complexity of the analyses performed on the data, and the usefulness of the results produced. The portal is designed so that a basic output of the genomics data queries can be obtained in less than one minute with only a few clicks of the mouse. This includes query of our knowledge base or pasting one's own gene list, selecting the genomics data to query, and retrieving the data in the form of a spreadsheet or an R ExpressionSet. At this point, the user can either use their own data analytical tools or perform basic manipulation and analysis of the retrieved data within the portal. Subsequent analyses are performed using the retrieved data and the simple interface that deploys R and Bioconductor procedures using the MySQL databases for organizing knowledge base and genomics data, and R scripts for performing analysis of retrieved data using the RServer infrastructure to connect Java with R. The relational databases storing genomics data are loosely based on the MySQL version of ArrayExpress, MaxD http://www.bioinf.man.ac.uk. Query gene lists are constructed and genomics datasets selected using Java-based interfaces. Java programs then call appropriate R scripts that query genomics databases (using RMySQL) and perform the analysis using various Bioconductor packages. The hardware infrastructure mirrors these three basic computational modules  are periodically updated by executing appropriate R scripts. For GO and KEGG updates we utilize most recent Bioconductor data objects. For L2L and miRBase lists, we download the current version from the primary web sites.• For gene lists constructed by analyzing public data (Disease gene lists and Mouse phenotype gene lists), the process is similar except it proceeds in two steps. The current version of primary data  is downloaded and re-analyzed using existing R scripts to construct the gene list which are then uploaded to the back end database.Processed primary genomics data is continually added, but there are no planned updates of the data that has already been uploaded. Each curated dataset is associated with a standardized R script that was used to process the raw data and upload the dataset to the back-end database. When an error is detected in any of the datasets, we generally remove the dataset, correct the error in the script used to upload the dataset and re-upload the dataset. This way, each manipulation of the data is documented by the R program that performed it. The download and processing of curated GEO data sets (GDS) was performed in batch using again specifically developed R programs, and new datasets will be periodically added as they become available using the same scripts.We demonstrate the utility of the Genomics Portals through a case study investigating a proliferation gene expression signature in rat mammary epithelium induced by different fatty acid diets . That stlimma package [All four gene expression datasets used in the analysis investigate proliferation-related biological processes. For each of the datasets, we assessed the enrichment of differentially expressed genes in specific comparisons among the proliferation genes identified in the rat dietary study Figure . For eac package  for the  package . The que-9).The Miller dataset  is a wel-8).The Fournier dataset  Genomic. Again, -8).The Herschkowitz dataset  Genomic. Query g-6). For the same cell line without re-expressing ESR1, there was no discernible effect on expression of genes in our list , n-Myc , Tcfp2l1 , c-Myc , and Klf4  all show signs of increased binding to regulatory regions of these genes.In addition to using gene expression data, we further characterized our proliferation signature using ChIP-seq data for E2F1 transcription factor (TF)  and by cA similar analysis of two epigenomics histone marks, H3k4me3 Figure  and H3k2By constructing Genomics Portals we sought to provide meaningful access to diverse genomics datasets currently residing in major public repositories for biomedical researchers without much technical bioinformatics expertise. There is a myriad of bioinformatics solutions that facilitate download and analysis of such datasets. For example, to process and populate our database we used R  and BiocThe general strategy of focusing on lists of functionally related genes as the basic unit for querying and analyzing genomics data is in-line with current thinking that specific functions of a living system are conferred by a coordinated action of a specific set of genes. In this paradigm, a single gene analysis is superseded by the joint analysis of all genes that are functionally related under certain conditions. The utility of such approaches has been demonstrated in numerous studies and confirmed in the case study we presented in this manuscript. Of course, a single gene can be treated as the list of size one and Genomics Portals allows queries and analysis based on the single gene as well. The second guiding idea in constructing the platform is the need to integrate functional data (gene expression), regulatory events data  and the knowledge base .Our decision to use R as the analytical engine within Genomics Portals provides us with virtually limitless possibilities for the analysis of data acquired in a given query. In the current implementation we sought to strike a balance between the key limiting factors such as the complexity of query interfaces and the computational complexity of the analyses performed on the data, and the usefulness of the results produced. This resulted in a highly streamlined interface with relatively few analytical options. Users who wish to perform additional off-line analyses can do so by downloading the data retrieved in the query. In future implementations we plan to "expose" more of the Bioconductor functionality through an additional "Advanced Analysis" web-interface while still keeping the current simple interface as the main option.Other natural extensions of current infrastructure will be allowing for simultaneous queries of multiple datasets and addition of data from other organisms. The implementation of simultaneous queries of multiple datasets will be straightforward. However, query times and computational resources required to perform such queries will be additive in the number of datasets queried. This option will be included once we ascertain that our computational infrastructure is sufficiently robust to provide the current level of interactivity at such increased level of functionality. Expanding the data coverage to any other organism covered by Entrez Gene identifiers will also be rather straightforward. Since genomics data for different species always reside in different databases, adding such data will almost certainly not affect the performance of the portal.The case study presented here is demonstrating the usage of only a small portion of the platform. Comprehensive online documentation is provided for complete description of the data and analytical options available through Genomics Portals.Genomics Portals represents a powerful new tool for gaining knowledge from results of new genomics experiments as well as for mining a large collection of primary genome-scale datasets.Project name: Genomics Portalshttp://GenomicsPortals.orgProject home page: Operating system: platform independentProgramming language: Java, MySQL, ROther requirements: NoneLicense: The tool is available online free of chargeAny restrictions to use by non-academics: NoneKS developed the database infrastructure, tools for populating databases and web-interfaces for querying databases, MP participated in the overall design, developed the protocols and wrote R scripts for performing the analyses and producing graphical displays, MM conceived the portal, developed initial scripts for processing and uploading data and guided the development of all aspects of the portal, JF processed and analyzed a significant number of public datasets, JC developed protocols for processing and uploading, and processed most of the ChIP-chip and ChIP-seq data, QL processed and analyzed a significant portion of curated datasets, ZH computed the predicted transcription factor binding sites, VJ developed and integrated the FTreeView browser, KG assisted with scoring computationally the likelihood of transcription factors binding, JM provided important feedback through incorporating the use of the portal in several graduate courses he is teaching. All authors read and approved the final manuscriptGenomics Portals User Manual. The online help file containing screenshots and step-by-step instructions on how to use Genomics Portals.Click here for file


Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases.Mutations in the dihydrofolate reductase  genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data.This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa.A database consisting of dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.Increasing prevalence of the  Global malaria control initiatives have a turbulent history driven by a number of biological, social, and economic factors. The Malaria Eradication Programme launched by the World Health Organization (WHO) in 1955 successfully eliminated malaria in many temperate and sub-tropical regions of the world, but little progress was made in sub-Saharan Africa. The programme confronted technical challenges including, but not limited to: anti-malarial drug resistance, insecticide resistance, lack of infrastructure to implement and sustain control measures, and a lack of community engagement and participation .The renewed vision for malaria control and eventual elimination focuses on the development of new tools and improved integration of existing interventions . FinanciP. falciparum became a major challenge for control programs [ACT is a vital component of the malaria control strategy and currently represents the last line of defense against infections that have developed resistance to previous anti-malarial therapies. Chloroquine (CQ) monotherapy was highly effective in treating malaria for several years, but eventual appearance of CQ resistance to programs -8. The oprograms ,10.Drug resistance continues to present a major threat to remaining effective anti-malarial therapies, such as ACT, that currently serve as a cornerstone of the malaria control strategy. Therefore, the success of control efforts relies heavily upon surveillance methods to adequately monitor and promptly respond to emerging resistance.Clinical efficacy trials that measure the therapeutic failure rate following administration of a particular drug are the gold-standard method for gauging the effectiveness of anti-malarial therapies. However, clinical efficacy trials are resource-intensive, making them difficult to use broadly for surveillance purposes, reviewed in . Translain vitro-based assays have been used as tools to examine the role of host-drug and parasite-drug interactions that influence clinical efficacy of anti-malarials. Specifically, pharmacological studies conducted within the context of clinical trials can enable an examination of pharmacokinetics and pharmacodynamics (PK-PD) and their role in therapeutic response. An understanding of PK-PD in different clinical settings and populations is essential to establish therapeutic drug dosages for target groups and distinguish between true therapeutic failures and other factors such as poor adherence, drug quality, absorbance, and elimination. PK-PD data is also essential if outcomes from different clinical efficacy trials are to be accurately compared.Pharmacological and In vitro parasite susceptibility assays enable a measure of intrinsic chemosensitivity of P. falciparum parasites to a particular drug by controlling for host factors such as acquired immunity, nutritional status, and compliance dosing. Most in vitro studies report the 50% inhibition concentration (IC50) of an anti-malarial drug, which in turn serves as an indicator of parasite tolerance to the drug. In vitro assays offer benefits, such as the ability to quantify the parasite inhibiting properties of partner drugs within combination therapies and identify temporal trends by comparing the drug sensitivity profiles of frozen reference parasite lines with samples in question. Both in vitro and pharmacological studies play an important role in differentiating drug resistance from other potential causes of decreased drug efficacy. Although these methods complement clinical efficacy trials to collectively identify the presence of drug resistance and help quantify its impact on drug efficacy, they do little to propose specific parasite resistance mechanisms.in vitro and pharmacological studies in that they provide the means to identify genetic markers that confer resistance. Association and genetic complementation studies have identified and validated genetic markers of resistance for older anti-malarial drugs, such as CQ, SP and chlorproguanil-dapsone [Molecular studies examining genetic correlates of resistance differ in scope from -dapsone -24. Mole-dapsone . Lessonsin vitro, and molecular studies each shed light on a certain aspect of drug efficacy and drug resistance, but each also has its own set of benefits and limitations. Lack of standardization across studies of each type and differences in conventions for reporting data are two pervasive problems that seriously limit the ability to compare studies conducted at different times and locations. In an effort to mitigate these and other issues, the WorldWide Anti-malarial Resistance Network (WWARN) has put forth a comprehensive plan to network clinical, in vitro, pharmacological, and molecular studies in a centralized database. The proposed structure and objectives for each of the four components of the database have been detailed in a series of articles [Clinical, pharmacological, articles ,26-28.et al [The need to standardize and network molecular data is particularly pressing. The advancement of high-throughput molecular techniques has enabled a rapid increase in the number of molecular studies conducted in recent years; however, a lack of meaningful ways to compare studies severely limits the potential utility of this valuable information. Databases that assemble the reported frequency of resistant parasite genotypes from multiple molecular studies can provide more comprehensive and easily accessible information. Drug resistance maps at the London School of Tropical Medicine and Hygiene  has asseet al  used a mA growth in the number of molecular studies reporting the frequency of known resistance markers presents new opportunities to assess the broader impact that reporting conventions and study methodology may be having on the data being produced. Some of the variables that have been proposed as potentially influential include age and clinical status of the sampled population, date and location of the molecular study, reviewed in , and repResistance to anti-malarial drugs for which validated molecular markers are available is already widespread; however, a database that specifically assembles studies reporting the frequency of parasite genotypes associated with anti-folate resistance is valuable for a number of reasons.® (CTX), is a low cost regimen that has been shown to decrease morbidity and mortality in HIV-infected individuals [P. falciparum genotypes following CTX treatment [in vitro [in vivo [First, SP use in combination with artemisinin-based derivatives ,34 and aividuals . Howeverividuals . Thus, treatment , continuin vitro  and has [in vivo ,43, even[in vivo ,45. MoniPlasmodium parasite life cycle [The anti-folate class of drugs consists of compounds that bind enzymes necessary for parasite folate biosynthesis. The most widely used anti-malarial drugs within this class are sulphadoxine-pyrimethamine (SP) and, more recently, chlorproguanil-dapsone. The pyrimethamine portion of SP and chlorcycloguanil, the active metabolite of chlorproguanil, bind the enzyme dihydrofolate reductase (DHFR) . Sulphadfe cycle .dhfr and dhps genes of P. falciparum parasites have been associated with decreased parasite sensitivity to the anti-folate drugs. A change from wildtype Ser108 to Asn108 (S108N) in dhfr is sufficient to cause low level pyrimethamine resistance both in vitro and in vivo [in vitro resistance to pyrimethamine by 100-fold relative to wild-type [dhfr can yield higher levels of SP resistance in vitro and in vivo [dhfr gene have evolved in different parts of the world  in combination with the dhfr triple mutant genotype  is a better predictor of clinical failure than either multiple mutant genotype alone [Mutations in codons Ser436 to Ala or Phe (S436A/F), Ala437 to Gly (A437G), Lys540 to Glu (K540E), Ala581 to Gly (A581G), and Ala613 to Ser or Thr (A613S/T) in  dapsone ,59,60. T in dhps . Multiplendently . Howeverpe alone .dhfr and dhps mutant genotypes in African P. falciparum populations; 2) highlight the significance and limitations of these studies; 3) use condensed tables, plots, and geostatistical illustrations to present frequency data; 4) use statistical models to describe changes in the prevalence of select highly resistant genotypes over the years; and 5) elucidate the impact of variables that may influence reported genotype