Children's exposure to secondhand smoke (SHS) has been causally linked to a number of childhood morbidities and mortalities. Over 50% of UK children whose parents are smokers are regularly exposed to SHS at home. No previous review has identified the factors associated with children's SHS exposure in the home.To identify by systematic review, the factors which are associated with children's SHS exposure in the home, determined by parent or child reports and/or biochemically validated measures including cotinine, carbon monoxide or home air particulate matter.Electronic searches of MEDLINE, EMBASE, PsychINFO, CINAHL and Web of Knowledge to July 2014, and hand searches of reference lists from publications included in the review were conducted.Forty one studies were included in the review. Parental smoking, low socioeconomic status and being less educated were all frequently and consistently found to be independently associated with children's SHS exposure in the home. Children whose parents held more negative attitudes towards SHS were less likely to be exposed. Associations were strongest for parental cigarette smoking status; compared to children of non-smokers, those whose mothers or both parents smoked were between two and 13 times more likely to be exposed to SHS.Multiple factors are associated with child SHS exposure in the home; the best way to reduce child SHS exposure in the home is for smoking parents to quit. If parents are unable or unwilling to stop smoking, they should instigate smoke-free homes. Interventions targeted towards the socially disadvantaged parents aiming to change attitudes to smoking in the presence of children and providing practical support to help parents smoke outside the home may be beneficial. Exposure to secondhand smoke (SHS), also known as passive smoking or environmental tobacco smoke, is the involuntary inhalation of other people's cigarette smoke. Children's exposure to SHS has been causally linked to increased risks of respiratory tract infections, middle ear infections, sudden unexplained death in infancy, and asthma Forty percent of children globally are exposed to SHS To our knowledge, studies which aim to understand the factors or characteristics associated with children's SHS in the home have not been previously reviewed. Consequently, we have carried out such a review of relevant studies conducted in children aged ≤18 years, examining factors associated with home SHS exposure. We aimed to identify factors, such as environmental or socioeconomic characteristics, which have been shown to be independently associated with children's SHS exposure in the home, and to determine potential characteristics that may be important for the development of effective future SHS and smoke-free home interventions.This systematic review was conducted and reported in accordance with the PRISMA guidelines secondhand smoke, environmental tobacco smoke, passive smoke/smoking, smoking in the home, smoke-free home, smoking rules, child, children, school child*, infant, baby, babies, parent, mother, father, predictor, association, factors, determinants.Electronic databases MEDLINE, EMBASE, PsychINFO, CINAHL and Web of Knowledge were searched to the end of July 2014 without date restrictions, using combinations of the following key words: The reference lists of papers identified as being relevant in the above electronic searches were also hand searched.Titles and abstracts identified from the searches were reviewed, and all studies meeting the following inclusion criteria identified: (a) English language studies examining the factors associated with SHS exposure in children aged ≤18 years, (b) reported a measure of child SHS exposure , (c) examined potential factors/associations for child SHS exposure .The age cut-off of ≤18 years for childhood was chosen to reflect variation in the legal age of adulthood across countries, with the majority of countries considering those aged 19 to be adults, and was considered appropriate as it is also the upper-limit at which adolescents are likely to remain in compulsory full-time education.Whilst biomarkers are able to provide a quantitative measure of SHS exposure, this may reflect exposure both in the home and elsewhere. However, there is strong evidence to suggest that biomarkers can be used as an appropriate measure for child domestic SHS exposure. Research has shown that children spend the largest proportions of their time either in school attendance or as leisure time inside the home Papers that were not original quantitative methodologies were excluded. Papers exploring associations with parental reported ‘smoke-free homes’ (e.g. their child was NOT exposed to SHS in the home) were also excluded; creating ‘smoke-free homes’ is a behaviour change, and thus it is likely that there are a number of complex reasons, barriers or facilitators related to implementing home smoking bans. The factors associated with these are therefore likely to be quite different to those associated with children's SHS exposure in the home.Following the title and abstract review, SO (first author) independently reviewed the full texts. A summary of each of the included studies is presented in a priori cut off point of seven points out of a possible 10 was used to identify papers considered to be of higher methodological quality, as has been used previously with the comparable original scale Studies that met the inclusion criteria were assessed for quality using a modified version of the Cochrane Collaboration Non-Randomized Studies Working Group recognised Newcastle-Ottawa Quality Assessment Scale There were 4013 papers identified through the systematic literature searches. After removal of duplicates, a further 2,316 articles were excluded based on title and abstract review. These included intervention studies to reduce child SHS exposure, studies examining the health risks associated with child SHS exposure and editorial papers. Sixty-five papers were considered as potentially eligible based on title and abstract review, and full-texts were obtained. Following the review of full-texts, 41 of these papers were included in the final review .Ten of the 41 studies were conducted in the UK England , eight iThirty of the papers reported studies which were of cross sectional design Using the modified Newcastle-Ottawa Quality Assessment Scale The majority of studies focused on school-age children of approximately 5–18 years 2.5] Eighteen studies used the following validated measures of child SHS exposure: salivary cotinine A number of other studies used only self-report measures such as parental Of the 41 included studies, the associations between 90 different variables and child SHS exposure were identified; these were grouped into five conceptually similar categories: (1) socioeconomic status (SES) , (2) parental characteristics , (3) family and home characteristics , (4) child characteristics , and (5) parental smoking characteristics . The size of effect of statistically significant associations reported between principle variables and SHS exposure in the home  are presented in The relationship between child SHS exposure and proxy measures of SES were examined in 11 studies; measures of SES used were the Registrar General's Social Class system Seven studies with mixed findings There was similarly inconsistent evidence for a link between employment status or occupation and child SHS exposure. Three studies found a significant association between employment and exposure to SHS in the home; in one study Twenty-six studies Parental race or ethnicity was examined in nine studies Parental age was not shown to be linked to child SHS exposure; eleven studies Of the 18 studies Eight studies examined an association between the number of cigarettes smoked by parents either per day The number of cigarettes smoked in the home was explored in a further four studies 2.5] 2.5) concentrations and child salivary cotinine among those mothers who strongly agreed that they would ask a smoker to smoke outside their house; however, this was only found in univariate analysis and there was no significant effect for other attitudinal questions. Three of the studies Four studies Thirteen studies There was no clear relationship between family size and exposure, which was investigated in 11 studies There was some evidence for an association with accommodation size or characteristics. Seven studies The association between child age and exposure was explored in 19 studies Nineteen studies Children whose parents are smokers, are of low SES or less educated were at an increased risk of SHS exposure in the home. There was also some evidence that children whose parents held more negative attitudes towards SHS were less likely to be exposed. Associations were strongest for parental cigarette smoking status; compared to children of non-smokers, those whose mothers or both parents smoked were between two and 13 times more likely to be exposed to SHS at home. These findings show that the best way to prevent child SHS exposure in the home is by encouraging smoking parents to quit.Literature in this review was synthesised narratively, which may introduce some bias if findings of one study are given inappropriate weight compared to others There were a number of limitations inherent in the studies included in the review. Using a modified Newcastle-Ottawa Quality Assessment Scale The finding that parental and other household member cigarette smoking status was associated with child SHS exposure supports previous research, which has shown the primary source of child SHS exposure to be smoking by parents In line with the findings of this review, sociodemographic characteristics are often linked to health inequalities. Low SES is frequently reported to be associated with poorer health outcomes, health morbidity and mortality In a recent review Future research is needed to explore SHS exposure specifically in very young infants; just three studies in this review explored factors associated with SHS exposure in this age group. Although other studies included infants of less than two years of age within their samples, this younger age group was not considered or reported independently of older children. Very young infants under two years of age may be particularly susceptible to the risks of SHS exposure as they have a higher respiration rate Children whose parents are smokers, are of low SES, less educated, or hold less negative attitudes towards SHS are at an increased risk of SHS exposure in the home. The largest observed risks were for children living in households with smokers; the best way to reduce child SHS exposure in the home therefore is for smoking parents to quit. If parents are unable or unwilling to stop smoking, they should instigate smoke-free homes. Interventions targeted towards socially disadvantaged parents aiming to change attitudes to smoking in the presence of children, and providing context specific practical support to help parents overcome barriers to smoking outside the home may reduce children's domestic SHS exposure.Table S1Study characteristics.(DOCX)Click here for additional data file.Table S2Associations identified and strength of effect.(DOCX)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


