Although underlying targeted sequencing methods are well established, necessary data handling and focused, structured analysis still remain demanding tasks. Here, we present a cloud-enabled autonomous analysis pipeline, which comprises the complete exome analysis workflow. The pipeline combines several in-house developed and published applications to perform the following steps: (a) initial quality control, (b) intelligent data filtering and pre-processing, (c) sequence alignment to a reference genome, (d) SNP and DIP detection, (e) functional annotation of variants using different approaches, and (f) detailed report generation during various stages of the workflow. The pipeline connects the selected analysis steps, exposes all available parameters for customized usage, performs required data handling, and distributes computationally expensive tasks either on a dedicated high-performance computing infrastructure or on the Amazon cloud environment (EC2). The presented application has already been used in several research projects including studies to elucidate the role of rare genetic diseases. The pipeline is continuously tested and is publicly available under the GPL as a VirtualBox or Cloud image at  During the last years, the rapid development of next generation sequencing (NGS) technologies Although the costs of human whole-genome sequencing dropped significantly, it still remains a time consuming and expensive method. Therefore, exome sequencing proved to be a valuable alternative for the investigation of several diseases including rare Mendelian disorders The current bottleneck of NGS projects is not mainly the sequencing of DNA itself, but lies in the structured way of data management and the targeted computational analysis of experiments To this end, several NGS analysis pipelines were published To overcome these challenges, an automatized pipeline, called SIMPLEX, for analyzing exome sequencing data has been developed. The pipeline is able to handle single-end (SE) as well as paired-end (PE) data and is able to process input data encoded in nucleotide space or color space. SIMPLEX combines published and in-house developed applications and is continuously, automatically tested. To facilitate the analysis of exome-seq data, especially in small scale laboratories, the pipeline is offered as a fully functional VirtualBox image that requires no additional installation of tools and databases. Furthermore, the pipeline provides a transparent usage of the high performance computing (HPC) infrastructure and offers quick access through a dedicated Cloud image.SIMPLEX is a comprehensive pipeline for investigating exome SE and PE sequencing data generated by deep sequencing devices from Illumina and ABI SOLiD. It exposes a wide variety of parameters to offer great flexibility for analyzing data according to the given biological problem and, at the same time, provides a well chosen set of standard parameters for unversed users. SIMPLEX requires as input the raw sequence reads, their corresponding base calling quality values, and a list of genomic positions specifying the complete exome. A default exome pipeline analysis with SIMPLEX includes all steps depicted in The pipeline is able to handle different input file formats which result from combining specific library preparation protocols with various sequencing platforms. Data produced by Illumina devices need to be in FASTQ file format  is necessary to correct for alignment artifacts by minimizing the number of mismatching bases across all reads. SIMPLEX uses the realignment algorithm of the Genome Analysis Toolkit (GATK) Systematic bias introduced by the initial base calling quality calculations are corrected using the base quality score recalibrator of GATK. It corrects the co-variation of the assigned quality value considering (i) the position within the sequence read, (ii) preceding and current nucleotide calls, and (iii) the probability of mismatching the reference genome. After performing base quality recalibration, the pipeline creates summarizing reports of this step.Using the improved quality values, a critical filtering step removes unmapped and improperly paired reads. Furthermore, it detects unwanted PCR-duplicates and excludes reads which do not overlap with exonic regions of the reference genome. These filters can be fine-tuned by setting individual parameters.BAM statistics module provides a quick summary of the performed alignment, including total number of reads, number of mapped and unmapped reads, and read coverage in relation to the genome size. Alignment summary metrics report high level metrics about the alignment, such as median read length, deletions/insertions rate, and number of bases of high quality aligned reads. Furthermore, the pipeline reports insert size metrics that are useful to evaluate the insert size distribution of PE data.The third step see  calculatThe next analysis component -, as text (TXT)-, and as Browser Extensible Data (BED) files, which can be displayed in Genome Browser tracks. In order to accelerate DIP identification, the pipeline evenly divides the input data and executes DIP calling in parallel.A raw set of SNP calls is determined by comparing the reference genome with the consensus sequence, which was previously deferred from the read alignment information by a Bayesian identifier. To improve runtime performance, SNP positions close to previously identified DIPs are ignored during sequence comparison. The identified SNPs are reported in VCF file format and are separated in homozygous and heterozygous variants. The subsequent SNP filter masks ambiguous SNP calls to create an improved SNP call set. This set is used as training data for variant score recalibration, which aims at improving the biological variant estimation.Variant annotation is a two-step process, that comprises (1) adding information to already known mutations and (2) providing de- novo information for unknown variants.The first component , which forms a dynamically extensible calculation back end and provides all necessary functionality to the pipeline users. This in-house developed API allows the delegation of numerical intensive calculations to a HPC infrastructure, which can be located in a dedicated server room or in a public cloud environment.The pipeline client, which can be started on a regular office PC, transfers the raw sequencing data over secure web access to the calculation back-end and coordinates the parallel execution of analysis steps. Already completed result files are transferred back to the client workstation, and available summary information is collected throughout the complete pipeline run. The client side can be terminated during execution and easily restarted with the same parameters in order to continue the previously interrupted run. The service allows multiple users to perform multiple analyses at the same time and secures access to data by a central usermanagement system.The client needs an adequate network connection to the server which is hosting JClusterService and an installed Java Runtime Environment. Since the result files of the pipeline can be several gigabytes, e.g. if mapped reads are fetched from the calculation back-end, increased storage capacity on the client side might be an additional requirement.A cloud image including SIMPLEX and all required programs has been created within Amazon’s Elastic Compute Cloud (EC2). This way, anyone with an Amazon EC2 account can instantiate the image with little to no effort and can run SIMPLEX without local compute facilities or advanced technical know-how. The EC2 cloud instantaneously provides the pipeline users with the full set of functions required to analyze exome sequencing data.The cloud image is based on CloudBioLinux Once started, the user has the choice to either transfer the data manually to the cloud storage to analyze it with SIMPLEX, or call the pipeline client from a local machine, which will then automatically transfer the raw data to the cloud image and fetch the desired results.The pipeline is currently controlled through a command line interface, where input files and pipeline parameters can be specified. Although only a few parameters are required to run SIMPLEX, there is a large number of optional parameters that allow in- depth customization for specific biological questions  the use of the EC2 Cloud image is recommended.A detailed user manual is available in the supplementary section see , where aThe presented autonomous pipeline for investigating exome sequencing data, SIMPLEX, allows researchers to analyze data generated by Illumina and ABI SOLiD NGS devices. It supports SE and PE data and takes advantage of HPC infrastructures or the EC2 Cloud to perform intensive calculations in a timely manner. The pipeline requires sequence reads, their corresponding base calling quality values, and a list of exon positions specifying the complete exome as input. The manifold results of the pipeline include a detailed summary report, files that can be used for viewing mapping results in Genome Browsers, and annotated lists of variants, which can be easily opened with office programs see .To assess the performance on real biological data, we downloaded raw sequencing data generated by a study of the Kabuki syndrome A performance summary of the pipeline is depicted in read-trimming , min-length-filter (filters out reads shorter than 25), n-max-filter , and quality-filter . On average, 98% of SE and 100% of PE reads passed all preprocessing steps, indicating that raw files had already been preprocessed before publication  mutations in all investigated samples. Moreover, 24,093 variants were annotated as either missense or nonsense mutations.All automatically generated summary reports were combined into a single