frequencies.The objective of this meta-analysis are to 1) provide a comprehensive database of reported frequencies for resistant dhps," "dihydrofolate reductase," "dhfr," "Plasmodium falciparum," "Africa," "sulphadoxine pyrimethamine," "antifolate," "drug resistance," "Fansidar." Boolean operators were used to search for articles using these terms in various combinations. All variations in the spelling of these terms were accounted for in the search. Additional articles were found through the Related Articles search feature on the Pubmed website and through the reference sections of candidate articles. The Drug Resistance Maps database [A literature search was done using the National Library of Medicine Pubmed/Medline search engine. Search terms included "dihydropteroate synthase," "database  was alsost, 2009 to be included.1) The article must have been published in English prior to Jan 1dhfr  and/or dhps  to be included.2) The article must explicitly state or provide interpretable mutation frequencies for any of the selected codons in in vitro cultured blood isolates were also excluded.3) The article must have provided mutant frequency data derived from pre-treatment blood isolates to be included. Genotype frequency data derived from patients receiving anti-malarial drugs within two weeks of enrollment and/or patients who were determined to have therapeutic levels of anti-folate drugs in their bloodstream at the time of enrollment were excluded. Genotype frequency data derived from 4) The study must have restricted its sampling to blood isolates of African origin from local patient populations to be included. Genotype frequency data derived from travelers were excluded.dhfr and dhps were extracted from published articles and were summarized using two separate databases called the Mutant Allele Frequency (MAF) database and Mutant Genotype Frequency (MGF) database  and dhps double mutant genotype  using a standardized system, similar to the format described by Kublin et al [Data reporting the frequency of mutations in in et al . Table 2One of the major methodological differences across studies was the reporting standards for mixed mutant genotypes. Mixed mutant genotypes are detected when two or more populations of parasites with distinct genotypes at particular codons of interest are present in a single infection. These infections are commonly referred to as "polyclonal" since multiple clonal populations of parasites are present in a single blood isolate. Varying classifications for mixed genotypes can have a significant impact on the determination of mutant genotype frequency; particularly in high transmission settings where polyclonal infections have been reported in greater than 50% of sampled isolates, reviewed in .A majority of studies included in this review used the Restriction Fragment Length Polymorphism (RFLP) technique to differentiate between the mutant and wildtype form at codons of interest. Since this is an indirect method to detect codon change it has some inherent limitations in identifying appearance of new mutations and in accurately genotyping mixed infections . A numbedhfr triple mutant genotype and the dhps double mutant genotype  that is generated by the ArcGIS software. The software determines the optimal (p) value for the map by minimizing the root mean square prediction error (RMSPE), which is a summary statistic that describes the error of the prediction surface. A (p) value of "0" means a given data point has equal influence on all parts of the prediction surface regardless of the distance from the point. Whereas, a large (p) value means the influence of a data point drops off very quickly as the distance from the point increases. The optimal (p) value for each map is shown in Figures An inverse distance weighted (IDW) algorithm was used to provide a snapshot of The prediction surface of each map is shaded using a gradient scheme consisting of quintiles. Areas of the prediction surface considered to have a high prevalence of mutant genotypes are denoted by darker shading, whereas areas considered to have a lower estimated mutant genotype prevalence are denoted by lighter shading. The IDW spatial representation does not account for the date of the study. Therefore, shaded areas of the maps can only serve as crude snapshots of mutant allele prevalence between 1993 and 2008 with decreasing reliability as distance from observed data points increases. The MARA  map descet al [Time plots were created , and also helps to avoid convergence issues in fitting the model . Year enThe fixed effect representing the age of sampled population are divided into five age categories: 1) adolescents and adults, 2) children and adolescents, 3) children under 6 years, 4) mixed ages, 5) N/A. For this study, individuals age 12 and under are considered children, between 12 and 18 years of age are considered adolescents, and age 18 or greater are considered adults. A study is assigned to age category 1, 2, or 3 if greater than 75% of the sampled population for that study fit the category description. If the age of the sampled population is described but did not meet the 75% threshold then it is assigned to the mixed ages category. The N/A category is used to describe studies that did not describe the age of the sampled population.The fixed effect representing mixed genotype categorization is divided into four categories based on the methodology used for reporting mixed genotypes: 1) separate, 2) combined, 3) excluded, and 4) N/A. The separate category is assigned to studies that report mixed genotypes separate from pure mutant or pure wildtype genotypes. The combined category is used to describe studies that counted mixed genotypes as pure mutant genotypes. The excluded category is used to describe studies that completely removed mixed genotype data. The N/A category is used to describe studies that did not describe mixed genotypes in the study. The criteria used to categorize studies based on reporting of mixed genotype data is more thoroughly described in Table ij Ybe the number individuals with the mutant genotype in the th jstudy of the th icountry. Conditional on the fixed and random effects, Yij ~ Binomial, where ij nis the number of individuals in the th jstudy of the th icountry. On the logit scale, the probability of mutation ij πis modeled as a linear combination of the fixed and random effects. Equation (1) represents the logistic regression model for dhfr triple mutant genotypes, and equation (2) represents the logistic regression model for the dhps double mutant genotypes.Let i = 1, ..., number of countries; j = 1, ..., number of studies in the th icountryi vis the intercept random effect of the th icountry, ij uis the intercept random effect of the th jstudy in the ith country, and β is a vector representing the fixed study level effects. The study and country random intercepts allow the intercept to differ between studies. For model (1) the country random effects i vare assumed to be normally distributed i v~ N, while the study random effects ij uare assumed to be normally distributed ij ~ Nu. For the analysis of dhfr triple mutant genotypes, σv, dhfr2 represents the heterogeneity between countries, and σu, dhfr2 represents the heterogeneity between studies within a country. In model (2) the study random effects uij are assumed to be normally distributed uij ~ N, and σu, dhps2 represents the heterogeneity between studies of the dhps double mutant genotypes. The variance components are estimated on the logit scale, and therefore do not have a direct interpretation. Statistical analysis was performed in R version 2.8.1 using the lmer function in the lme4 package, which uses Laplace approximation to the likelihood to obtain parameter estimates [p-values less than 0.05 are considered statistically significant.Here, α is the fixed intercept, stimates . The sigstimates ; p-valuedhfr and/or dhps genotypes. Of the remaining articles, several were excluded on the basis of their location outside of Africa. Few studies were excluded on the basis of other criteria . Data was successfully extracted from 100 articles that yielded genotype frequency information for 220 studies conducted in 31 African countries and published prior to 2009. Of these 220 studies, 207 studies reported the frequency of one or more mutations of interest within the dhfr or dhps gene. These mutations include dhfr A16V, C50R, N51I, C59R, S108N/T, I164L and dhps S436A/F, A437G, K540E, A581G, A613 S. Studies reporting the frequency of these individual mutations are summarized in the mutant allele frequency (MAF) database  or the