A number of rhythmic protocols have emerged for non-invasive brain stimulation (NIBS) in humans, including transcranial alternating current stimulation (tACS), oscillatory transcranial direct current stimulation (otDCS), and repetitive  transcranial magnetic stimulation (rTMS). With these techniques, it is possible to match the frequency of the externally applied electromagnetic fields to the intrinsic frequency of oscillatory neural population activity (“frequency-tuning”). Mounting evidence suggests that by this means tACS, otDCS, and rTMS can entrain brain oscillations and promote associated functions in a frequency-specific manner, in particular during  stimulation. Here, we focus instead on the changes in oscillatory brain activity that persist after the end of stimulation. Understanding such aftereffects in healthy participants is an important step for developing these techniques into potentially useful clinical tools for the treatment of specific patient groups. Reviewing the electrophysiological evidence in healthy participants, we find aftereffects on brain oscillations to be a common outcome following tACS/otDCS and rTMS. However, we did not find a consistent, predictable pattern of aftereffects across studies, which is in contrast to the relative homogeneity of reported online effects. This indicates that aftereffects are partially dissociated from online, frequency-specific (entrainment) effects during tACS/otDCS and rTMS. We outline possible accounts and future directions for a better understanding of the link between online entrainment and offline aftereffects, which will be key for developing more targeted interventions into oscillatory brain activity. These techniques act on the stimulated tissue by inducing a subthreshold polarization, which does not trigger action potentials, but rather change the resting membrane potential and thus lead to a change in the firing rate or pattern of the stimulated neurons.- otDCS involves a current that has either an anodal or cathodal polarity with respect to the target electrode, i.e., the direction of current flow is either directed inward (toward the electrode) or outward (away from that electrode), respectively. The current intensity oscillates at a specific frequency either between 0 and maximum amplitude  form (rTMS) refers to the application of trains of magnetic pulses at specific frequencies. The TMS mechanism of action is qualitative different from tACS and otDCS. Magnetic stimulation exploits time-varying magnetic fields generated within a coil positioned on the scalp to induce electric currents in the brain. The rapidly changing induced electric field affects the transmembrane potentials and may lead to a depolarization of nerve cells, induce the generation of action potentials, and result in phase-reset of ongoing oscillations  for at least 1 min following stimulation, i.e., when entrainment-echoes likely have ceased. In the following, we will refer to these lasting changes (regardless of the specific measure used) as aftereffects unless otherwise specified. For details please refer to the corresponding Table Oscillatory transcranial electrical stimulation is often accompanied by aftereffects on neural synchrony Table . Of the In terms of duration of tACS/otDCS aftereffects, only few data are available as analyses were mostly restricted to short stimulation-free intervals between successive blocks of stimulation  as their origin.As an index of control over brain oscillations, we assessed whether a consistent relationship exists between the externally applied stimulation frequency and affected frequencies in intrinsic oscillatory activity. Specifically, we surveyed the literature for evidence that stimulation frequency can predict the observed changes in intrinsic frequencies , and vice versa, whether effect frequencies are preferentially associated with specific stimulation frequencies. Patterns that would indicate that the aftereffects primarily originate from direct interactions with oscillatory brain activity are (i) a match between stimulation and effect frequencies  stimulation over frontal areas  mostly during sleep   has been reported to generally induce broadband EEG power modulations, with the exception of rTMS over motor areas which seems to affect activity in a more restricted  frequency band  a complex pattern of gamma power and connectivity modulation was observed , consisting of per se, these results are relevant for two reasons. They suggest that rTMS, and possibly NIBS in general , is likely to lead to aftereffects in brain oscillations that are largely frequency-unspecific, and by extension, represent confounds for those attempting to control brain oscillations and associated functions through direct interaction via externally applied stimulation.In summary, many rTMS-studies did not analyse the aftereffects at stimulation frequency. Of those that did, a considerable fraction did not report any effects in this frequency band  to allow strong conclusions. Collectively, these findings are promising but given that the neural and behavioral measures were generally obtained up to several hours apart, it is debatable whether the behavioral changes reflect a neural aftereffect or rather the interaction with an ongoing process in the acute phase of the experiment.The most promising evidence for behavioral aftereffects that correspond to the reported tACS/otDCS-induced EEG modulations has been provided by those studies using frontal 0.75 Hz tACS/otDCS aiming to either enhance or disrupt slow waves during sleep, a rhythm that has been associated with memory consolidation  frequency changes in EEG/MEG and associated behavior. For rTMS, the reported EEG/MEG aftereffects are broadband throughout, and range across multiple canonical frequency bands. Collectively, these data illustrate that despite some promising reports of controlled interventions into brain oscillations beyond stimulation, the overall evidence so far is weak. Below, we discuss some of the challenges that need to be overcome in order to test whether such interventions are viable. We suggest ways to optimize existing stimulation protocols, and outline future research directions to close the gaps in the existing evidence.The most promising attempts to induce aftereffects in directly targeted brain oscillations and their associated functions have been made in tACS/otDCS studies. However, collectively, the tACS/otDCS results are mixed, with a considerable number of studies reporting effects that are independent of stimulation frequency. This illustrates that neural oscillations are responsive to electrical stimulation in general, in line with frequently reported changes in oscillatory brain activity after NIBS with other non-rhythmic protocols such as transcranial direct current stimulation  vs. low (< 1 Hz) rTMS frequencies, respectively] account for the variability in oscillatory aftereffects? This alternative explanation is also unlikely, given for instance that alpha-power enhancement after motor cortex stimulation has been observed across a wide variety of rTMS frequencies that cross the excitatory-inhibitory parameter divide  for controlled interventions, we believe it is worthwhile to design future rTMS studies to examine more explicitly the opportunity that direct interventions with brain oscillations may provide  and limit their EEG/MEG analyses to one or few, broad frequency bands. This often effectively precludes the assessment of (sub-)harmonic changes that would be expected to be limited to narrow frequency bands.Third, we note a substantial variability in stimulation montages/sites across studies, even for a given brain oscillation/network of interest. Especially for tACS/otDCS, little is known about optimal electrode sites and configurations, and how different montages might lead to different results with otherwise identical stimulation parameters. A systematic effort is required to compare different montages with respect to an intended manipulation, using guidance from modeling of effective field strength in the cortex  stimulation, we find mixed evidence for the controllability of NIBS-induced aftereffects on brain oscillations. We argue that while online mechanisms must be at the origin of offline effects, there is little evidence that the offline effects can be considered proxies of the online effects. Future studies will need to investigate the link between these online and offline effects to advance our understanding of rhythmic NIBS techniques and their potential for neuromodulation via controlled intervention into brain oscillations. Because a growing number of studies indicate that neurological and mental diseases are characterized by abnormal pattern of specific brain oscillations (the so called oscillopathies; Schnitzler and Gross, This work was supported by a Wellcome Trust Award to Gregor Thut and Joachim Gross . AV was supported by a PhD Studentship from the College of Science and Engineering, University of Glasgow.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer and handling Editor declared a current collaboration and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Jack feasibility trial, funded by the NIHR evaluates a film-based educational intervention aimed at increasing boys’ and girls’ intentions to avoid teenage pregnancy (ISRCTN11632300). The trial is ongoing and has recruited eight post-primary schools and 831 young people. As part of the trial, we compiled validated and non-validated instruments to measure sexual behavior data including engagement in sexual intercourse, contraception use, drugs and alcohol usage, diagnosis of STIs; data regarding knowledge, attitudes, skills and intentions relating to avoiding teenage pregnancy; and socio-economic status. The questionnaire was piloted and appropriately modified after pupils took part in focus groups to identify procedural challenges in completing the questionnaire. We discovered difficulties in questionnaire completion due to understanding of terminology and ensuring privacy in different classroom layouts. We will also discuss the issues young people raised when completing the questionnaire, for example by examining the challenges encountered with specific questions such as ‘Have you ever drunk alcohol?’ and present a qualitative description of issues surrounding understanding. We will highlight the importance of feasibility and pilot work for school-based trials in sexual health.It cannot be taken for granted that young people will understand and interpret questions in the way we expect. This is true even when using validated measures, developed and used with the appropriate age group. It can cause problems for trials that need to minimise “noise” when measuring outcomes. We will describe our observations and lessons learnt from developing a questionnaire for young people in a sexual health trial. The