document see . AmongstSIMPLEX is a novel pipeline for the consistent analysis of exome sequencing data, covering the complete workflow from read filtering and mapping to annotated lists of detected variants. Due to its support for SE and PE data in nucleotide and color space, it is universally applicable to several different platforms and biological problems. Since the installation of all required analysis tools and the targeted analysis itself are still daunting tasks for many researchers, SIMPLEX is provided as a ready to use VirtualBox image and a fully configured Cloud image, which allows users to quickly and comfortably add more computational power. Detected variants are annotated with additional information, including multiple scores which are useful for getting more accurate functional predictions of mutations In principal, it is possible to extend SIMPLEX to support the analysis of whole-genome data by simply changing a few configuration parameters. However, since currently many labs  are focusing on exome sequencing data due to the lower data amount and straightforward interpretation of the results, we focused on this data type and included specific steps such as filtering exonic regions, calculation of exome capture specificity, and detection of poorly captured exons.All external tools that are integrated into SIMPLEX were carefully selected and thoroughly evaluated. Several programs for the read preprocessing steps were tested for their suitability, including TileQC full projectAlignment of reads is performed by the Burrows-Wheeler based aligner BWA Post processing is done either by GATK or by a custom made component which performs several filtering steps. First, it applies mapping and properly-paired filters, followed by PCR duplicate removal and exome filtering. Results of each filtering step are documented in log files and are assembled in a summary report.GATK was chosen for variant calling, as it supports several NGS platforms and is suitable for both, individual and multi sample, analyses. In addition, the toolkit is shipped with a set of additional SNP analysis tools, including SNP quality evaluation, SNP filtering, and standardized downstream recalibration of variant quality scores. Moreover, it has already been successfully used in large-scale projects like the 1000 Genomes Project SeattleSeq Annotation is not available for local installation, and SNPs3D Additional information for detected variants, such as RefSeq names, GO ids, and KEGG ids, are added using the GATK annotator. However, as those annotations depend on existing dbSNP ids, they are not suitable for newly discovered variations. Therefore, SIMPLEX includes Annovar Currently several NGS analysis pipelines are available which differ in their provided feature sets and functionality The performance assessment of the presented pipeline was done with data from the Kabuki study described by After the initial analysis, we checked for MLL2 loss-of-function mutations in each individual which could be observed in 8 out of 10 subjects see . This nuexome coverage, filtering results, or exome capture specificity. All detected variants are annotated with additional information allowing researchers to easily and quickly discriminate silent mutations from variants that are potentially causing diseases. Furthermore, result files can be used in downstream analyses to additionally identify driver mutations using tools such as Auto-Annovar or VAAST We have developed and validated SIMPLEX, a highly configurable pipeline for the analysis of NGS exome data, covering the complete workflow from sequence alignment to SNP/DIP identification and variant annotation. Due to the pipeline’s flexible design SIMPLEX is supporting SE and PE data as well as input from various sequencing platforms. The pipeline is optimized for HPC infrastructures and can be used in the Amazon EC2 Cloud to speed up the analysis process. Complex methods and commands are abstracted from investigators to facilitate the use of NGS technologies in laboratories even without a specialized bioinformatics staff. SIMPLEX outputs highly readable reports including summary documents that list key figures such as Due to the modular design of the pipeline, it is possible to integrate any command line tool by simply extending a XML file and creating a few Java wrapper classes . The whole pipeline code is open-source and can be extended to one’s needs. We hope to encourage the community to create extensions and submit them to the productive development branch. Regarding the long-term use and sustainability of the pipeline, the application is under heavy use in clinical research environment, which guarantees active development and improvements.In conclusion, SIMPLEX is a tool designed to be readily used for life science researchers to quickly obtain biological insight into genetic events investigated by exome sequencing. The presented system was successfully applied in a recent study to elucidate the cause of the rare genetic disease epileptic encephalopathy and amelogenesis imperfecta (Kohlschütter-Tönz syndrome) Table S1SIMPLEX parameters and their description.(PDF)Click here for additional data file.Table S2Kabuki syndrome study summary.(XLS)Click here for additional data file.Table S3Comparison of SIMPLEX, Atlas, and Treat pipelines.(PDF)Click here for additional data file.Table S4Kabuki syndrome study - SNV statistics grouped by individuals.(PDF)Click here for additional data file.Table S5Kabuki syndrome study - unique occurrences of MLL2.(PDF)Click here for additional data file.Table S6Kabuki syndrome study - summary of autoannovar results.(PDF)Click here for additional data file.Supplementary Material S1SIMPLEX user manual.(PDF)Click here for additional data file.Supplementary Material S2QA report after raw sequence preprocessing for sample SRR063831.(PDF)Click here for additional data file.


Snail-borne infections provide a worthy challenge for any young parasitologist looking for an exciting career.If this request had been made during the presidential address at the ASTMH meeting in Philadelphia in 2011, even though the room was filled beyond capacity, only a few people would have stood up. Yet, 300 million disadvantaged people suffer from snail-transmitted infections, with consequences ranging from life-threatening cholangiocarcinoma to subtle morbidity effects that stunt physical and mental development. The disability-adjusted life year (DALY) scores for these diseases have long been underestimated. The term “The recent World Health Organization (WHO) announcement of a global effort to eliminate human schistosomiasis by 2025 integrated control strategies that also target the intramolluscan larval stages of parasites. For example, what determines the population structure and geographical distribution of snails that define endemic areas for parasite transmission, and how will global warming affect these from the field is lacking for how long infected snails continue to shed cercariae, and the number of cercariae produced per snail. Deciphering properties of immunity and virulence that have evolved to influence snail–parasite compatibility reveals determinants of host competence that will facilitate monitoring, predicting, and ultimately modifying transmission of schistosomiasis and other snail-borne parasites.So, how can study of relevant snails contribute to eliminating schistosomiasis and other snail-borne parasites? A detailed grasp of the role of snails in transmission is essential for developing Biomphalaria); the involvement of antigenic variation by Schistosoma to survive in snails; and the epigenetic modification of snail host chromosomes during the course of infection Biomphalaria's role in schistosomiasis transmission will be forthcoming. Microarray platforms Biomphalaria snails B. glabrata genome sequence is fully available Bulinus, Lymnaea, and OncomelaniaExciting science can be done! Snails (and trematodes parasites) are lophotrochozoan protostomes, an animal lineage to which little attention has yet been paid—fundamental discoveries consequently lie ahead in a field that is not cluttered by many competing research groups. Recent novel basic insights into host–parasite interactions include the discovery of somatic diversification of immune molecules in invertebrates  influencing snail-trematode interactions. The characterization of regulators of parasite transmission in natural snail population can contribute to the development of novel, ecologically friendly snail control methods , and open up new lines of study such as introduction of snail transgenes capable of disrupting larval growth/differentiation. With so many snail-transmitted infections still at large, and so many obvious approaches awaiting investigation, we sincerely hope that the decline in snail-related funding, with a concomitant decline in the number of trained investigators, can be reversed. The availability of young workers even able to identify medically relevant snails has dropped to a shockingly low level.To conclude, it is an unchanging reality that snails are essential for the continued flourishing of snail-borne parasites, including those that cause schistosomiasis. Given the recent call for global elimination of schistosomiasis, it is imperative we pursue a broader agenda that incorporates basic and applied snail research. From such efforts can emerge integrated and more sustainable control strategies. This will also help to arrest the alarming decline in young investigators, particularly in endemic countries. Given the considerable attention currently focused on other parasitic diseases such as malaria, could it be that the greatest opportunities to make significant new advances in parasitology now lie in other fields that have been truly neglected?