dhps double mutant genotype (A437G and K540E), or both. These data are summarized in the multiple mutation (MM) database  of the dhfr triple mutant genotype in parts of Cameroon, Uganda, Kenya, Tanzania, and Malawi.The map describing the prevalence of the a Figure  shows thdhps double mutant genotype across Africa , displays the variability of the probability of mutation between countries. Having a random intercept near zero implies that the study's country level random intercept in the logistic regression model is close to the overall fixed continent intercept, α. Countries with smaller random country effects are estimated to have a lower probability of mutation at the start of the study time period compared to countries with larger random effects. The study level random effects are nested within the country effects, and therefore are centered at the country level random effects. The study level random effects (denoted by a hollow circle) are displayed as i v+ ij uand show the variability of the probability of mutation between studies within the same country. The y-axis of Figure Multilevel mixed-effects regression was used to assess the association of several variables on the reported frequency of both the dhfr triple mutant genotype and has a positive parameter estimate. Therefore, the model suggests that, conditional on the random effects, the reported frequency of this genotype in Africa is increasing over time. Of the three remaining variables that were examined, only mixed mutant categorization had a significant (P = 0.01) association with the reported frequency of the dhfr triple mutant genotype. The combined, excluded, and N/A categories all had a positive parameter estimates relative to the separate category; however, only the combined and excluded categories significantly differed from the separate category. This suggests that studies reporting mixed mutant genotypes as pure mutants or excluded them completely tend to have significantly higher reported frequency of the dhfr triple mutant genotype than studies that did not describe mixed mutant genotypes. The age of the sampled population and the type of study  were not significantly associated with reported mutant genotype frequency. A summary of model results for the dhfr triple mutant genotype can be found in Table For the fixed effects, the year of the study is significantly associated (P < 0.01) with the frequency of the dhps double mutant genotype  were excluded from the statistical analysis because they were conducted earlier and could be too influential. A data shortage for the dhps double mutant genotype analysis led to convergence issues that did not allow for estimation of a country level random effect. However, the study level effect was still included in the model as a random intercept effect in order to allow for variation in the probability of mutation at the start of the study time period. As in Figure dhps double mutant genotype is highly significant (P < 0.01) and is estimated to be 8.83. This is much larger than the between or within country variation observed in the analysis of the dhfr triple mutant genotype because the variance for the dhps double mutant genotype could not be partitioned. This suggests that there is significant variability in the reported frequency of the dhps double mutant genotype among different studies at the start of the study time period.The model for the dhps double mutant genotype and has a positive parameter estimate. Therefore, the model suggests that, on average, mutant prevalence is increasing over time in Africa. The three remaining variables that were examined; mixed mutant categorization, age of sampled population, and type of study, were not significantly associated with the reported frequency of the dhps double mutant genotype. A summary of model results for the dhps double mutant genotype can be found in Table The year of the study is significantly associated (P <0.01) with the reported frequency of the dhfr and/or dhps genotypes in Africa to be included in this meta-analysis. The studies were conducted as early as 1993 and as late as 2006; however, the distribution is skewed (mean year: ~2001 for studies reporting either multiple mutant genotype) towards recent years reflecting an increase in the number of studies reporting the frequency of the dhfr triple mutant and/or dhps double mutant genotypes. This observation is in line with the increase in the number of anti-malarial clinical efficacy trials reported in the literature in recent years [dhfr triple mutant genotype and nearly 8,000 individuals for the dhps double mutant genotype. The clear emphasis on reporting the dhfr triple mutant genotype is likely due to its emergence some 10-20 years earlier than the dhps double mutant genotype in Africa [dhps double mutant genotype (437G/540E), on the other hand, emerged much later in the early to mid-1990's [Broad inclusion criteria allowed most studies that report the frequency of nt years  and liken Africa  and its d-1990's  and is odhfr triple mutant genotype  are more likely to yield mixed genotypes than parasite populations that have a low diversity for a particular gene. Thus, differences in the way mixed genotypes are reported have a more pronounced effect on the prevalence rates reported for a gene that has greater diversity in a given population than one that does not. It has been shown that the dhps  mutant genotype is either ubiquitously present or absent in most of the populations where it has been examined [dhps double mutant genotypes along with another dhps mutant genotype are less likely to occur. However, the dhfr triple mutant genotype has historically been found alongside a higher prevalence of sensitive, single, and double mutant dhfr genotypes circulating in the population [dhfr genotype became fixed in the population. This phenomenon may explain why reporting conventions for mixed genotype data was significantly associated with the reported frequency of the dhfr triple mutant genotype but not the dhps double mutant genotype.This meta-analysis shows that there are some important differences in the reported frequency of the n Africa , pervasin Africa , and humn Africa , may expexamined ; therefopulation ,88; therin vitro studies, and standardized interpretation of existing mixed genotype data [dhfr triple mutant genotype and dhps double mutant genotype, there are also some similarities - The prevalence of both alleles is increasing on average in Africa. On average, the age and clinical status of the sampled population are not significantly associated with the reported frequency of either mutant genotype. This is in contrast to smaller regional studies that have shown association between age, treatment failure rate, and the prevalence of genetic markers for resistance [The specific reasons responsible for the differential impact of reporting conventions for mixed genotype data on the reported frequency for either mutant genotype examined in this meta-analysis are difficult to identify and may also be an artifact of confounding variables that were not accounted for in the statistical regression used herein. Regardless, the fact remains that polyclonal infections commonly occur in many of the high transmission settings across Africa and the characterization of mixed genotypes is an issue that must be addressed. Novel methods for analyzing mixed infections , multivaype data  have allsistance ,91. The dhfr triple mutant and dhps double mutant genotypes but could not be examined in this analysis. This was due to lack of power from limited datasets and inability to link these data to reported frequency data where this information was not specifically reported.One of the major limitations in this meta-analysis was the small number of variables that could be assessed in the statistical models. There are a number of clinical and epidemiological variables such as transmission, regional drug administration policies, and target populations  that may better explain the reported frequency of the dhfr and dhps multiple mutant genotypes included in this analysis were conducted over a period of 13 years. In most cases, the data from a particular country only spans a fraction of this 13 year period; therefore, there is a regional bias where data from a particular year is really a reflection of certain regions, not the continent as a whole. The sample size of studies included in the analysis may have also contributed to regional and temporal bias since the regressions were weighted. The mean study size was 90 isolates for studies reporting the dhfr triple mutant genotype and 85 isolates