Despite being relatively rare, drug-induced liver injury (DILI) is a serious condition, both for the individual patient due to the risk of acute liver failure, and for the drug development industry and regulatory agencies due to associations with drug development attritions, black box warnings, and postmarketing withdrawals. A major limitation in DILI diagnosis and prediction is the current lack of specific biomarkers. Despite refined usage of traditional liver biomarkers in DILI, reliable disease outcome predictions are still difficult to make. These limitations have driven the growing interest in developing new more sensitive and specific DILI biomarkers, which can improve early DILI prediction, diagnosis, and course of action. Several promising DILI biomarker candidates have been discovered to date, including mechanistic-based biomarker candidates such as glutamate dehydrogenase, high-mobility group box 1 protein and keratin-18, which can also provide information on the injury mechanism of different causative agents. Furthermore, microRNAs have received much attention lately as potential non-invasive DILI biomarker candidates, in particular miR-122. Advances in “omics” technologies offer a new approach for biomarker exploration studies. The ability to screen a large number of molecules  simultaneously enables the identification of ‘toxicity signatures,’ which may be used to enhance preclinical safety assessments and disease diagnostics. Omics-based studies can also provide information on the underlying mechanisms of distinct forms of DILI that may further facilitate the identification of early diagnostic biomarkers and safer implementation of personalized medicine. In this review, we summarize recent advances in the area of DILI biomarker studies. Drug-induced liver injury (DILI) is one of many forms of adverse drug reactions that appear in a small proportion of patients. DILI is generally classified as intrinsic if predictable based on dose and pharmacological properties [for example acetaminophen (APAP) overdose] or idiosyncratic when unpredictable by the same features. The latter is believed to be a consequence of interactions between drug properties, host factors, and environmental conditions in a susceptible individual. Hence, no functional animal models are currently available for idiosyncratic DILI, unlike the presence of well-established murine models for APAP hepatotoxicity. DILI can have a profound impact on patient health due to its potential to cause acute liver failure, although most idiosyncratic DILI cases have a favorable outcome with full recovery after withdrawal of the culprit drug. DILI presents a clinical challenge as it can mimic almost any acute or chronic hepatobiliary condition and there are currently no specific biomarkers or diagnostic test available for this condition. A clinical diagnosis of idiosyncratic DILI therefore relies heavily on exclusion of alternative causes and a compatible drug history. The lack of DILI specific biomarkers also affects the drug development process and can result in early termination of drug candidates with assumed idiosyncratic hepatotoxicity potential. This can have considerable economic consequences and can prevent a large targeted recipient group from benefitting from a drug that may only be harmful to a small proportion of patients. New more specific and sensitive DILI biomarkers could enable better monitoring of patients receiving new drugs and minimize liver injury through early detection and subsequent cessation of dosing.A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention . While oAlanine aminotransferase is thought to be more specific for liver injury than AST, as it is present mainly as a cytosolic protein in the liver and in low concentrations elsewhere, while AST is present in both cytosolic and mitochondrial forms in liver, heart, skeletal muscle, kidney, brain, pancreas, and lung tissue as well as in white and red blood cells . Human ADrug-induced liver injury is generally classified into three patterns of liver injury , based originally on histological features. Hepatocellular type of injury is characterized by hepatocyte necrosis and inflammation, while bile stasis, portal inflammation and bile duct injury are commonly found in cholestatic injury. Mixed injury is, as the name suggests, a combination of these two liver injury types. As biopsies not normally form part of the DILI diagnostic process, the pattern of injury is mainly deduced based on liver profile analytical values. More precisely, the relationship between ALT and ALP values at DILI onset are used to determine the type of liver injury according to the R formula, R =  ÷ ALP/ULN; . An R va1.An ALT value alone offers little predictive value with regards to the clinical course of DILI, but can provide a prognostic indication when combined with TBL. This is today referred to as Hy’s Law and is based on clinical observations made by the late Hyman Zimmerman that DILI patients with hepatocellular type of liver injury and jaundice have a 10–50% higher risk of mortality or need for liver transplantation. Hy’s Law has since been defined more specifically as AST or ALT > 3 xULN and TBL > 2 xULN in the absence of ALP elevations and alternative reasons for ALT and TBL elevations, and is endorsed by the American Food and Drug Administration as criteria for severe DILIThe validity of Hy’s Law has now been confirmed in various large DILI cohorts . However2,3,4 has resulted in the discovery of several promising candidates (Table 1). Many of these biomarker candidates were initially identified using APAP models, but could also be promising for aspects of idiosyncratic DILI.The heightened interest in developing new more sensitive and specific DILI biomarkers for early prediction and diagnosisGlutamate dehydrogenase (GLDH) is a mitochondrial enzyme found primarily in the liver and to a lesser degree in kidneys and trace amounts in skeletal muscles. This enzyme is more tissue-specific than ALT and AST. Healthy individuals present a stable and measurable level of serum GLDH that is unaffected by age and gender . A rise High-mobility group box 1 (HMGB1) is a chromatin-binding protein with proinflammatory activity mediated through Toll-like receptor and receptor for advanced glycation endproducts signaling . It is pMicroRNAs (miRNAs) are small (20–24 nucleotides) non-coding RNAs involved in post-transcriptional regulation of gene expression. Organ damage usually results in miRNAs being released into the bloodstream and to some degree also in urine. MiRNAs are relatively stable in biofluids, a feature that has contributed to that circulating miRNAs have received much attention lately as potential non-invasive DILI biomarker candidates. To date several studies have reported changes in serum miRNA concentrations during liver injury . MiR-122Emerging data also indicate that miRNA-122 could have a prognostic value, with higher early serum levels reported in APAP overdose patients who met the King’s College Criteria for liver transplantation . HoweverWhile miRNAs can enter the blood stream passively during cell necrotic and/or apoptotic cell death, active release also occurs in a regulated manner via exosomes. Exosomes are one of several forms of membrane-surrounded structures released by almost all types of cells. The complete role of exosomes besides transportation and delivery of various substances, such as signaling molecules and cellular waste, is yet to be elucidated . The conThe introduction of omics technologies offers a new approach for biomarker exploration studies. The ability to screen a large number of molecules  simultaneously enables the identification of ‘toxicity signatures’, which could be used to enhance preclinical safety assessments and disease diagnostics. Omics-based studies can also provide information on the underlying mechanisms of distinct forms of DILI that could further facilitate the identification of early diagnostic biomarkers. Metabolomics is recognized as a promising technique, typically through mass spectrometry or nuclear magnetic resonance spectroscopy. This technique enables the evaluation of global metabolic changes and subsequently the identification of specific metabolic profiles associated with for example diseases or treatment responses. Metabolomics has been successfully applied to identify potential biomarker candidates in many types of liver diseases, including non-alcoholic fatty liver disease, steatosis, fibrosis, cirrhosis, hepatocellular carcinoma, and cholangiocarcinoma (reviewed by in vitro models. An ultra-performance liquid chromatography/time-of-flight tandem mass spectrometry analysis of galactosamine-treated rat sera found differences in several metabolites such as glucose, amino acids, and membrane lipids compared with control rats, with some of these correlating with the degree of histologically determined liver damage (However, in the field of hepatotoxicity, metabolomics