In a systematic review of the literature, Julia Hussein and colleagues seek to determine the effect of referral interventions that enable emergency access to health facilities for pregnant women living in developing countries. Pregnancy complications can be unpredictable and many women in developing countries cannot access health facilities where life-saving care is available. This study assesses the effects of referral interventions that enable pregnant women to reach health facilities during an emergency, after the decision to seek care is made.Selected bibliographic databases were searched with no date or language restrictions. Randomised controlled trials and quasi experimental study designs with a comparison group were included. Outcomes of interest included maternal and neonatal mortality and other intermediate measures such as service utilisation. Two reviewers independently selected, appraised, and extracted articles using predefined fields. Forest plots, tables, and qualitative summaries of study quality, size, and direction of effect were used for analysis.Nineteen studies were included. In South Asian settings, four studies of organisational interventions in communities that generated funds for transport reduced neonatal deaths, with the largest effect seen in India (odds ratio 0·48 95% CI 0·34–0·68). Three quasi experimental studies from sub-Saharan Africa reported reductions in stillbirths with maternity waiting home interventions, with one statistically significant result (OR 0.56 95% CI 0.32–0.96). Effects of interventions on maternal mortality were unclear. Referral interventions usually improved utilisation of health services but the opposite effect was also documented. The effects of multiple interventions in the studies could not be disentangled. Explanatory mechanisms through which the interventions worked could not be ascertained.Community mobilisation interventions may reduce neonatal mortality but the contribution of referral components cannot be ascertained. The reduction in stillbirth rates resulting from maternity waiting homes needs further study. Referral interventions can have unexpected adverse effects. To inform the implementation of effective referral interventions, improved monitoring and evaluation practices are necessary, along with studies that develop better understanding of how interventions work.Please see later in the article for the Editors' Summary Every year, about 350,000 women die from pregnancy- or childbirth-related complications. Almost all of these “maternal” deaths occur in developing countries. In sub-Saharan Africa, for example, the maternal mortality ratio  is 500 and a woman's life-time risk of dying from complications of pregnancy or childbirth is 1 in 39. By contrast, the MMR in industrialized countries is 12 and women have a life-time risk of maternal death of 1 in 4,700. Most maternal deaths are caused by hemorrhage (severe bleeding after childbirth), post-delivery infections, obstructed (difficult) labor, and blood pressure disorders during pregnancy, all of which are preventable or treatable conditions. Unfortunately, it is hard to predict which women will develop pregnancy complications, many complications rapidly become life-threatening and, in developing countries, women often deliver at home, far from emergency obstetric services; obstetrics deals with the care of women and their children during pregnancy, childbirth, and the postnatal period.It should be possible to reduce maternal deaths  in developing countries by ensuring that pregnant women are referred to emergency obstetric services quickly when the need arises. Unfortunately, in such countries referral to emergency obstetric care is beset with problems such as difficult geographical terrain, transport costs, lack of vehicles, and suboptimal location and distribution of health care facilities. In this systematic review , the researchers assess the effectiveness of interventions designed to reduce the “phase II delay” in referral to emergency obstetric care in developing countries—the time it takes a woman to reach an appropriate health care facility once a problem has been recognized and the decision has been taken to seek care. Delays in diagnosis and the decision to seek care are phase I delays in referral, whereas delays in receiving care once a women reaches a health care facility are phase III delays.The researchers identified 19 published studies that described 14 interventions designed to overcome phase II delays in emergency obstetric referral and that met their criteria for inclusion in their systematic review. About half of the interventions were organizational. That is, they were designed to overcome barriers to referral such as costs. Most of the remaining interventions were structural. That is, they involved the provision of, for example, ambulances and maternity waiting homes—placed close to a health care facility where women can stay during late pregnancy. Although seven studies provided data on maternal mortality, none showed a sustained, statistically significant reduction (a reduction unlikely to have occurred by chance) in maternal deaths. Four studies in South Asia in which communities generated funds for transport reduced neonatal deaths (deaths of babies soon after birth), but the only statistically significant effect of this community mobilization intervention was seen in India where neonatal deaths were halved. Three studies from sub-Saharan Africa reported that the introduction of maternity waiting homes reduced stillbirths but this reduction was only significant in one study. Finally, although referral interventions generally improved the utilization of health services, in one study the provision of bicycle ambulances to take women to the hospital reduced the proportion of women delivering in health facilities, probably because women felt that bicycle ambulances drew unwanted attention to them during labor and so preferred to stay at home.These findings suggest that community mobilization interventions may reduce neonatal mortality and that maternity waiting rooms may reduce stillbirths. Importantly, they also highlight how referral interventions can have unexpected adverse effects. However, because the studies included in this systematic review included multiple interventions designed to reduce delays at several stages of the referral process, it is not possible to disentangle the contribution of each component of the intervention. Moreover, it is impossible at present to determine why (or even if) any of the interventions reduced maternal mortality. Thus, the researchers conclude, improved monitoring of interventions and better evaluation of outcomes is essential to inform the implementation of effective referral interventions, and more studies are needed to improve understanding of how referral interventions work.http://dx.doi.org/10.1371/journal.pmed.1001264.Please access these Web sites via the online version of this summary at maternal mortality, including the WHO/UNICEF./UNFPA/World Bank 2008 country estimates of maternal mortalityThe United Nations Children's Fund (UNICEF) provides information on maternal health, including information about Millennium Development Goal 5, which aims to reduce maternal mortality ; the Millennium Development Goals, which were agreed by world leaders in 2000, are designed to eradicate extreme poverty worldwide by 2015The World Health Organization provides information on Immpact is a global research initiative for the evaluation of safe motherhood intervention strategiesVeil of Tears contains personal stories from Afghanistan about loss in childbirth; the non-governmental health development organization AMREF provides personal stories about maternal health in AfricaMaternal Death: The Avoidable Crisis is a briefing paper published by Médecins Sans Frontières (MSF) in March 2012 The importance of referral in an obstetric emergency is related to the unpredictability of pregnancy complications and their potential to progress rapidly to become severe and life threatening. For example, a serious haemorrhage can lead to death of a woman and the unborn fetus within minutes or hours The three delays model provides a conceptual framework of the factors influencing the timely arrival to appropriate care in obstetric emergencies A large number of interventions to overcome phase II delays are being implemented within maternal mortality reduction programmes. Many report improvements, especially in terms of health service indicators such as referral rates and utilization. Reviews of the literature have so far not attempted to systematically collate and appraise the reported effects. This systematic review aims to assess the effects of emergency obstetric referral interventions that overcome phase II delays, enabling pregnant women to reach health facilities in developing countries.PRISMA guidelines  and a stStudies of referral systems for emergency maternity care from published and grey literature were included if they were randomised controlled trials (RCTs) or other quasi experimental study designs—i.e., studies lacking randomisation such as controlled before-after (CBA) studies and interrupted time series (ITS)—provided comparison groups were available. Participants were pregnant and post partum women with an obstetric complication, referred as an emergency from the community or from a primary care centre to a higher level comprehensive emergency obstetric care facility. Interventions included aimed to overcome delays in reaching the appropriate facility (phase II delay), which improved emergency referrals antenatally, during labour, or up to 42 d after delivery. Examples of interventions of interest were technologies such as telephones, radios, and vehicles for transport; financing and incentive schemes; guidelines and protocols to help health professionals make decisions on referrals; reorganisation of care systems ; and mobilisation of community members  to actively participate in referral activities (such as accompany the women or drive vehicles). Interventions that addressed phase II delays, combined with those that improved phase I and III delays, were included. Studies from low- and middle-income developing countries as classified by the World Bank Studies with no comparison group, and interventions that referred the newborn, women without maternity related conditions, non-emergency cases, and between hospitals were excluded. Interventions that only improved phase I and III delays were omitted. Studies in refugee, war zone, and mass casualty settings were not included as these special settings were likely to confound the effect of obstetric referral. Interventions