for studies reporting the dhps double mutant genotype. However, studies examined as few as 3 isolates and as many as 453 isolates . Since studies that have small sample sizes are more likely to be skewed by rare events [Another limiting factor that must be considered when making statements regarding trends across the continent is the confounding effect of space and time. The studies reporting the frequency of the e events , and theFinally, accessibility to studies and the interpretation of data from included studies that use different reporting conventions represented noteworthy sources of bias and error, respectively. The literature search in this study was limited to the Pubmed database and Cochrane Library. Molecular studies that were not available through these sources are not reported. Studies that were included still required a level of interpretation due to differences in methodological and reporting conventions; some of which were specifically examined in this meta-analysis. However, many of these aspects simply could not be addressed within the scope this study. Since there is no established format for the collection and dissemination of molecular data, there was difficulty in determining if molecular data for the same samples was published multiple times in different articles. Efforts were made to filter out articles that reported data from isolates that were already described in a different article; however, there was uncertainty for some articles and it is possible that molecular data for some isolates were reported twice and others were excluded unnecessarily.dhfr and dhps genotypes in Africa that are known to confer anti-folate resistance. These data collectively show that the dhfr triple mutant genotype consisting of  is fixed in several parts of Africa and has been increasing, on average, over the time period from 1993 to 2006. The dhps double mutant genotype consisting of (A437G and K540E) also increased in prevalence, on average, during the same time period. However, this genotype appears to only be fixed in parts of East Africa while its prevalence in West Africa remains low. Continued drug pressure with SP may contribute to further selection of quadruple dhfr mutant genotypes and triple dhps mutant genotypes in Africa; similar to what has been observed in Asia and South America. The increasing prevalence and fixation of these mutant genotypes has historically been associated with a loss of anti-folate drug efficacy and may pose a threat to settings in Africa where anti-folates are still used to treat clinical malaria.This database, despite its limitations, provides a centralized source for information describing the frequency of mutant P. falciparum infection in women with acquired immunity and usually low density asymptomatic parasitaemia. Thus, the treatment efficacy of SP for clinical malaria in young children cannot be directly extrapolated to its usefulness for IPTp [dhps codon 581 in women receiving IPTp. In a subsequent study involving young children from Tanzania, emergence of triple mutant dhps allele with mutations in codons 437, 540 and 581 has been confirmed and this coincided with the high rate of therapeutic failure [dhps mutation at codon 581 to other parts of Africa has been presented. These findings suggest that the spread of the highly resistant triple mutant dhps allele along with triple/quadruple mutant dhfr alleles in different parts of Africa could further compromise the effectiveness of SP for the treatment of falciparum malaria and may have implications for IPTp. Therefore, systematic molecular surveillance studies coupled with assessment of drug effectiveness, including in pregnant women receiving IPTp, will be critical in the future to correlate molecular surveillance data with SP efficacy.SP is currently used in sub-Saharan Africa for IPTp. In this context, the drug is used to clear for IPTp . Howeverfor IPTp  in Muhez failure . In the A continuation of efforts to conduct systematic molecular surveillance will further provide valuable information needed to construct future iterations of resistant genotype maps and databases; however, standardized methodology and reporting conventions are critical to improve the resolution and statistical power of centralized information systems. Challenges confronted during this meta-analysis suggest that patient specific data, standardized characterization of mixed genotypes, more specific date and locality descriptions, and better design and distribution of molecular studies are all critical objectives that must be pursued in order to fulfill the vision of the centralized resistance network proposed by WWARN.The authors declare that they have no competing interestsVU and JWB conceived the original format and objectives of the meta-analysis. KMH produced an initial version of the database and genotype frequency maps. LMS and SS developed search criteria, data extraction methods, and tabular design. SS extracted and compiled the frequency data examined in the meta-analysis. SKM performed all statistical analyses and worked with SS and VU to write the first draft of the manuscript. All authors have read and approved the final draft of the manuscript.Mutant Allele Frequency (MAF) Database. The data provided show the mutant allele frequency reported in each study, organized by country in alphabetical orderClick here for fileMutant Genotype Frequency (MGF) Database. The data provided show the mutant genotype frequency reported in each study, organized by country in alphabetical orderClick here for fileArticle Database. The data provided reference each article from which frequency data are extracted, organized by study identification numberClick here for file


The lung consists of at least seven compartments with relevance to immune reactions. Compartment 1 - the bronchoalveolar lavage (BAL), which represents the cells of the bronchoalveolar space: From a diagnostic point of view the bronchoalveolar space is the most important because it is easily accessible in laboratory animals, as well as in patients, using BAL. Although this technique has been used for several decades it is still unclear to what extent the BAL represents changes in other lung compartments. Compartment 2 - bronchus-associated lymphoid tissue (BALT): In the healthy, BALT can be found only in childhood. The role of BALT in the development of the mucosal immunity of the pulmonary surfaces has not yet been resolved. However, it might be an important tool for inhalative vaccination strategies. Compartment 3 - conducting airway mucosa: A third compartment is the bronchial epithelium and the submucosa, which both contain a distinct pool of leukocytes . This again is also accessible via bronchoscopy. Compartment 4 - draining lymph nodes/Compartment 5 - lung parenchyma: Transbronchial biopsies are more difficult to perform but provide access to two additional compartments - lymph nodes with the draining lymphatics and lung parenchyma, which roughly means "interstitial" lung tissue. Compartment 6 - the intravascular leukocyte pool: The intravascular compartment lies between the systemic circulation and inflamed lung compartments. Compartment 7 - periarterial space: Finally, there is a unique, lung-specific space around the pulmonary arteries which contains blood and lymph capillaries. There are indications that this "periarterial space" may be involved in the pulmonary host defense.All these compartments are connected but the functional network is not yet fully understood. A better knowledge of the complex interactions could improve diagnosis and therapy, or enable preventive approaches of local immunization. The lung is a complex organ and anatomically or functionally it can be divided into different cellular compartments such as the bronchoalveolar space, bronchus-associated lymphoid tissue (BALT) and the bronchial mucosa, the lung parenchyma or interstitium, the draining lymph nodes, the intravascular cell pool and the periarterial space Figure  1]. The. The1]. BAL) is the most frequently used diagnostic method to gain insight into the lung, yielding leukocytes, secretions, and epithelial cells from the alveoli and the airway mucosa. From experience a number of diagnoses are supported by a specific cytology, for example in asthma, infections, sarcoidosis, tumors [The bronchoalveolar lavage  The intravascular pool of leukocytes. This is because of its interaction with lung vessels and capillaries, which controls the entry of leukocytes into different lung compartments and also the integrity of the alveolo-capillary barrier. B) The bronchial mucosa of the conducting airways, which is the first line of defense if microbes or allergens enter the lung. C) The periarterial space, because periarterial inflammation is an important part of the intrapulmonary immune response and a mechanism which keeps the alveoli and therefore the gas exchange area functional. All lung compartments contribute to the innate and the adaptive immune response. The most important questions might be: How is the influx of leucocytes into lung compartments regulated? Which patterns of migration between those compartments are important for immune control? What are the mechanisms for the initiation of perivascular inflammation and -at least equally important - its cessation and how could all these processes be blocked or stimulated by drugs?The authors declare that they have no competing interests.TT and RP have worked together for many years in research on the host defense of the lung, and have continuously discussed morphological issues of the lung and their functional relevance.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/9/39/prepub