studies performed to date are mainly limited to intrinsic DILI in animal or r damage . A similr damage . Recent r damage . Metabolr damage . It shour damage , while cr damage . Likewisr damage . HoweverAlanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin are currently the only approved DILI biomarkers in clinical practice. While fulfilling an important role in disease diagnostics, these biomarkers are not specific for hepatotoxicity, as an increase in any of these biochemical values is detected during practically all liver conditions. Furthermore, these biomarkers lack sensitivity as they appear once liver damage has developed and are consequently of limited use for predicting potential liver injury at a very early stage. This is of particular importance in clinical trials during drug development. The presence of DILI biomarkers with enhanced specificity and sensitivity may detect drug candidates with hepatotoxicity potential earlier in the drug development process and subsequently reduce the number of late stage drug attrition and postmarketing drug withdrawals. Furthermore, such biomarker would improve patient safety, due to offering improved monitoring during drug treatment initiation, as well as a faster and more accurate DILI diagnosis.The lack of specific DILI biomarkers is to a large extent a consequence of the limited mechanistic understanding of this condition. The arrival of the “omics era” has brought hope and anticipation of scientific breakthroughs on the mechanistic side of DILI. Substantial progress has been made recently with the identification of new mechanistic-based biomarker candidates, such as HMGB1, K18, and miR-122. However, further efforts to explore and refine new improved DILI biomarker panels are needed. To be valid in clinical practice such non-invasive biomarkers not only need to demonstrate substantial specificity and sensitivity, but also need to be reasonably stable in extracted body fluids to allow reliable detection, as well as being easily assayed. The ability to validate new DILI biomarker candidates is restricted due to the current lack of fully functional animal models for idiosyncratic DILI. Although improved cell culture system such as induced pluripotent stem cell-derived hepatocytes are emerging, large DILI patient cohorts are the foremost approach for biomarker validations. This highlights the importance of collaborative efforts to establish DILI registries, which can circumvent the low DILI incidence rate generally encountered in individual hospital units. The Pro-Euro-DILI Registry is a recently established international DILI registry with serial biological sample collections from DILI onset to normalization of enrolled patients, intended for future DILI biomarker studies and validations .All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


In contrast to the dominant drug paradigm in which compounds were developed to “fit all,” new models focused around personalized medicine are appearing in which treatments are developed and customized for individual patients. The agricultural biotechnology industry (Ag-biotech) should also think about these new personalized models. For example, most common herbicides are generic in action, which led to the development of genetically modified crops to add specificity. The ease and accessibility of modern genomic analysis, when wedded to accessible large chemical space, should facilitate the discovery of chemicals that are more selective in their utility. Is it possible to develop species-selective herbicides and growth regulators? More generally put, is plant research at a stage where chemicals can be developed that streamline plant development and growth to various environments? We believe the advent of chemical genomics now opens up these and other opportunities to “personalize” agriculture. Furthermore, chemical genomics does not necessarily require genetically tractable plant models, which in principle should allow quick translation to practical applications. For this to happen, however, will require collaboration between the Ag-biotech industry and academic labs for early stage research and development, a situation that has proven very fruitful for Big Pharma. Historically, the pharmaceutical industry has developed drug treatments that target the widest segment of the population. Although this business model has been very successful, there is a need to update this “one size fits all” approach to drug development. Genetic variability in the human population renders some individuals less responsive to certain therapies . More im1. Although 90% of CF patients have an in-frame deletion that results in the mislocalization of the CFTR gene product, a small fraction of CF patients (~5%) have a missense mutation G551D-CFTR that has correct CFTR localization but reduced chloride channel activity  gene have been identified in cystic fibrosis (CF) patientsactivity . Using tactivity . For exaactivity . Ivacaftactivity . During Table 1; Roundup Ready crops that have been engineered for resistance to glyphosate, the active ingredient in the herbicide Roundup , is slowly transported and quickly metabolized by maize . Given tArabidopsis have been identified that are resistant to the picolinate auxin picloram but not 2,4-D . From the perspective of plant breeding, genetic manipulation of growth regulators has been central to both horticulture and agriculture. Perhaps the best example involves the impressive yield increases of the green revolution of the 1960s (Figure 1; Aside from its role in herbicide discovery, the Ag-biotech industry also has a long history in the development of growth regulators that enhance useful plant attributes (he 1960s , driven he 1960s . In parahe 1960s . CompounTable A1 in Appendix; These and many other examples of chemical applications for horticultural or agronomic crop improvement were discovered anecdotally by testing known growth regulators on various plant species (Pseudomonas identified compounds that promote pathogen resistance in Arabidopsis by invoking the hypersensitive cell death pathway in response to pathogen attack (in planta (Plant hormones such as auxin, GA, and ABA continue to be excellent targets for the Ag-chemical industry, and have a long-standing history of being manipulated in plant biotechnology . Given tn attack . The inhn attack , whereasn planta . Whethern planta .Arabidopsis genome appears to be involved in metabolism, and close to 200,000 enzymes have been annotated across 17 species for which information is available2. The plant metabolome may represent an area of untapped potential through which chemical biology can facilitate the development of novel plant growth regulators.Though hormones present an obvious point to manipulate plant output through chemical biology, metabolites can also serve as signaling molecules that impact plant growth and development . By defiOnce a metabolite has been discovered to influence the development of an important plant trait, chemical screens can uncover modifiers of this response. For example, the presence of glutamate influences root system architecture by restricting primary root elongation and promoting the proliferation of lateral roots . ModifyiDirecting metabolic output through genetic engineering has been a goal of plant scientists for some time. The development of Golden Rice, engineered to synthesize β-carotene in its seeds , demonstDespite the difficulties bringing Golden Rice to the field, it has demonstrated that metabolic engineering can promote nutritional value in crops and raises questions about the ability to use chemicals analogously to genetic engineering in directing the metabolic output of plant. If enzymes make good drug targets then it should be possible to uncover chemicals that can direct metabolic flux by modulating biosynthetic pathways. Presumably, blocking metabolism at crucial time-points during plant development can promote the accumulation of specific metabolites that could have economic or nutritional value. Methods that enable quick assessment of metabolite abundance would facilitate screens in search of compounds that promote the accumulation of metabolites of interest. Though we are not necessarily advocating for increased application of chemicals to food products, we believe that targeted manipulation of plant metabolism through chemical biology does have the potential to promote nutritional value in crops and to enhance the accumulation of rare or expensive natural products in some species.Arabidopsis were then tested across agriculturally important species. Some characteristics of the model plant Arabidopsis, including its small size and rapid growth, make it an obvious choice as the subject of phenotype-based high-throughput chemical screening (Arabidopsis should be assayed in other plants to assess the utility of these leads in commercial applications. Focus on the development of compounds that modify traits in important species might encourage collaboration between the Ag-biotech sector and academic research groups, a relationship that has stimulated innovation in the pharmaceutical industry (Similarity between crop species is beneficial to plant researchers because treatments that are effective in one species are likely to be useful in a related species. In principal, this should facilitate translation from laboratory science to real-world applications. Despite this, there seems to be a paucity of published examples in which leads from high-throughput screens in creening . As sequindustry .In many ways, the Ag-chem industry is facing a similar situation to the pharmaceutical industry, in which exorbitant costs of drug development have become prohibitive. This has resulted in a stagnating supply of innovative new products coming through the research and development pipeline . The incThis relationship has allowed the burden of high-risk projects to be taken by the research institute, whose incentives and measures of success may differ from that of the corporate partner . An acadA similar strategy would benefit the Ag-biotech industry, in which academic chemical biology labs could make use of the available high-content screening platforms to develop new herbicides and agricultural chemicals. The prevalence of herbicide-resistant weeds, coupled with the increased abiotic stresses crippling agricultural