to change traditional birth attendants' (and other lay carers of pregnant women) decision making for referral were not included (unless there was active engagement of the carer in enabling transfer to a facility), as we considered these to mainly affect decision making. Isolated interventions to introduce first-aid practices to stabilise or treat the woman during referral  were excluded as these procedures do not affect the delay in reaching a facility but enable improvement of the woman's condition while awaiting transfer. First aid is, however, normal practice during referral and if it was provided as part of an intervention to reduce phase II delays, the study would be included.The outcomes of interest were guided by the conceptual framework . These wThe electronic search strategy was based on terms related to referral, transport, or transfer, in obstetric emergencies and conducted between July and November 2010. The full search strategy is available as Appraisal of titles, abstracts, and full text articles was conducted independently by two reviewers on the basis of the inclusion and exclusion criteria. Disagreements were resolved by discussion among all members of the review team.Quality assessment was guided by Effective Public Health Practice Project criteria Synthesis of the studies involved categorising interventions by their characteristics, narrating and summarising study quality, size and direction of effect, and presenting data visually as odds ratios, forest plots, and tables. Data extraction and analyses was done with Review Manager Version 5. In our analysis, the effect estimates of cluster RCTs were corrected to account for this design using inverse variance. Denominators used were based on the total numbers of pregnant women, live births, or total births (in the case of stillbirths) in the catchment areas of the respective studies.The search results are summarised in The studies were categorised using the EPOC taxonomy of interventions for practice change The six organisational interventions involved surmounting obstacles to emergency transport, especially those of cost. All targeted women and community members including traditional birth attendants (TBAs) There were seven structural interventions that involved the use of maternity waiting homes, radios, and ambulances to overcome phase II delays One intervention in Bangladesh included both organisational and structural characteristics Four interventions were described in more than one paper. In the organisational intervention from Bangladesh All studies collected community-based data and provided information on outcomes at the population level, with the exception of the four maternity waiting home studies Seven papers provided maternal mortality data . The thrOf the quasi experimental studies reporting maternal mortality, the mixed organisational-structural intervention Neonatal deaths in four RCTs There is less consistency in the data on stillbirths . Two RCTData on health facility utilisation were available from nine studies. Six We focused our review on interventions that aimed to overcome phase II delays. We were not able to establish the effectiveness of referral interventions on maternal mortality or other intermediate or process outcomes such as uptake of care. Although we found some reductions in neonatal mortality and in stillbirths, inference of effect due to interventions that specifically overcome phase II delays is not possible due to design limitations of the individual studies.Reduction in neonatal deaths were found in four South Asian RCTs of community mobilisation interventions, which included the generation of funds for transport, to overcome phase II delays There is a possibility that maternity waiting homes in sub-Saharan Africa may reduce stillbirth rates. Unlike the RCTs on community mobilisation, this group of studies on maternity waiting homes Transport and communication interventions were another group of studies investigated in this review The outputs and intermediate effects of the various referral interventions were arranged in Other reviews on referral interventions are available Much remains unknown. Does function of the health system, terrain, or whether the intervention targets antenatal, intrapartum, or post partum periods matter when selecting which interventions to implement? The studies that met our inclusion criteria were set in rural areas—we can make no conclusions on urban settings or any other contextual factors. Investigating phase II delays in isolation may be an oversimplification as phase I and III factors will have effects on phase II and referral interventions work through complex mechanisms. However, the literature base on referral interventions as a whole is very large and until efforts are made to break down the various components into manageable parts, progress in understanding referral interventions cannot be made. Despite the wealth of literature describing means to improve women's access to maternity care during emergencies, know-how for effective implementation remains limited. The 19 out of over 600 potentially relevant studies that met the criteria for inclusion in our study, and the findings of other recent reviews on referral linkages The limitations inherent in the studies included in this review mean that findings should be interpreted with caution. We found that complex, community-targeted interventions reduce neonatal mortality but not how the referral components contributed. The reduction in stillbirths observed in studies of maternity waiting homes makes this a potentially promising intervention that needs further investigation. While continuing to invest in implementing referral interventions within maternal and newborn health programmes, we urge health planners to ensure that the interventions are rigorously monitored and evaluated, and operations research studies designed with controls and comparisons. There should be awareness that referral interventions may have adverse effects. Future research should aim to understand how the interventions work and why, by using methods that provide understanding of causal pathways and mechanisms of action.Text S1PRISMA checklist.(DOC)Click here for additional data file.Text S2Review protocol.(PDF)Click here for additional data file.Text S3Search strategy.(DOC)Click here for additional data file.


Bone relies on multiple extracellular signaling systems to maintain homeostasis of its normal structure and functions. The amino acid glutamate is a fundamental extracellular messenger molecule in many tissues, and is used in bone for both neural and non-neural signaling. This review focuses on the non-neural interactions, and examines the evolutionarily ancient glutamate signaling system in the context of its application to normal bone functioning and discusses recent findings on the role of glutamate signaling as they pertain to maintaining healthy bone structure. The underlying mechanisms of glutamate signaling and the many roles glutamate plays in modulating bone physiology are featured, including those involved in osteoclast and osteoblast differentiation and mature cell functions. Moreover, the relevance of glutamate signaling systems in diseases that affect bone, such as cancer and rheumatoid arthritis, is discussed, and will highlight how the glutamate system may be exploited as a viable therapeutic target. We will identify novel areas of research where knowledge of glutamate communication mechanisms may aid in our understanding of the complex nature of bone homeostasis. By uncovering the contributions of glutamate in maintaining healthy bone, the reader will discover how this complex molecular signaling system may advance our capacity to treat bone pathologies. Although not widely known, a fourth family of iGluRs termed the delta receptors exists, with two subtypes identified (GluRδ1 and GluRδ2), although they may only be secondarily involved in glutamate signaling , which form ion-gated cation channels when activated, and metabotropic glutamate receptors (mGluRs), which are coupled to intracellular G-proteins and regulate the production of intracellular second messengers. An excellent review by Dingledine et al.  providesConsiderable evidence exists to suggest that glutamate signaling in normal bone is involved in a variety of processes, including cell differentiation effects and mature cell functions. Glutamate concentrations within the bone environment are modulated by a variety of cell types, through glutamate-specific transporters which are expressed by the bone-resorbing osteoclasts . For example, glutamate concentrations in synovial fluid were reported to increase more than 50-fold in patients with RA compared to controls, from 6.25 to 326 μM (McNearney et al., in vivo studies will require targeted manipulation of glutamatergic signaling to evaluate such consequences on bone homeostasis. Bone may be especially susceptible to glutamate interference as bone cells express the necessary receptors and transporters to transmit and receive glutamate signals for their normal functions. Glutamate transport appears critical for feedback control between Oc and Ob and disruption of this process may be relevant in osteoporosis. In osteoarthritis, glutamate receptor expression is altered compared to normal bone cells, and extracellular glutamate concentrations are significantly increased in affected joints in RA and could impact inflammatory responses. In arthritis, glutamate-sensitive nociceptors may be stimulated by this locally elevated signal, and may contribute to arthritis pain. Cancers growing in bone significantly disrupts bone metabolism and causes severe pain. As cancer cells are known to secrete glutamate, this errant extracellular signal may deregulate the tightly coupled glutamatergic bone remodeling process and may directly stimulate nociceptors. Pharmacologic modulation of the transporter secreting glutamate from cancer cells is offered as a potential strategy to reduce both metabolic disruption and pain in bone cancer, and in a wider context, suggests that the glutamatergic communication system holds significant potential as a therapeutic target in a number of bone-related disorders. As glutamate signaling is such a fundamental biological process, identifying how it functions in normal bone is vital to advancing our capacity to develop glutamate-based treatments for bone diseases.Healthy bone is maintained in an exquisitely balanced state by numerous intercellular communication systems. Evidence that glutamate signaling is a significant participant in bone homeostasis continues to accumulate, and disruptions in glutamatergic mechanisms may contribute to