Head injury is one of the leading causes of death and disability in traumatic accidents. Post-operative contralateral epidural hematomas after surgery for acute subdural hematoma seem to be rare. In this case, expansion and spontaneous resolution of a fractural epidural hematoma contralateral to the side of acute subdural hematoma is presented. The importance of immediate post-operative computed tomography is also highlighted to detect delayed traumatic mass lesions. Despite recent advances in diagnostic and therapeutic modalities, acute SDHs remain among the most lethal traumatic lesion of the brain, with a mortality rate of approximately 50-74% . OutcomeWe report a 19-year-old Caucasian male of Iranian Nationality with acute right-sided traumatic SDH (due to car accident) with presenting Glasgow Coma Scale score of 6/15 and CT evidence of severe brain swelling and associated midline shift and contralateral small fractural EDH in the temporoparietal region . The patSeveral neurosurgical procedures have been reported to be associated with the incidence of contralateral EDHs. Some of these procedures include evacuation of acute or chronic SDHs and ventriculo-peritoneal shunt insertion. Different authors have reported the incidence of contralateral EDH after evacuation of ipsilateral acute SDH differently (from very rare to 5.7%) ,4-6.Intra-operative brain swelling, post-operative neurologic deterioration, papillary dilation contralateral to the SDH, grand mal seizures, and intractably elevated ICP have been proposed as some hints to the detection of this pathology ,7. TheseSeveral causes have been proposed for delayed formation/expansion of contralateral EDH including loss of tamponade effect, vasomotor mechanisms, and coagulopathy , with thPaying attention to persistent high or elevating ICP after surgery for evacuation of an acute SDH is critical. Although, most instances of increased ICP after SDH evacuation seem to be due to brain edema, this phenomenon should raise the suspicion of evolution of a contralateral hematoma (as in our case). The presence of skull fractures, though not evident in the initial CT  is said to be a prominent risk factor for the malady .Another point to be mentioned is the low incidence of this complication in the elderly, which is elucidated to be due to tight adherence of the dura to skull in this patient population . The diaPrompt detection of acute contralateral EDH in these patients is of critical importance since its treatment will lead to a more efficient ICP management which is directly related to the patient outcome . Less seThe treatment of contralateral EDH is done according to the CT findings. In the present case, there have been several cues to the expansion of the contralateral EDH: intraoperative brain swelling, contralateral fracture and persistent raised ICP after the first surgery. Second surgery was rejected by the patient’s family, and this revealed the natural course of the malady. It seems that rapid decompression and loss of tamponade effect and upsetting of the ICP equilibrium led to the expansion of the contralateral fractural EDH. Yet, progressive brain swelling and evolution of right PCA infarct causing increased ICP caused expulsion of the hematoma through the fracture line underneath the scalp.Expansion/evolution of contralateral hematomas should always be considered after ipsilateral decompression of acute SDH especially in cases of intra-operative brain swelling and skull fractures. A high degree of clinical suspicion is mandatory to confer best outcome to these patients.


Ectopic pregnancy in the interstitial part of the fallopian tube is a rare event. This condition presents a challenge for clinical as well as radiological diagnosis. Although routine two-dimensional ultrasound can be suggestive, three-dimensional ultrasound is highly accurate in diagnosis. Hence, the authors report a rare case of interstitial ectopic pregnancy diagnosed preoperatively by three-dimensional ultrasound and managed laparoscopically. Interstitial ectopic pregnancy is defined as the ectopic gestation developing in the uterine part of the fallopian tube. It is a rare event constituting only 5% of all tubal ectopic pregnancies and is associated with a high rate of complications. The condA 37 year-old para-2 woman with presenting complaints of irregular bleeding over 2–3 weeks following a menstrual delay of one week, was referred to our department for transvaginal ultrasonography. She had a history of prolonged cycles in the past but none of any previous ectopic pregnancy, pelvic inflammatory disease or the utilization of assisted reproductive techniques. Ultrasound examination revealed an empty uterine cavity with the evidence of a 4–5 week-old gestation sac (GS) located eccentrically on the left side of the uterine fundus . The gesin vitro fertilization and ovulation induction. However, in our index case, none of the above risk factors were noted suggesting the role of natural causes as well.Risk factors associated with the higher incidence of interstitial ectopic pregnancy include uterine anomalies, pervious ectopic pregnancy or salpingectomy, pelvic inflammatory disease, Interstitial ectopic pregnancy is associated with a higher risk of shock and hemoperitoneum than other forms of ectopic pregnancy, as well as with a higher risk of maternal mortality due to delayed diagnosis and high vascularity of the myometrium. The presTreatment options for interstitial ectopic pregnancy include local injection or systemic therapy with methotrexate, local injection of potassium chloride, conservative laparoscopic surgery and uterine artery embolism and in emergency situations, cornuectomy or hysterectomy.6 EvidencTo conclude, the diagnosis of an interstitial ectopic pregnancy is usually difficult and delayed resulting in high morbidity and mortality. In the present case, the diagnosis of an interstitial ectopic pregnancy was suggested on the basis of 3D transvaginal ultrasonography prior to the development of any complication, resulting in early management with a favorable outcome. This case thus highlights the role of 3D ultrasonography in cases of irregular bleeding or suspected early pregnancy, to diagnose or rule out unusual sites of ectopic pregnancy for their early safe management.


Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Dα-catenin remains relatively stable, while Arm and DE-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of DE-cadherin from the plasma membrane during apoptosis.We define early and late apoptotic stages and find that early in apoptosis Drosophila apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.This study defines the specificity of caspase cleavage of Arm in  C. elegans to man  embryos were made under conditions previously shown to keep caspases active in these extracts . Males of this stock were crossed to females of the recombinant th109mat15::Gal4/TM3 [ftz::lacZ] stock; this cross allows expression of the UAS transgene with onset of zygotic gene expression in the early embryo.Fly work and embryo collections were performed after standard protocols. Apoptotic cells were recorded in embryos homozygous for the DIAP1 null allele  domains . As contHAJM and TK conceived the project. TK designed and performed the experiments, prepared the figures and assisted in drafting the manuscript. HAJM planned and participated in the design of the experiments and performed some of the experiments. HAJM drafted and edited the manuscript. Both authors read and agreed on the final draft.