output are putting pressure on the Ag-chem industry to develop innovative methods of crop protection that sidestep the need for genetic modification. Modern genomic analysis should enable researchers to quickly understand the mechanism of resistance, and scientists now have the tools available to develop tailored chemical treatments that target specific classes of weeds and other pests. Taking a lead from Big Pharma, the private sector and academic laboratories should collaborate to establish translational research programs that promote innovation and open new opportunities to sustain agricultural productivity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ability of B cells to produce high-affinity antibodies and to establish immunological memory in response to a wide range of pathogenic antigens is an essential part of the adaptive immune response. The initial step that triggers a humoral immune response involves the acquisition of antigens by B cells via their surface immunoglobulin, the B cell receptor (BCR). BCR-engaged antigens are transported into specialized lysosomal compartments where proteolysis and production of MHC class II-peptide complexes occur, a process referred to as antigen processing. Expression of MHC class II complexes at the B cell surface allows them to interact with T cells and to receive their help to become fully activated. In this review, we describe how B cells rely on conserved cell polarity mechanisms to coordinate local proteolytic secretion and mechanical forces at the B cell synapse enabling them to efficiently acquire and present extracellular antigens. We foresee that the mechanisms that dictate B cell activation can be used to tune B cell responses in the context of autoimmune diseases and cancer. B lymphocytes are tailored to mount antibody responses upon recognition of foreign antigens and display critical roles as antigen-presenting cells that can shape immune responses. The ability of B cells to achieve complete activation and become specialized antibody-secreting cells relies on their capacity to capture external antigens, process and present them as peptide fragments onto MHC class II molecules to CD4+ T cells . This inin vivo studies based on intra-vital microscopy revealed that larger antigens (with a molecular mass >70 kDa) such as viral aggregates, immune complexes, or antigen-coated particles, which have limited access to the follicle, are able to reach the B cell zone. These antigens are found tethered to the surface of presenting cells, such as macrophages  is rapidly recruited to the B cell synapse where internalized antigens are efficiently cleaved to generate MHC class II-peptide complexes that will be presented to CD4+ T cells. The ability of B cells to coordinate vesicle trafficking at the immune synapse relies on conserved polarity proteins and is critical for B cells to become fully competent as antigen-presenting cells.in vitro , directional cell migration to the maintenance of tissue integrity . Cell poin vitro . Importa in vivo ; whereas in vivo . The latallenges .Recently, the ancestral polarity protein Par3 was found to be enriched at the site of BCR–antigen interaction where it was proposed to act as a landmark of polarity used to guide the further recruitment of key molecules required to form a functional immune synapse . Indeed,Drosophila, Scrib was shown to regulate retromer-dependent sorting events required for cargo recycling to the cell surface, which is critical to control epithelial organization  regulate apical polarity . Conversnization . In T cenization . Moreovenization . Whethernization . Interesnization , where iWhether polarized membrane trafficking to the B cell synapse involves other intracellular compartments, such as the ER and mitochondria has not been addressed. Indeed, the recruitment of internal membranes could assist membrane spreading as well as provide accessory molecules that locally remodel cortical cytoskeleton and receptor endocytosis, thereby shaping the B cell synapse to acquire its signaling and antigen extraction functions.Does the polarized phenotype induced during B cell synapse formation have an impact on later stages of B cell activation? Early polarization not only guides antigen extraction and presentation but evidence suggests that it can also influence later events, such as migration toward the T cell zone, establishment of germinal centers, as well as asymmetric cell division. Indeed, asymmetric T cell division was shown to result from prolonged synaptic interactions between T cells and antigen-presenting cells. This process was guided by generating a segregation of ancestral polarity proteins, aPKCz and Scribble, giving rise to different T cell progeny with effector or memory fates . Asymmetin vitro; however, their role in mechanical extraction of antigens remains to be addressed. Nevertheless, given that silencing of polarity proteins in B cells impairs both B cell development and activation in vivo it has become apparent that they are essential for proper humoral responses. Therefore, identifying new proteins that bridge cell polarity to membrane trafficking and coordination of mechanical forces shall allow a better understanding on how B cells acquire their antigen-presenting functions. Elucidating these molecular pathways can also provide valuable therapeutical targets for immunomodulation, in particular, in the context of autoimmune diseases and cancer.The current view on how B cells acquire antigens has rapidly evolved from classical receptor-mediated endocytosis toward the formation of a dynamic platform, the immune synapse. This process involves the coordination of complex cellular pathways that regulate local secretion of proteases as well as mechanical forces used for extraction of extracellular antigens. Recent data have exposed a critical role for conserved polarity proteins in orchestrating cytoskeleton remodeling and membrane trafficking at the synaptic interface of B cells. These proteins were shown to regulate local protease secretion at the immune synapse 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.


This article reviews the different MRI techniques available for the diagnosis, treatment and monitoring of brain metastases with a focus on applying advanced MR techniques to practical clinical problems. Topics include conventional MRI sequences and contrast agents, functional MR imaging, diffusion weighted MR, MR spectroscopy and perfusion MR. The role of radiographic biomarkers is discussed as well as future directions such as molecular imaging and MR guided high frequency ultrasound. Brain metastases are the most common central nervous system tumours in adults with a rising incidence due to the increased availability and utilisation of brain imaging and prolonged survival from primary cancers -3. MRI iAccurately identifying the number, location and size of brain metastases is important to determine which interventions, if any, are appropriate for a patient. Multiple scoring systems used to predict prognosis take into account the number of lesions, for example the Recursive Partitioning Analysis or RPA classification -14. WithCould novel sequences and agents therefore increase detection without escalating the contrast dose? Different gadolinium based agents, all of which may have slightly differing relaxivity profiles have been compared and these studies are summarised elsewhere. At present gadobutrol appears to identify the greatest number of lesions with the greatest contrast to noise ratio whilst having a lower risk of nephrogenic systemic fibrosis . MR seqd agents ,24. Mored agents ,26. The d agents .A patient presenting with no known primary cancer and a solitary ring enhancing brain lesion may be suspected of having a brain metastasis, a primary cerebral tumour such as glioblastoma or a cerebral abscess, and despite careful history and exam, misdiagnoses may still occur . Distingsupported but not consistently repeated by studies examining DTI metrics such as fractional anisotropy which may be superior to ADC alone [Distinguishing metastases from high grade glioma has proven more difficult using DWI although in theory the region of vasogenic oedema around metastases should show greater free diffusion than the more cellular, infiltrated region around a high grade glioma. Studies have varied in their cutoff for distinguishing the two pathologies as well as the methodology of how to take the reading  and there is no agreement about reliability -43. DiffDC alone -47.31P MRS[Further advanced techniques have therefore been combined with diffusion imaging in this context to try and increase sensitivity, most commonly MR spectroscopy . Single 31P MRS.Further information on physiological activity can be gathered by combining with MR perfusion studies. MRI perfusion permits the generation of maps of relative cerebral blood flow (rCBF) and volume (rCBV) which are measures of vascularity. High grade glioma pathologically shows neovascularisation and infiltration of surrounding brain, hence the peritumoral rCBV is higher than for a metastasis ,56. Usinregardless in order to move forward treatment. The role and evidence for MRI in surgical planning is therefore the next area to consider.In summary, multimodal MR used singly or in combination has improved our ability to distinguish metastases from primary cerebral tumours or abscesses. Despite the application of these advanced MR techniques of diffusion, perfusion and spectroscopy to solve this clinical problem  it remaiImage guided surgery allows the surgeon to plan the safest route to the tumour preoperatively and to maximise safe resection intraoperatively. Conventional post contrast T1 weighted images with thin slice protocols are generally acquired for image guidance software registration and the increased spatial resolution of such sequences compared to the usual diagnostic scans  has been highlighted in the discussion of detection. Diffusion imaging allows images of white matter tracts and their relationship to tumour to be delineated. DTI acquisitions, in addition to generating quantitative parameters, can permit tractography by estimating the directionality of fibre tracts, permitting the tracking of fibre bundles in 3D space. The methodology for this post processing varies and a number of non-proprietary software solutions in addition to those