a variety of bone diseases. However, owing to their apparent fundamental importance in numerous organ systems, future The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obesity is clinically diagnosed by a simple formula based on the weight and height of a person (body mass index), but is associated with a host of other behavioral symptoms that are likely neurological in origin. In recent years, many scientists have asked whether similar behavioral and cognitive changes occur in drug addiction and obesity, lending many to discuss the potential for “food addiction”. Advances in understanding the circuitry underlying both feeding behaviors and drug addiction may allow us to consider this question from the viewpoint of neural circuits, to complement behavioral perspectives. Here, we review advances in understanding of these circuits and use them to consider whether drawing comparisons to drug addiction is helpful for understanding certain forms of obesity. Drug addiction is a chronic, relapsing disorder that is characterized by physical signs such as tolerance and withdrawal, as well as emotional and behavioral symptoms such as sensations of craving and compulsive reward-seeking. Tolerance describes a phenomenon in which higher doses of a drug are required to achieve an effect, while withdrawal signs describe a range of physiological and emotional consequences that occur when an addict stops taking a drug. The behavioral changes associated with drug addiction can be broadly grouped into three main categories  that cause extreme weight gain  of the hypothalamus constitutes a variety of diverse cell-types that are ideally situated to integrate blood-borne signals released from peripheral tissues, as the ARC rests at the base of the brain adjacent to the third ventricle and median eminence. Specifically, two distinct ARC subpopulations, orexigenic agouti-related protein (AGRP) and anorexigenic proopiomelanocortin (POMC) neurons have been substantially linked to alterations in food intake. Both heterogeneous subtypes are conversely stimulated and inhibited by the fat-derived hormone leptin  promoter fragment were co-transduced with channelrhodopsin-2 (ChR2) and simultaneously photostimulated, completely reversing the AgRP→PVN-evoked increase in food intake. Finally, by applying combinatorial opto- and chemogenetic manipulations with pharmacology, alternative downstream circuits of AGRP neurons were implicated in eliciting feeding behavior. Recently, it was revealed that AGRP axonal projections to the bed nucleus of the stria terminalis (BNST), lateral hypothalamus (LH) or paraventricular thalamus (PVT), in addition to the PVN, are sufficient to drive feeding  hypothalamus evoked feeding in a similar magnitude to direct somatic AGRP activation, implicating a crucial role for neurons in this brain site in directing appetite signaling  or indirect pathway medium spiny neurons (iMSNs), respectively  and emotional stressors (outlined below). An animal model of this is stress-induced reinstatement of food seeking. In this paradigm, animals are trained to lever-press for food, after which this is extinguished but can be reinstated with stressors, including the pharmacological stress mimic yohimbine (and α2-adrenergic antagonist). Optogenetic inhibition of the medial PFC during yohimbine treatment impaired this reinstatement, similar to reports with cue induced reinstatement of cocaine, suggesting that similar processes may underlie both results  have been linked to obesity and multiple forms of drug addiction  availability compared with lean people and animals (Wang et al., Reduced D2R function may be predicted to elevate activity in iMSNs, as D2R is a Gi coupled receptor. Therefore, it is possible that obese individuals consume foods that over-stimulate dopamine release to inhibit these overactive iMSNs and escape from pervasive negative emotional states. Consistent with this hypothesis, animals that express ChR2 in iMSNs exhibit aversion to stimulation of these cells (Kravitz et al., In addition to dopamine receptors, alterations in dopamine producing neurons in the VTA may contribute to the emergence of negative emotional states. Through their inputs to the VTA, efferents emanating from the laterodorsal tegmentum and the lateral habenula elicit positive and negative states in mice, respectively (Lammel et al., Finally, in humans, the amygdala has been linked to both anxiety-disorders (Etkin et al., In recent years, the drug addiction paradigm has been applied to the neural circuits mediating behaviors associated with obesity. This perspective has sparked important insights, while still recognizing that obesity has important differences from drug addiction. Primarily, food is necessary for survival, which makes parsing the adaptive and maladaptive components of feedings a challenge when thinking of potential therapies, as obese people cannot develop strategies to avoid food altogether, as a drug addict might towards drugs of abuse. Given the ability of feeding behaviors to be both necessary for survival and harmful in excess, understanding the neural circuits related to food addiction calls for tools of utmost precision, such as manipulations facilitated by optogenetic and chemogenetic approaches.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our study aimed to assess adult women’s knowledge of human papillomavirus (HPV) and cervical cancer, and characterize their attitudes towards potential screening and prevention strategies.Women were participants of an HIV-discordant couples cohort in Nairobi, Kenya. An interviewer-administered questionnaire was used to obtain information on sociodemographic status, and sexual and medical history at baseline and on knowledge and attitudes towards Pap smears, self-sampling, and HPV vaccination at study exit.Only 14% of the 409 women  had ever had a Pap smear prior to study enrollment and very few women had ever heard of HPV (18%). Although most women knew that Pap smears detect cervical cancer (69%), very few knew that routine Pap screening is the main way to prevent ICC (18%). Most women reported a high level of cultural acceptability for Pap smear screening and a low level of physical discomfort during Pap smear collection. In addition, over 80% of women reported that they would feel comfortable using a self-sampling device (82%) and would prefer at-home sample collection (84%). Nearly all women (94%) reported willingness to be vaccinated to prevent cervical cancer if offered at no or low cost.These findings highlight the need to educate women on routine use of Pap smears in the prevention of cervical cancer and demonstrate that vaccination and self-sampling would be acceptable modalities for cervical cancer prevention and screening. Cervical cancer is a preventable disease, yet the number of cases globally is expected to almost double by the year 2025 Traditionally, Pap smear, combined with treatment of cervical precancer and early stage cancer, has been successful in preventing up to 80% of invasive cervical cancer cases in developed countries Uptake of self-sampling has been shown to be successful and HPV testing from self-collected samples is highly sensitive for detection of cervical intraepithelial neoplasia grade 2/3 in both clinic and home settings With the increase in technology and opportunity to prevent cervical cancer worldwide, it is important to understand attitudes and barriers of screening among women at high-risk of invasive cervical cancer in low-resource settings. Therefore, our study aimed to assess women’s knowledge of HPV and cervical cancer, and their attitudes towards potential screening strategies, including routine Pap smears, self-sampling for HPV DNA testing, and HPV vaccination. These data will be critical for successful implementation and high uptake of community-level cervical cancer screening programs.Women were recruited into a cohort of HIV-1-discordant couples identified in voluntary counseling and testing centers in Nairobi, Kenya, from May 2007 to October 2009. Couples were eligible to enroll in the study if they reported ≥3 sex acts in the previous 3 months, planned to stay in Nairobi in their current relationship for at least 2 years, and if one member of the couple was HIV-1-infected and the other HIV-1 susceptible. Women who were pregnant and participants using antiretroviral therapy at the time of enrollment were excluded. At enrollment and at quarterly follow-up visits, the HIV status of the uninfected partner was determined using the Determine® HIV–1/2 Rapid Test  or Bioline Recombigen HIV Test , with confirmation by the Vironostika® HIV Uni-form II Ag/Ab ELISA kit . In the HIV-1-infected partner, CD4+ T-cell counts were taken at enrollment and every six months using a FACSCaliber flow cytometer .At enrollment, clinical staff administered a questionnaire to obtain information on sociodemographic characteristics, sexual history and behavior, history of Pap screening, and medical history. Questionnaires were presented in English or Kiswahili, depending on participant preference, and were administered individually to ensure confidentiality. A medical examination was also conducted during biannual study visits, which included collection of a cervical Pap smear. All participants with abnormal cervical cytology were followed up with a repeat Pap test or colposcopy and biopsy as recommended.At the final study visit, up to two years after study enrollment, clinical staff administered an extended questionnaire to obtain additional information on male circumcision, pregnancy history, domestic violence, ARV medication history, and cervical cancer and screening. Relevant questions fell into three categories to assess women’s: 1) knowledge of HPV and the causes and prevention of cervical cancer, 2) attitudes towards Pap smear screening after having had biannual screening in the study, and 3) attitudes towards routine Pap smear screening, self-sampling and HPV vaccination. Specifically, participants were asked if they had ever heard of HPV and if they knew the cause of cervical cancer or how to prevent it. Participants gave detailed histories of Pap smear screening prior to the study or the reasons why they had never had any screening, and were also asked about future intentions for Pap smear screening. Women rated their level of pain during study-conducted Pap smears, beliefs of cultural acceptability, and feelings of necessity of Pap smear screening. Finally, women were asked if they would feel comfortable collecting their own vaginal samples at home and if they would consider HPV vaccination if it were offered at no or low cost. No intervention or education on cervical