Left-sided gall bladder without situs inversus viscerum is a rare albeit recognized clinical entity. We report our experience of two cases of left-sided gall bladder in two women aged 36 and 48 who underwent laparoscopic cholecystectomy for chronic calculous cholecystitis. Left-sided gall bladder may provide an unusual surprise to the surgeons during laparoscopy as routine pre-operative studies may not always detect the anomaly. Awareness of the unpredictable confluence of the cystic duct into the common bile duct (CBD) and selective use of intraoperative cholangiography aid in the safe laparoscopic management of this unusual entity. A left-sided gall bladder is a rare congenital anomaly defined as a gall bladder attached to the lower surface of the left lateral segment of the liver, i.e. to the left of the interlobar fissure and round ligament. This gall bladder is situated under the left lobe of the liver between segments III and IV or on segment III to the left of the falciparum ligament. Left-sided gall bladder is a para physiologic condition that, when identified before surgery, must be properly evaluated with the use of computed tomography (CT) or magnetic resonance imaging (MRI), when incidentally discovered during surgery must be promptly recognized by the surgeon who must be aware of the unpredictable confluence of the cystic duct into CBD. If in doubt, the surgeon should perform an intraoperative cholangiography. We report two adult patients in each of whom left-sided gall bladder was found incidentally, intraoperatively and managed successfully.A 36-year-old female presented with complaints of recurrent right upper abdominal pain associated with vomiting and flatulent dyspepsia for five months. There was no history of jaundice or hospitalization. On examination normal heart sounds were audible on left side of the chest. Abdominal examination was normal. Hemogram, kidney function tests and liver function tests were normal. Ultrasonography of the abdomen showed partially distended gall bladder with large calculus measuring 2.1 cm seen in gall bladder lumen at fundus with normal wall thickness and outline. Intrahepatic biliary radicles (IHBR) and CBD were normal. A laparoscopic cholecystectomy was planned and performed using standard 4-port technique. Pneumoperitoneum was created by closed veress needle technique. Laparoscope was introduced through a 10 mm umbilical port. Actual position of the gall bladder could not be made out because of adhesions with the omentum. 10 mm epigastric port was made and omental adhesions separated. Gall bladder was found on the left of falciform ligament . Rest ofA 48-year-old female was admitted in the hospital with complaint of pain in the right upper abdomen for three to four months with nausea and vomiting. There was no history of fever, jaundice and hospitalization. Liver function tests were normal. Ultrasonography showed cholelithiasis with gall bladder wall thickening. A laparoscopic cholecystectomy was performed using standard 4-port technique. The gall bladder was lying to the left of the falciform ligament . The carMalposition of the gall bladder occurring in the absence of situs inversus is very rare anomaly. Two types of malpositions are known 1) medioposition (2) sinistroposition .[ medioposIn medioposition, the gall bladder is displaced medially to lie on the undersurface of the quadrate lobe (segment IV) but is still on the right side of the round ligament. In sinistroposition, the gall bladder lies under the left lobe (segment III) to the left of the round ligament. Hochstetter first described a case of sinistroposition in 1886 and since then 105 cases have been reported till 1997. In a mulIn sinistro position, the cystic artery always crosses in front of the CBD from right to the left. The cystic duct may open on the left or right side of the common hepatic duct or on to the left hepatic duct directly.As routine pre-operative studies may not detect the anomaly, it may provide the surgeons with an unusual surprise during laparoscopy. When incidentally discovered during surgery it must be promptly recognized by the surgeon, who must be aware of the unpredictable confluence of the cystic duct into CBD and should limit the use of diathermy and avoid division of structures until a clear picture of the bile duct and blood vessels is obtained.In both our cases the condition was promptly recognized, cystic artery and cystic duct were isolated, clipped and divided and retrograde dissection of the gall bladder done. Post-operative course of the patients was uneventful in both the cases.In selective cases adopting a French position, modification of the port sites (use of accessory ports) and the use of falciparum ligament lift may facilitate the procedure. If in doubt, the surgeon should perform an intraoperative cholangiogram to further define the biliary system. Open surgery should be considered if the anatomy still remains unclear.Left-sided gall bladder without situs inversus is a rare clinical entity and can provide an unusual surprise to the surgeons during laparoscopy. Awareness of unpredictable confluence of cystic duct into the CBD, selective use of intraoperative cholangiography and modification of the technique aid in safe laparoscopic management of this unusual anomaly.