supplied by manufacturers are available. Further refinements to the methodology, including use of higher order algorithms to resolve ambiguous directionality on voxels may further increase reliability . FunctioIn cases of multiple metastases or solitary lesions with no known primary, MRI may give useful diagnostic clues as to the original tumour. Some primaries may have particular signal characteristics even on conventional MR. For example melanoma metastases may show high signal on non-enhanced T1 sequences due to the effect of melanin and mucinous metastases may show low T2 signal compared with the expected hyper-intensity on this sequence. The metabolic profile has been investigated for metastases of differing primary tumours as well as the surrounding brain with no abnormal spectra reported outside of the lesion itself. Metabolic features on MRS have shown limited value in predicting primary type. However, raised mobile lipid content has been proposed as a weak sign of a colonic origin for metastasis .Others hIn summary, MRS may be used to distinguish primary tumour origin. However more than one advanced MR metric may need to be combined in order to robustly generate models that differentiate the primary lesion and though these studies would likely be retrospective, ideally some image guided correlation of regions of interest on the advanced MR with the final tissue samples is needed, as has been performed for glioma .Beyond diagnosis, MRI may be used to monitor response to treatment as part of clinical and radiological follow up. This may be immediate, as in post-operative MRI to determine if there has been a complete resection or delayed, i.e. has the metastasis responded chemo- or radio-therapy. In general, conventional sequences are utilized but one area of particular clinical interest in following cases up is ongoing or increasing enhancement following surgery or stereotactic radiosurgery to a metastasis and whether this represents radio-necrosis or recurrence . StandarAs described in many studies here, various metrics or measurements may be taken from the different advanced MR images described using the device workstation or non-proprietary software. A biological marker or “biomarker” is strictly any such reading which is used for diagnostic or prognostic purposes. Even conventional MR may provide such markers and it has been shown that for solitary metastases in one series, the degree of oedema on the preoperative T2 weighted scan was related to the degree of angiogenesis, brain invasiveness and overall survival with reduced oedema surprisingly being a worse prognostic factor . The rolAs cancer diagnostics and therapeutics become increasingly related three areas of emerging MR technology with practical applications to brain metastases stand out. Integration of cancer staging via PET-MR offers one potential means of incorporating further functional data in real time without losing all the knowledge already acquired about the behaviour and characteristics of metastases on MRI . Novel cpractical application of advanced MRI techniques including diffusion weighted imaging, tractography, perfusion studies, functional MRI and MR spectroscopy in common clinical scenarios relating to brain metastases. Previously the literature has predominantly focused on applying these modalities to intrinsic brain tumours such as glioma but there appears to be an increasing recognition of the burden of metastatic brain disease and the need for novel applications of MR technology to solve the sort of practical clinical problems described and provide more prognostic information. It may be that this occurs by development of further conventional sequences and better contrast media, including at a molecular level, or by combination of the existing possibilities to generate multimodal datasets. Evidence of practical, clinical utility specific to metastases needs to be gathered at each stage to justify the development of - and determine the best use of - these expensive resources.This article has summarised the current evidence for the MRI: Magnetic resonance imaging; ADC: Apparent diffusion coefficient; ROI: Region of interest; MRS: Magnetic resonance spectroscopy; DWI: Diffusion weighted imaging; FLAIR: Fluid attenuated inversion recovery; rCBV: Relative cerebral blood volume; CBF: Cerebral blood flow; fMRI: Functional magnetic resonance imaging; RPA: Recursive partitioning analysis; GPA: Graded prognostic assessment; SRS: Stereotactic radiosurgery; DTI: Diffusion tensor imaging; DSC: Dynamic susceptibility contrast.The authors declare that they have no competing interests.RZ drafted the article with assistance from KD, MB and MR. MR and MB assisted with finding figures. CW, MDJ, MR significantly edited the article. All authors read and approved the final manuscript.


Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL) plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL) and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression. Atherosclerosis is characterized biochemically by the accumulation of excess cholesterol in the artery wall. This process is initiated by migration of low density lipoproteins (LDL) and other apolipoprotein B (apoB)-containing lipoproteins across an injured artery endothelium, where they are retained through a charge-charge interaction with matrix proteoglycans in the subendothelial space . The most common mutation seen in CESD patients is a splice junction mutation at exon 8 of LIPA, which leads to ~3–5% of normally spliced LAL protein and LAL activity ; the TFEB nuclear localization was reduced at 24 h of oxLDL incubation, however, indicating this response of LIPA is likely mild and transient  is upregulated in response to nutrient restriction Sandri, , and hasLIPA expression increases with liver-X-receptor (LXR) and peroxisome proliferator-activated receptor (PPAR) agonists. LIPA is not known to contain an LXR or PPAR response element in its promoter region. Bowden et al. , which interacts with PPARγ, is a transcriptional target of TFEB  cleavage activating protein (SCAP) in the endoplasmic reticulum, and downregulation of SREBP-dependent new cholesterol synthesis and LDL receptor expression by the cell  proton pumping activity in the lysosomal membrane, without a change in the amount of v-ATPase protein. The resultant increase in pH within the lysosome corresponded with a time-dependent inhibition of CE hydrolysis and accumulation of apoB, indicating a general decline in lysosomal hydrolytic activity. After 7 days of incubation with mildly oxLDL or aggLDL, the majority of lysosomal vesicles within THP-1 macrophages had pH >4.8. Vacuolar ATPase activity could be partially recovered if isolated lysosomes were treated with cyclodextrins in order to remove membrane cholesterol. As LAL is only active at acidic pH, with maximal activity ~pH 4 and very little activity above pH 4.5  stress caused by an excess of FC in the ER membrane. Garver et al.  and JeliAs the rate of release of FC from the lysosome is a regulator of ABCA1 expression, lysosomal dysfunction in atherosclerosis may also lead to decreased expression of ABCA1 in artery wall cells. Choi et al.  have rep−/− mice was able to reverse the pathogenic storage of lipids in multiple tissues. Du et al. (Recombinant human LAL (rhLAL) is currently in phase 3 clinical trials to treat LAL deficiency in Wolman disease and CESD. This treatment is potentially life-saving in Wolman disease. Phase 2 clinical trials in patients with CESD using rhLAL demonstrated safety and improvements in serum lipid profiles  gene has been identified as a susceptibility gene for coronary artery disease by several genome-wide association studies (Consortium, Although rhLAL treatment seems to be a viable therapeutic strategy for premature atherosclerosis in CESD patients, it is unclear what the effects would be of rhLAL treatment in patients having atherosclerosis unrelated to a deficiency in LAL. The lysosomal acid lipase A (ex vivo using acidic pH adjustment, they are representative of potentially functional LAL present in vivo but not necessarily actual LAL activity. Therefore, although the amount of artery wall LAL may increase in response to increased cellular influx of apoB-100 containing lipoproteins, the hydrolytic activity of LAL may decrease over time as lysosomal function is impaired. Sequestration of cholesterol in the lysosomal membrane has been implicated as a cause of lysosomal dysfunction, specifically through the inhibition of v-ATPase proton pumping. Increasing lysosomal pH reduces the activity of LAL. The result of this is a time dependent switch of lysosomal accumulation of FC to CE. Early stage atherosclerosis may involve normal and perhaps increased LAL activity leading to cytosolic lipid droplet accumulation, whereas later stages of atherosclerosis may have an acquired dysfunction in LAL hydrolytic activity leading to lysosomal lipid sequestration. Therefore, later stages of atherosclerosis, where LAL function is inhibited, may be considered to be morphologically similar to Wolman disease and CESD, albeit for an entirely different reason.The role of LAL in the progression of atherosclerosis is complex. Modified forms of LDL such as oxLDL and aggLDL lead to lysosomal accumulations of first FC and later CE, indicating an acquired loss of LAL hydrolytic activity. In agreement with this observation, tissue studies have indicated increased lysosomal lipid accumulations in addition to cytosolic lipid droplets at later stages of atherosclerosis. Activity assays of atherosclerotic tissue homogenates have indicated an increase in LAL and also other lysosomal enzymes. As these studies are conducted in vivo strategy. Further research is necessary to know whether manipulation of arterial cell LAL activity is likely to alleviate or aggravate the consequences of cholesterol accumulation in the artery wall.Outside of alleviating premature onset of atherosclerosis associated with genetic deficiency in LAL, it is hard to predict whether augmentation of LAL is a good therapeutic strategy. There are indications that selective upregulation of the autophagic-lysosomal pathway may be able to recover lysosomal function and LAL hydrolysis. It is unclear, however, whether induction of this pathway beyond normal response is a viable 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.