cancer and screening was provided during the study unless medically indicated, and all responses were based on women’s one-time self-report.Written informed consent was obtained from all participants. The study was conducted according to procedures approved by the University of Washington Institutional Review Board and the Kenyatta National Hospital Ethics and Research Committee.A descriptive summary of knowledge and acceptability overall and stratified by HIV-status is presented. The numbers and percentages of each response are presented. Measures of Pap smear acceptability, necessity and pain-level are based on a scale of 0 to 100 and women’s responses are summarized by the mean, median and interquartile range. Exact logistic regression was used to calculate unadjusted odds ratios and 95% confidence intervals to describe the associations between baseline socioeconomic indicators and medical and sexual history and: 1) having ever had a Pap smear prior to study enrollment, 2) having ever heard of human papillomavirus, 3) knowing that Pap smear screening is conducted to prevent invasive cervical cancer, and 4) feeling that Pap smears are completely acceptable (rating of 100) versus less than completely acceptable. All analyses were conducted using SAS version 9.2 .We interviewed 409 women from HIV-1-discordant couples, 268 (65%) of whom were HIV-positive at baseline with a median CD4+ T-cell count of 463  reported having had a Pap smear prior to the study. Of those who reported having a Pap smear prior to enrollment in the study, most had been done as part of routine care (42%) or as part of a prior research study (19%) and the remaining were conducted for unknown reasons (23%). Those who had never had a Pap smear reported that they did not get screened because they did not know what a Pap smear was or why they needed one (77%). After having at least two Pap smears as part of the study protocol, nearly all women (93%) said that they would seek out Pap screening in the future, with the hospital being the most commonly cited place at which they can be screened. Three-quarters of women said they would be willing to pay up to 400 Kenyan shillings for a Pap smear (approximately 5 US dollars).When asked about methods of screening and prevention, including self-sampling for HPV testing and HPV vaccination, the majority of the women (82%) reported that they would be comfortable using an at-home cervico-vaginal self-sampling device. In fact, 84% of all women said they would prefer this method over having a sample collected in a clinic. Despite their willingness to consider self-sampling, the majority of women (91%) had concerns regarding self-sampling, with the ability to properly collect the sample being the most commonly cited concern (67%). HIV-negative women were slightly more likely to report no concerns (16%) as compared to HIV-positive women (5%). Nearly all HIV-negative (94%) and HIV-positive women (94%) said they would get a vaccine to prevent cervical cancer if offered to them in the future at no or low cost.When asked about the level of acceptability, pain, and necessity that they felt towards Pap smear screening, nearly all women (95%) gave Pap smears the highest possible rating for necessity . About hWomen age 30 years and older , with at least a secondary education , who had ever heard of HPV  or knew that Pap smears were used to prevent invasive cervical cancer  were more likely to have ever had a Pap smear prior to enrolling in the study . Women wWomen with at least a secondary education compared to less than a secondary education , those who had ever had a Pap smear prior to enrollment in the study compared to never previously screened , and those who knew compared with those who did not know that Pap smears were conducted to as part of preventing cervical cancer  were more likely to have heard of HPV. Similarly, women with at least a secondary education compared to less than a secondary education  and those who had ever versus never heard of HPV  were more likely to know that Pap smears can identify cervical abnormalities before cancer develops in order to prevent the development of invasive cancer. A woman’s history of Pap smear screening , knowledge of HPV  and knowledge that Pap smears are a tool to prevent invasive cervical cancer  were associated with giving routine Pap smear screening the highest possible rating for acceptability. Although education was associated with a history of Pap smear screening, knowledge of HPV and knowledge that Pap smears are used in the prevention of cervical cancer, education was not associated with acceptability of Pap smears .The majority of women knew that Pap smears are used to detect cervical cancer (69%), but very few knew that routine Pap screening is the main way to prevent cervical cancer (18%). Most women did not know the cause of cervical cancer (78%) and only 18% had ever heard of HPV. However, it was reassuring to find that most women held positive attitudes towards future Pap smear screening, self-sampling, and HPV vaccination. Our findings highlight the need to educate and reinforce to women that routine Pap smear screening is a key part of preventing invasive cervical cancer so that the fear of being diagnosed with cancer is not a barrier to screening.Despite the fact that knowledge of HPV was very low in our cohort, almost all women reported that they would be willing to be vaccinated against HPV if the vaccine was available at no or low cost. This finding is similar to other studies Consistent with previous studies that document low uptake of Pap smear screening in sub-Saharan Africa, previous Pap smear screening was low in our cohort, with most women (86%) reporting never having had a Pap smear prior to joining the study Without an intervention or educational campaign, it is likely that the strongest predictor of future screening is having been screened in the past. Identifying the characteristics of women who have and have not had previous screening can help to target screening and outreach efforts. In this cohort, women with less education, who were HIV-positive, or didn’t have knowledge of HPV or Pap smears, were less likely to have had previous Pap smear screening. Given the cross-sectional study design, it is unclear whether women learned of HPV during Pap smear screening or whether they learned about HPV from other sources and thus sought out cervical cancer screening. However, in a setting such as Nairobi, where Pap smear screening is available, although access and campaigns are limited, and where HPV testing is now being advertised, it is likely that women who had previous Pap smears for a research study, routine care or diagnostic purposes learned of HPV during or in response to screening. In addition, these findings are consistent with a previous study of cervical cancer patients and non-patients in Nairobi, which found a lower level of education and lack of knowledge of cervical cancer to be associated with a decreased likelihood of having had previous pap screening Women’s knowledge and attitudes were self-reported when they exited the study so there was no follow-up to confirm their feelings or intentions. Pap smear screening conducted during the study may have influenced women’s perception towards Pap smear acceptability, as compared to women who have never been screened. However, after having at least one Pap smear during the study, women reported little physical discomfort associated with Pap smear screening and most responded positively toward future use of a self-sampling device and HPV vaccination. All women in our cohort were in stable, HIV-discordant partnerships and so their responses may not be generalizable to other HIV-negative and HIV-positive women in the general population. However, this is a very relevant population of women since they are of screening age, HIV-positive or at risk for HIV, and many are mothers with daughters potentially eligible for HPV prophylactic vaccination. Furthermore, very little data exist on women’s knowledge and attitudes towards cervical cancer and prevention, especially from East Africa where the incidence and mortality rate of cervical cancer are one of the highest worldwide Despite low levels of knowledge of HPV and that the fact that Pap smears are a tool to prevent, not just detect, cervical cancer, women reported a high-level of acceptability for cervical cancer prevention measures, including Pap screening, HPV vaccination, and self-sampling. Our findings highlight the need for education regarding the cause and prevention of cervical cancer. Currently over 100 sites in Kenya offer regular screening, however, awareness is low and cervical cancer screening coverage for all women age 18–69 years is only 3.2%


TAZ gene on chromosome Xq28, also known as G4.5, are responsible for the BTHS phenotype resulting in a loss-of-function in the protein product tafazzin. Clinical management of BTHS has also seen improvement. Patients with neutropenia are susceptible to life-threatening bacterial infections with sepsis being a significant concern for possible morbidity and mortality. Increasingly, BTHS patients are suffering from heart failure secondary to their CMP. Left ventricular noncompaction (LVNC) and dilated CMP are the most common cardiac phenotypes reported and can lead to symptoms of heart failure as well as ventricular arrhythmias. Expanded treatment options for end-stage myocardial dysfunction now offer an opportunity to change the natural history for these patients. Herein, we will provide a current review of the genetic and molecular basis of BTHS, the clinical features and management of BTHS, and potential future directions for therapeutic strategies. © 2013 Wiley Periodicals, Inc.Barth syndrome (BTHS) is an X-linked recessive disorder that is typically characterized by cardiomyopathy (CMP), skeletal myopathy, growth retardation, neutropenia, and increased urinary levels of 3-methylglutaconic acid (3-MGCA). There may be a wide variability of phenotypes amongst BTHS patients with some exhibiting some or all of these findings. BTHS was first described as a disease of the mitochondria resulting in neutropenia as well as skeletal and cardiac myopathies. Over the past few years, a greater understanding of BTHS has developed related to the underlying genetic mechanisms responsible for the disease. Mutations in the  TAZ gene (G4.5) located on chromosome Xq28 which encodes for tafazzin. Tafazzin is a phospholipid transacylase which is located in the inner leaflet of the mitochondrial membrane and plays an important role in the remodeling of cardiolipin (CL). CL is known to be critical in maintaining mitochondrial structure