The prevalence of AD in an East-Hungarian agricultural area is nearly as high as that reported for populations residing in industrially developed countries, with a higher prevalence during childhood. Data suggest that premature abruption of breast feeding maybe one of the major factors among other environmental factors that is contributing to the development of AD.The prevalence of atopic dermatitis has significantly increased in developed countries during the past several decades. Surveys performed in Hungary also show a growing number of atopic dermatitis (AD) cases, although, a carefully designed case-controlled studies have not been performed. Therefore, we investigated the prevalence of AD in individuals under 19 years of age within the agricultural area of East-Hungary. Combined data obtained with Schultz-Larsen questionnaire on 1158 children were analyzed, and 25% of the index persons were examined by dermatologist. The mean prevalence of AD determined by questionnaires appeared to be 17.5% in the entire study population. Result of dermatological examination verified the validity and sensitivity of the questionnaire. A negative correlation was found between the severity of the disease and the length of breast feeding period. (Spearman's correlation coefficient = − 0.2247,


We face various problems while measuring the Clinical attachment level (CAL) from Cemento-enamel junction (CEJ). This study aims to record and compare the CEJ location measurements using a xed reference point (FRP) [Custom made stent] before and after ap elevation.A custom made stent and UNC-15 probe were used. Recording of CEJ location was made using a UNC-15 (Hu-Friedy) probe, before (close CEJ) and after (Open CEJ) the reflection of the flap from the lower edge of the stent in those subjects who were indicated for flap surgery, at baseline.We used statistical analysis involving intra-group comparison done by Paired-‘t’ test. The close and the open CEJ measurements demonstrated a, statistically, non-significant difference. The equi-measurements of close and open CEJ numerical data were remarkably lower than the under and overestimation of measurements. Thus, despite certain disadvantages of stent, the FRP provides a simple solution for CAL measurement.The results of this study confirms the objective of the study and strongly suggests that CAL measurements done without FRP is subjected to great variation and the diagnostic and prognostic interpretation of CAL should be viewed seriously in periodontics. One of the most important parameters to assess periodontal destruction is loss of connective tissue attachment to the tooth root surface. The Cemento-enamel junction (CEJ) acts as a static landmark to measure CAL or periodontal destruction. Thus, thIn the past, recording of pocket depths was the only method to measure periodontitis. However, merely probing the pocket and recording the pocket depth is insufficient as increase in pocket depth could be due to a gingival enlargement without destruction of underlying periodontal tissue. In some cases, recession of marginal gingival may accompany attachment loss, thus masking ongoing disease progression if the pocket depth measurements are taken alone.If the CEJ is not present due to a restoration, or difficult to determine, it can be recorded from margin of a restoration or a stent and the recording thus made is referred as Relative attachment level (RAL).The assessment of attachment levels due to its fixed reference point (FRP) provides better information relating to gain or loss of attachment to the root surface and in assessing the disease progression as compared to pocket depth measurements. Though attachment level measurements too have certain inherent shortcomings like its high level of tactile sensitivity and time consumption, it still represents “Gold Standard” by which clinicians record disease status.There are several studies discussing reliability of CAL using CEJ and FRP in assessment of periodontal therapy; however Medline search reveals no data on location of CEJ using stent before and after reflection of flap.The routine assessment of CAL is subjected to considerable over or under estimation due to problems associated to exact location sub-gingival location of CEJ, the non-visibility and tactile error are the major setback in identifying true CEJ levels. To determine the CAL measurement error, the prime objective of this study is to locate CEJ before (close) and after (open) flap reflection using a fixed reference point so as to ascertain the number of times the close CEJ was under, over or equi-estimated with the open CEJ measurements.For this study, subjects belonging to both the sexes were selected from the outpatient department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India. The age group of the patients selected ranged from 16 to 45 years. The inclusion criteria was - patients who required periodontal flap surgery. The exclusion group consisted of patients with a history of known systemic diseases/pregnant and lactating women. Total no. of sites measured was 232.rd on the buccal and the lingual side.[A custom made stent and UNC-15 probe were used. The stent was made with the cold cure acrylic by the sprinkle method. It covered the occlusal/incisal 1/3Comparison of the measurements of close and open CEJ locations, i.e., before and after the reflection of flap was made Tables –5. The mThe comparison of Open V/s Close CEJ measurements at individual sites revealed no significant difference between the methods. The discrepancy of measurement at different levels for various sites demonstrated higher % of overestimated values for mesiobuccal, distobuccal, midbuccal, mesiolingual and midlingual sites. For six distolingual sites, a higher % of the close CEJ measurements was underestimated 50%). On the whole, the equi-measurements of close and open CEJ numerical data was remarkably lower than the under and overestimation of measurements Tables –5.0%. On thThe assessment of attachment levels provides better information relating to gain or loss of attachment to the root surface and in assessing the disease progression as compared to pocket depth measurements.The recording of CAL using close CEJ measurements is considered to estimate risk and progression of periodontitis; as well the outcome of periodontal therapy. The relative attachment level is an alternative to CAL and not a substitute as the FRP is always coronal to CEJ and the true attachment level measurements are from CEJ to base of the pocket (BOP). The RAL measurements are good enough in non-surgical periodontics. However, the CAL measurements are ideal for surgical periodontics wherein it is convenient to record CEJ during flap exposure. The FRP provided by the custom stent serves as a good tool to understand the over/under estimation of CEJ location before and after flap reflection.There are several papers discussing reliability and validity of CAL following periodontal therapy, however, the lack of data on the comparison of close versus open CEJ measurements prompted the present study.The open CEJ measurements are the true measurements as the location of CEJ is visualized after flap reflection. On comparison of close and open measurements of CEJ i.e. the measurements made before and after the reflection of the flap, different levels of differences was seen between the two measurements. With regard to mean values and variability of the measurements, there was no significant difference between close and open measurements. In terms of statistics, the non significant difference between open and close CEJ measurements could be due to nullifying effect of overestimated and underestimated values of close CEJ measurements. This can be appreciated by the fact that out of the total 232 sites examined, the close CEJ measurements were overestimated by 31.1% (72 sites) and underestimated by 43.6% (101 sites) and only 25.4% (59 sites) matched equally (without any difference). The comparison of open Vs close CEJ measurements at individual sites revealed no significant difference between the methods. The discrepancy of measurement at different levels for various sites demonstrated higher percetage of overestimated values for mesiobuccal, distobuccal, midbuccal, mesiolingual and midlingual sites. For six distolingual sites, a higher per cent of the close CEJ measurements was underestimated (50%). On the whole, the equimeasurements of close and open CEJ numerical data was remarkably lower the under and overestimation of measurements [Tables A custom-made plastic occlusal stent used in this study measures attachment levels in lieu of the traditional approach which uses the cementoenamel junction (CEJ) as a fixed reference point. Bulk to the stent on the occlusal surface contributes to its strength. It also makes sense to inscribe a line on the master cast at the apical edge of the stent; therefore if the stent is lost or broken, a new one can be fabricated. While this is likely to create some error, it is better than losing the data altogether.et al,[The reproducibility of periodontal measurements is better using a stent. Also in a study by Watts T, where four sets of measurements were performed for the gingival margin, probing depth and CEJ by an examiner using a constant force probe with and without stent; stent to CEJ showed the least exact reproducibility (53%) of the simple measurements. In anothet al, recommenOne electronic probe with the capability to detect the CEJ has been described, but this probe has been primarily used as a research tool only. In contrFuture studies should concentrate on recording of interproximal CEJ which is highly variable and diffcult to measure.Periodontal therapy is often based upon information regarding longitudinal attachment level data. Accurate, reliable measurement of attachment level is an important aspect of periodontal diagnosis and recommended treatment. If periodontists/practitioners could more accurately determine where real change in attachment level has occurred, they may ultimately improve the diagnosis and treatment of periodontitis.The rapid expansion and molecular outburst of periodontics is of little clinical interest and utility. There are several unanswered day to day clinical measurements that are basically considered for assessment of disease progression and periodontal treatment. If the clinical research concentrates on these basic clinical entities, the subject periodontics would scale better heights. By ignoring these problems and inclining towards molecular aspects, a great imbalance is created in understanding the growth of clinical periodontics.A great deal of drugs and materials are used for treatment of periodontics. However, the clinical measurements are still confounded with problems. Solving these should be the major interest of the clinical periodontal research. Despite certain disadvantages of stent, the fixed reference point provides a simple solution for CAL measurement.