Although malaria has been the leading cause of fever for many years, with improved control regimes malaria transmission, morbidity and mortality have decreased. Recent studies have increasingly demonstrated the importance of non-malaria fevers, which have significantly improved our understanding of etiologies of febrile illnesses. A number of non-malaria febrile illnesses including Rift Valley Fever, dengue fever, Chikungunya virus infection, leptospirosis, tick-borne relapsing fever and Q-fever have been reported in Tanzania. This study aimed at assessing the awareness of communities and practices of health workers on non-malaria febrile illnesses.Twelve focus group discussions with members of communities and 14 in-depth interviews with health workers were conducted in Kilosa district, Tanzania. Transcripts were coded into different groups using MaxQDA software and analyzed through thematic content analysis.The study revealed that the awareness of the study participants on non-malaria febrile illnesses was low and many community members believed that most instances of fever are due to malaria. In addition, the majority had inappropriate beliefs about the possible causes of fever. In most cases, non-malaria febrile illnesses were considered following a negative Malaria Rapid Diagnostic Test (mRDT) result or persistent fevers after completion of anti-malaria dosage. Therefore, in the absence of mRDTs, there is over diagnosis of malaria and under diagnosis of non-malaria illnesses. Shortages of diagnostic facilities for febrile illnesses including mRDTs were repeatedly reported as a major barrier to proper diagnosis and treatment of febrile patients.Our results emphasize the need for creating community awareness on other causes of fever apart from malaria. Based on our study, appropriate treatment of febrile patients will require inputs geared towards strengthening of diagnostic facilities, drugs availability and optimal staffing of health facilities. Understanding the awareness of the community on non-malaria febrile illnesses is crucial, especially during the recent decline of malaria episodes of malaria. This study conducted focus group discussions with communities to assess their awareness of non-malaria febrile illnesses. In addition, in-depth interviews with health workers were conducted to explore their views and practices related to diagnosis and management of these illnesses. We identified that the awareness of the study participants on non-malaria febrile illnesses was low and the majority believed that most instances of fever are due to malaria. Moreover, the participants could not mention the right causes of fever and many had inappropriate beliefs about possible causes of fever. Health workers from our study looked for non-malaria febrile illnesses when a febrile patient had negative mRDT result or there was persistence of fever following completion of anti-malarial dosage. Shortages of diagnostic facilities were identified as one of the impediments to proper diagnosis of febrile illnesses. These findings indicate the need for creation of public awareness on causes of fever other than malaria. We recommend appropriate measures be taken by the government and other stake holders to improve health care services delivery particularly at primary health care facilities. Febrile illnesses due to different etiological agents are the common causes of morbidity and mortality in developing countries The diagnosis of non-malaria febrile illnesses poses a challenge since many of these illnesses may have similar symptoms with malaria and thus making their clinical diagnosis difficult Following a long tradition of regarding malaria as the leading cause of fever, it is important for the community to understand the other causes of fever apart from malaria particularly during this period when the episodes of malaria related fevers are reported to decrease The study was conducted in Kilosa district which is one of the six districts in Morogoro region, located in eastern Tanzania. The district borders with Tanga and Manyara regions to the north and Mvomero district and Mikumi National Park to the east. On the western border are Dodoma and Iringa regions whereas to the south it borders with Kilombero district. The district lies between latitudes 6° south and 8° south and longitudes 36°30′ east and 38° west. The area has semi humid climate with an average rainfall of 800 mm annually. The early rains start in November and end in January followed by heavy rainfall between March and May. The district experiences a long dry season from June to October and the average annual temperature is 24.6°C. The district has an area of 14,245 square kilometers and has a population of 438,175 people Kilosa was selected due to its possession of intensive human activities with livestock as well as its proximity to wildlife from the Mikumi National Park , what waThis study was specifically conducted in 6 divisions, namely Kimamba, Kilosa town, Magole, Masanze, Rudewa and Ulaya. Within these divisions, 12 wards namely Dumila, Chanzuru, Magomeni, Kilosa, Msowero, Zombo, Ulaya, Kimamba, Mkwatani, Kasiki, Masanze and Rudewa were purposively selected based on (i) geographical representation within the district e.g. Zombo is in the south western part of the district, whereas Dumila ward is in north-eastern part , (ii) thWe conducted a cross-sectional study in which qualitative data collection methods were used. Focus group discussions (FGDs) with members of the communities were conducted to assess their knowledge, attitude and perception of community members on non-malarial febrile illnesses. In-depth interviews (IDIs) with health workers were conducted in order to obtain their views about practices related to diagnosis and management of non-malarial febrile illnesses.Parents, guardians or caregivers aged between 18–59 years for children of less than 10 years were eligible to participate in FGDs. This group of participants was targeted because febrile illnesses have been shown to be common in children and contribute to high proportion of hospital admissions globally, with significant morbidity and mortality For IDIs, only health workers who were on duty and attended patients (prescribers) in the health facilities during the study period were eligible to participate into the study. Health workers from 12 health facilities located in Dumila, Chanzuru, Ulaya, Zombo, Kilosa, Magomeni, Msowero, Kimamba, and Mkwatani wards were interviewed. Two health workers were interviewed from each health facility and only one health worker was interviewed at a time. In-depth interviews with health workers on average lasted for one hour and 2 IDIs were conducted per day.Focus group discussions and IDIs were conducted by a skilled and experienced social scientist who was assisted by an observer and note taker. All discussions and interviews were conducted based on a prepared semi-structured interview guide that consisted of questions corresponding to the research topic. To ensure accuracy of the information, the data collection tool was translated from English to Kiswahili and then back-translated. The interview guide for FGDs consisted of questions about their knowledge on non-malaria febrile illnesses, health care seeking behaviors and their recommendations on non-malaria febrile illnesses . Health FGDs and interviews were transcribed verbatim and translated from Swahili to English. Thereafter, the transcripts were converted into rich text format and imported into MaxQDA, a software for qualitative data analysis Ethical approval was obtained from Institutional Review Board of Ifakara Health Institute (IHI/IRB/No: 01-2013) and Medical Research Coordinating Committee of Tanzania's National Institute for Medical Research (NIMR/HQ/R.8a/Vol.1X/1472). A written informed consent was obtained from each respondent and participant prior to IDIs or FGDs. To protect identification of the respondents and FGD participants, all personal information that could identify the study participants were only used during the analysis and omitted from the final reports. The participants were assured of anonymity in the presentation and publishing of the data.In total, 93 participants were interviewed during FGDs, of which 56 were women and 37 were men. Most of the study participants 87 (93.5%) were subsistence farmers and the majority 73(78.5%) had primary level of education. The demographic characteristics of the study population are shown in Participants' understanding of fever.Awareness of the community on non-malaria febrile illnesses.Treatment seeking behaviors for febrile illnesses.Health workers' practices towards non malaria febrile illnesses.Capacity for diagnosis and management of febrile illnesses in health facilities.The study participants were asked to explain what they know about the term “fever” (“homa” in Swahili language). There were different levels of understanding among the participants. The responses provided by majority of the participants did not associate fever with high body temperature. They described fever as an illness condition such as malaria, colic, rheumatism and sleeping sickness or associated it with symptoms such as headache, coughing, rashes and body pain. Others reported that they did not know the exact meaning of the term fever. There were few participants who described fever as a raise of body temperature (hot body).“I personally, don't know what fever is.”(FGD-women)“Cold all over the body, severe headache, joint pain; to me, that is fever.”(FGD-women)When the participants were asked to mention the causes of fever in children, things such as change of weather  and sunlight were listed by many participants. The participants believed that exposure to a cold environment or prolonged stay under the sun by itself can lead to fever. Only a few participants mentioned the right cause of fever which included illnesses like measles, tuberculosis (TB), typhoid fever and UTIs and other participants associated fever with symptoms/clinical signs such as flu, coughing and diarrhea.