as well as being involved in mitochondrial apoptosis [Koshkin and Greenberg, Barth syndrome  was first described in 1983 as an X-linked disease of neutropenia, skeletal myopathy, and cardiomyopathy (CMP) in a large Dutch pedigree with high infant mortality [Barth et al., Tafazzin is a phospholipid transacylase which is located in the inner leaflet of the mitochondrial membrane and plays an important role in the remodeling of cardiolipin (CL). CL is known to be critical in maintaining mitochondrial structure as well as being involved in mitochondrial apoptosis.BTHS is a rare disorder with less than 500 patients in the BTHS Registry from around the world. There are no known ethnic or racial predilections. It is estimated that there are ∼10 new cases of BTHS diagnosed in the US each year. Although the actual prevalence is unknown, the Barth Syndrome Foundation reports an estimate of 1/300,000–400,000 live births. There are increasing data to suggest that BTHS may be underdiagnosed with a possible prevalence as high as 1/140,000 live births [Cantlay et al., Although rare, the clinical implications of BTHS can be devastating. Heightened awareness of this disease is needed globally as timely diagnosis may afford patients access to appropriate therapies and improved outcome.TAZ) gene, also known as G4.5, located in the long q arm region of chromosome Xq28, is responsible for BTHS [Bione et al., http://www.barth-sydrome.org). Less than 20% of boys carry de novo mutations not identified in maternal somatic DNA but gonadal mosacisim has been reported [Chang et al., The tafazzin . Tafazzin catalyzes the remodeling of immature CL after initial synthesis to mature CL which is predominantly L4-CL. In BTHS with abnormalities in tafazzin, less mature CL is produced resulting in the ratio of MLCL:L4-CL being increased [Schlame et al., Neuwald, . CL has CL has multiple roles as mentioned above, including maintenance of mitochondrial structure and involvement in mitochondrial apoptosis but also is needed to optimize cellular energy production [Klingenberg, Patients may have some or all of the reported associated clinical features of BTHS. The most widely reported of these are CMP, skeletal myopathy, growth delay, neutropenia, and increased urinary excretion of 3-methylglutaconic acid (3-MGCA). In an attempt to better characterize the clinical features of BTHS, the BTHS Registry in conjunction with the Barth Syndrome Foundation (BSF) was created in 2006. Some of the data collected from the Registry are described below which includes self-reported or medical chart abstraction data on 73 subjects. Based on these and other data, Roberts et al.  recognizIn an attempt to better characterize the clinical features of BTHS, the BTHS Registry in conjunction with the Barth Syndrome Foundation (BSF) was created in 2006. Some of the data collected from the Registry are described below which includes self-reported or medical chart abstraction data on 73 subjects.Neutropenia is very common in BTHS and may be persistent or intermittent. However, there is a wide range of clinical findings with some patients having extremely low levels of neutrophils and others having only mild involvement to normal levels of circulating neutrophils. Neutropenia was reported in 69.1% of 73 patients from the BTHS Registry [Roberts et al., Neutropenia may be the initial finding in an undiagnosed BTHS patient and may be seen prior to birth.Myelocyte arrest is seen on bone marrow aspiration with neutrophils showing membrane-bound vacuoles.The exact mechanism behind neutropenia in BTHS remains unclear.The clinical implications of neutropenia can be life-threatening secondary to possible bacterial infection and resultant sepsis. Clinical vigilance must be high as neutropenia may develop in someone who otherwise has had low normal to mild decrease in circulating levels. Ongoing surveillance is critical for a good outcome as levels may fluctuate dramatically in a single patient. Neutropenia is responsible for other morbidity leading to respiratory infections, dermatologic infections, and mouth ulcers.Growth delay is a well-described clinical feature of BTHS. The BTHS Registry evaluated 764 growth points on 60 patients and superimposed these points on the CDC growth curves for normal age appropriate males [Roberts et al., Cardiac disease is common in BTHS. BTHS Registry data describe 69 of 73 subjects reporting a history of CMP [Roberts et al., DCM is a common cardiac phenotype, characterized by decreased left ventricular (LV) systolic function, increased LV mass, and an increased LV end-diastolic dimension.Left ventricular noncompaction (LVNC) is also commonly seen either alone or in conjunction with other CMP phenotypes and is characterized by trabeculations in LV with associated wall motion abnormalities.In addition, there is an increased risk of arrhythmias in BTHS, some of which may be life-threatening. These may be a result of abnormal mitochondrial function but also may be a function of the associated cardiac phenotype as ventricular arrhythmias are well reported in DCM, LVNC, and HCM [Jefferies and Towbin, TAZ gene were reported in a female infant [Cosson et al., Previously, it has been thought that female carriers of would not exhibit clinical features of BTHS due to the presence of a normally functioning wild-type gene in the setting of an X-linked disease. However, clinical findings of BTHS with genetic confirmation of deletions of exons 1 through 5 in the TAZ can be performed at commercial laboratories both nationally and internationally and facilitate a diagnosis. Most available commercial panels for testing of patients with either LVNC or DCM typically include TAZ screening.Historically, BTHS has been diagnosed based on clinical presentation relying heavily on identifying boys with neutropenia, CMP, skeletal myopathy, and elevated urinary levels of 3-MGCA. However, based on the highly variable clinical presentations in BTHS, utilizing this approach make reliable diagnosis problematic. Armed with a greater understanding of CL species in BTHS, Houtkooper et al.  developePreviously, BTHS was often considered a lethal infantile and early childhood disease [Christodoulou et al., Interestingly, a clear change in survival has recently been reported based on birth year with patients born after 2000 having a 70% survival rate compared with a 20% survival rate in those born before 2000This suggests improved management strategies are having a measurable effect on outcome.Granulocyte colony stimulating factor (G-CSF) has been widely used in BTHS with reasonable success. G-CSF is typically used in times of neutropenia concomitant with appropriate prophylactic antibiotics if clinically indicated. Treatment is often used to raise counts to near normal and may not be continued indefinitely given the wide variability in counts that may be seen. G-CSF may be given subcutaneously either twice weekly or every other day based on severity. BTHS Registry data report nearly 50% of the cohort had been on G-CSF at some point in their history with 27.5% being actively treated [Roberts et al., Although decreased levels of growth hormone (GH) have been reported in BTHS patients less than 15 years, levels typically are higher than normal controls in the late teens and early twenties. As a result, GH supplementation has not been routinely used unless documented central GH deficiency has been established. Arginine depletion has been implicated in possibly contributing to low growth rates in BTHS. Low levels of arginine have been documented in the serum of BTHS patients and may result in limitations in protein synthesis [Coman et al., Treatment of associated myocardial dysfunction is paramount to alleviating symptoms as well as prolonging life. Medical and surgical options for DCM in BTHS have increased over the past few years and are largely driven by treatments used for adults with HF as well as children with DCM from other causes. Medical therapies must be directed at the particular cardiac phenotype being managed. Typical therapy for DCM would consist of angiotensin converting enzyme inhibitors (ACEi) or alternatively angiotensin receptor blockers (ARB) in combination with approved beta blockers [Jessup and Brozena, Although much attention is given to associated CMP with BTHS, clinically significant arrhythmias can be a major cause of mortality. The risk of ventricular arrhythmias is well known and may be precipitated by associated metabolic acidosis or concomitant LV systolic dysfunction [Yen et al., TAZ knockdown model was developed [Acehan et al., Ongoing efforts to better understand BTHS in humans have been historically limited secondary to the inability to develop a mammalian model. However, in 2010 an inducible Advancements in the treatment of cardiac dysfunction and heart failure continue to be reported. Medical and technological breakthroughs that are developed and refined in adult ischemic and nonischemic CMP populations will be available to treat BTHS patients with cardiac disease and potentially prolong life without the need for cardiac transplantation.BTHS, although a rare disease, has seen evidence of impactful research and clinical intervention over the past few years. Diagnostic strategies are improving and therapeutic options, especially for cardiac disease, are expanding. Perhaps most importantly, the wide variability in BTHS phenotypes is being recognized and thoughtful, longitudinal care plans are being developed to avoid potentially catastrophic scenarios such as sepsis, decompensated heart failure, and SCD.Cardiovascular disease in BTHS may come in the form of myocardial dysfunction or life-threatening arrhythmias. Management of systolic dysfunction may be effective with oral therapies but in some patients, advanced therapies may be necessary in the form of intravenous medications, MCS, and/or cardiac transplantation. The use of advanced device therapies such as ICDs has been reported and may be effective in managing patients with significant risk of life-threatening arrhythmias. Importantly, ongoing cardiac surveillance must be performed regularly as development of these sources of morbidity and mortality may develop at any time.On a broader note, mitochondria have become a therapeutic target in the treatment of advanced heart failure in those patients without BTHS [Bayeva et al., Active investigation continues to occur in the basic, translational, and clinical research domains directed at better diagnostic and therapeutic strategies for the care of BTHS patients. Efforts must be increased to heighten awareness of this disease to facilitate earlier diagnosis and intervention and improve outcome.


Focal and differential therapy represent an approach to improve the therapeutic ratio of prostate cancer treatments. This concept is a shift from treating the whole gland to intensely treating the portion of the gland that contains significant tumor. However, there are many challenges in the move towards focal approaches. Defining which patients are suitable candidates for focal therapy approaches is an area of significant controversy, and it is likely that additional data from imaging or detailed biopsy methods is needed in addition to traditional risk factors. A number of methods have been suggested, and imaging with multiparametric MRI and transperineal template mapping biopsy have shown promise. The approach of differential therapy where the entire prostate is treated to a lower intensity and the tumor areas to high intensity is also discussed in detail. Radiation therapy is a well suited modality for the delivery of differential therapy. Data in the literature using external beam radiation, high dose rate brachytherapy, and low-dose rate brachytherapy for differential therapy are reviewed. Preliminary results are encouraging, and larger studies and randomized controlled trials are needed to validate this approach. Prostate cancer is the most common malignancy in men in the US, with an estimated 241,740 cases to be diagnosed in the US in 2012 , a large Focal therapy represents an approach to improve the therapeutic ratio by maximizing tumor control while minimizing side effects. Whereas traditional therapies typically treat the entire prostate gland, this concept is a shift towards intensely treating the portion of the gland that contains significant tumor. This targeted treatment strategy has the potential to reduce the chance of injury to adjacent organs and resultant side effects while maintaining excellent oncologic outcomes. However, there are many challenges in the move towards focal therapy including identifying appropriate candidates, methodology of identifying intraprostatic tumor, and whether the remaining prostate gland should receive some form of treatment.Defining which patients are suitable candidates for focal therapy approaches is an area of significant controversy. Patients in the low-risk group, with the excellent outcomes described above, would appear to be the most logical choice for deintensification of treatment. Strict low-risk criteria were used by Katz et al.  to attempt to identify patients who could adequately be treated by focal therapy . They reThe current standard of care for prostate biopsy is to use transrectal ultrasound (TRUS) guidance to take 6–12 transrectal needle biopsies throughout the prostate in a systematic fashion. Although a reasonable approach when whole gland treatment is being considered, a comparison with prostatectomy specimens has shown that this method misses a number of clinically significant cancers and is inadequate to identify candidates for focal therapy . IncreasNewer imaging modalities offer a noninvasive method of identifying tumor foci. Ultrasonic tissue type imaging based on spectrum analysis of radiofrequency signals has been developed by Feleppa et al., to help identify prostate cancer. When compared to biopsy data, this method has shown a receiver-operator characteristic curve (ROC) of 0.87 , 15. ThiMagnetic resonance imaging (MRI), with the inclusion of diffusion and dynamic contrast, has shown even more promise in identifying intraprostatic tumors. Multiple studies have shown significant correlation between MRI abnormalities and radical prostatectomy specimens for determining size and location of cancer foci –19. Cancpure focal therapy) is too drastic and risks missing biologically significant tumor. Prostate cancer can be multifocal in up to 87% of patients, and the prostate often contains three or more foci of cancer, and sometimes as many as twelve, on radical prostatectomy specimens [A major dilemma in the implementation of focal therapy is whether the shift from treating the entire gland uniformly to purely treating the tumor only , for unilaterally boosted versus bilaterally boosted patients; however grade 4 toxicity was seen only in patients who had bilateral brachytherapy boost . The authors concluded that hemi-irradiation of the prostate with HDR brachytherapy could be considered when patients have rectal examination, MRI, and biopsy findings that suggest unilateral involvement only. Differential therapy using purely HDR brachytherapy has been investigated by Pouliot et al. at UCSF in a treatment planning study with 10 patients [High-dose-rate (HDR) brachytherapy has been used as part of a differential therapy approach, by Schick et al., to boost the dominant intraprostatic tumor in 77 patients . The patpatients . MRI andLow-dose-rate (LDR) brachytherapy is also an effective method for differential radiation therapy. Todor et al. suggested dual-isotope seed implants as a technique to vary the dose throughout the prostate gland . They usP ≤ 0.0009). In addition, consistent with the previously mentioned studies, there was statistically significant reduction in dose to the prostate gland outside the tumors seen with the differential dose brachytherapy. The experimental brachytherapy has been very well tolerated, as no grade 3-4 toxicities have been noted with a followup range of 4–16 months.Ultrasound spectrum analysis as a method for identifying intraprostatic lesions is particularly attractive for brachytherapy. Most brachytherapy is ultrasound based, and images can be acquired at the time of the procedure and readily incorporated into the treatment plan . At our Pure focal and differential therapy represents exciting approaches to improve the therapeutic ratio of prostate cancer treatment by decreasing complication risks while maintaining or perhaps even improving cancer control rates. Newer imaging modalities offer accurate and noninvasive methods for identifying intraprostatic tumors and can be incorporated into treatment planning. Radiation therapy is well suited for differential therapy, where treatment intensity is increased towards the tumors and decreased in the remaining prostate. Preliminary data using external beam radiotherapy, HDR brachytherapy, and LDR brachytherapy for differential therapy have been encouraging and shown that these techniques are feasible and safe. Further areas of study include defining the optimal degree of dose escalation to the tumors and dose reduction to the prostate that provide the most favorable outcomes for patients and refining the technical procedures used to deliver this dose. In addition, the possibility exists for using low-dose radiotherapy as an adjunct to HIFU, cryotherapy, and other focal treatment techniques. Additional clinical studies, and ultimately, large randomized controlled trials are needed to validate these approaches.