“The change of weather causes fever, such as when it's cold, windy or sunny.” (FGD-men)“I don't know whether fever is caused by heat or sunlight. So often you may offer drugs to your children and they could finish their dosages, but the fever does not disappear.” (FGD-women)“TB and flu are the main causes of fever.” (FGD women)meno ya plastiki in Swahili language), breastfeeding after long-term sunlight exposure of the mother as well as cessation of pulsation in the fontanel were mentioned by some participants.Moreover, inappropriate beliefs were perceived as causes of fever by the participants. For example, the presence of false teeth “When there is cessation of pulsation in the fontanel, it causes pain and this will definitely lead to fever.” (FGD-women)When the FGD participants were asked to mention the causes of fever other than malaria, several of them listed diseases or symptoms which were neither associated with fever nor non-malaria febrile illnesses. The commonly reported diseases/symptoms were headache, colic, hernia and abdominal pain. However, some participants admitted to be unaware of such illnesses. Only a small number of the participants mentioned the correct non-malaria febrile illnesses such as typhoid fever, UTIs (dirty urine), pneumonia, measles and tuberculosis (TB) and sleeping sickness. Both men and women participants had similar level of knowledge, but participants older than 30 years were more knowledgeable than those who were younger. Moreover, the participants from FGDs conducted in rural areas had limited knowledge in comparison with participants from urban and semi-urban areas.“A headache can cause fever.” (FGD-women)“Frankly we don't know, unless you explain to us.” (FGD-women)“Hernia.” (FGD-men),“Others include colic, this can cause fever in a child.”(FGD-women)It was also revealed that despite the decrease of malaria, the participants believed that most instances of fever were due to malaria. This was noted when several participants mentioned only malaria as the cause of fever. The participants explained that when their children get fever, they mostly associated it with malaria. This was also acknowledged when the participants were asked to describe the meaning of the term fever (theme 1). A considerable number of the participants explained fever is malaria:“To be honest, I don't know other diseases. We are aware of malaria. When a child gets sick, we know that it is malaria.”(FGD-women)“As we said earlier, we don't know other causes of fever apart from malaria, I don't know.” (FGD-women)During the interviews, health workers were asked to give their views about the knowledge of the community members on non-malaria febrile illnesses. All health workers reported that majority of community members were not aware or had little knowledge on these illnesses. Health workers explained that fevers were perceived to be caused by malaria by several members of communities. This situation was reported to be a challenge to health workers particularly when they want to obtain a comprehensive history of illness from patients since patients explain to health workers that they are suffering from malaria. Health workers identified this wrong perception as an impediment to proper management of patients and emphasized the need for change of attitude.“In short they are not aware of other causes of fever. What they know is that fever is synonymous with malaria. They believe that fever is caused by malaria alone.” “Parents don't have knowledge on non-malaria fevers. What they think is that, all children with fever have malaria, mRDT results may be negative, but their parents will not believe that they had no malaria.”“They don't have sufficient knowledge because once a person is ill or has fever, they think of malaria. People are still ignorant.” (IDI-registered nurse)In our study the most common reason for unawareness of the community on non-malaria febrile illnesses reported by the majority of health workers was lack of health education. Health workers pointed out that health education is offered at health facilities but it has not reached a wider community. Several community members live in remote villages and they rarely visit health centres for health care. Health workers emphasized that health education on diseases associated with non malaria fevers will help to create awareness to members of the community.“Health education doesn't reach the majority of people since it is only offered here at the health centre, and others live in remote villages.” (IDI-registered nurse)Findings from the FGDs revealed that community members from the study population sought treatment from both the health facilities and traditional healers. When queried on their health seeking behavior, the majority of the study participants reported sending their febrile children to the nearby health facility. However, when asked for any alternative treatment, almost half of the participants reported to seek treatment from the traditional healers. Therefore, this indicates that health facilities and traditional healers were both utilized by the participants. Even though several participants reported seeking treatment from health facilities, but interviewed health workers explained that community members rarely visit health facilities. They said the majority of community members live in remote areas and thus long distances pose as an impediment for visiting health facilities. In addition, health workers stated that the habit of seeking treatment from traditional healers was practiced by some members of the community. They further pointed out that sometimes children were sent to health facilities when they were terminally ill. Likewise, some parents/guardians admitted to health workers that delays to send their children to health facilities was due to prior consultations made from traditional healers.“It is not true that all go to the health facility as my colleague has put it. Some don't have even the idea of going to a dispensary, what comes into their mind is a traditional healer.”(FGD-women)‘Some bring them when they are in a critical situation as they believe in superstition, so they move around with their patients and sometimes they lose them. Sometimes they come to hospital after they have visited traditional healers but found that their children's illness has become worse.”“This ward has many people who live in the remote areas. Once they get ill, they first consult traditional healers since they believe that they have been bewitched. You can just hear “do you know somebody's baby? he is dead!………,he got ill and he was sent to a traditional healer, and when they thought of bringing him to the health facility, he passed away.”Our findings have also shown that seeking treatment from the traditional healers was sought when there was a persistent fever following treatment from health facilities. If children still had fever after completion of anti-malarial dosage, majority of the participants opted to consult traditional healers for further treatment. Only a few mentioned taking their children back to the health facilities.“As we said earlier, in this village, they believe in superstitions. They would say ‘I took my child to the hospital but the fever is still recurring’, then they would think of going to a soothsayer to ascertain the problem.”(FGD-women)“This happens when a child has gone to the hospital for seeking treatment. Having got treatment, they come back home, but if the fever recurs, they think of going to the traditional healers.”(FGD-women)This study found that self-medication was commonly practiced by several participants. Many participants reported purchasing drugs from pharmacy/drug shops without prior medical prescription. They explained that they prefer using anti-malarial drugs for the treatment of fever in children. The practice of self-medication was reported by FGD participants from wards located near the health facilities as well as wards which were far off from health facilities. During the interviews with health workers, all of them acknowledged that self-medication was commonly practiced by members of the communities. Heath workers further pointed out that some members of the communities would still visit health facilities if they had found no improvement following self-medication. Health workers explained that the habit of self-medication delays provision of prompt and proper treatment and in most cases results into death. The common reported reasons which influenced many members of the community to opt for self-medication were poor health services from health facilities, shortages of drugs, lack of diagnostic facilities, long distance to the nearby health facility and inability to afford health care charges.“It is a long way from the health centre to the patient's residence There are long queues and poor health services because of the shortages of medical staff, therefore the option is to buy Sulphadoxine-pyrimethamine (SP) drug, and if the drug could not help the child, that's when we decide going to the health facility.” (FGD-women)“If the hospital is long distant, you simply buy medicines for the children. But if the situation gets worse, you send them to the health facility.”(FGD-men)“A few individuals bring their children here when they are ill but most parents buy drugs from a pharmacy. There are shortages of drugs at the facility and hence they think there is no alternative way than buying drugs from the pharmacy without a laboratory test.”(IDI-registered nurse)In our study, we found that the diagnosis of febrile patients was mostly done by mRDTs or clinical symptoms/signs as presented by patients with the assistance of Integrated Management of Childhood Illness guidelines. Interviewed health workers explained that mRDTs were used to distinguish malaria from non-malaria fevers and the majority (10/14) prescribed anti-malarial drugs only to patients with mRDT positive result. When patients had negative mRDT result, health workers reported looking for other causes of fever based on clinical signs and history of the fever. Only a few health workers (4/14) stated initiating anti-malarial drugs even if patients had negative mRDT, as one health worker said:“…..for instance, we get many patients whom we think do have malaria, but after applying mRDT, the results are different. However, we also offer anti-malarial drugs to those with negative mRDT results and it helps them.” Moreover, when mRDTs were not available, majority of health workers relied only on clinical manifestations of the patient. When asked to describe symptoms or signs which guide them to make a conclusive diagnosis of febrile illnesses such as malaria, typhoid fever or urinary tract infections, most health workers (12/14) mentioned presence of fever, vomiting, headache, loss of appetite and diarrhea as typical symptoms of malaria. With regards to UTIs, pain during urination was mentioned by majority of health workers as a definitive symptom whereas fewer health workers (2/14) considered urine coloration (from yellowish to milky colour) and small urine volume as symptoms of UTIs. For typhoid fever, symptoms such as abdominal pain and diarrhoea were commonly listed by the majority of health workers.“I check symptoms such as fever, or if the parent has told me ‘his child is vomiting’, or has loss of appetite, oh I see! this is definitely malaria and because of these symptoms I initiate anti-malarial treatment. However, even though there is no urine test we think of urinary tract infection as the cause of fever, therefore, we offer anti-malarial drugs and septrin syrup, and this helps!.”(IDI registered nurse)It was also revealed that when mRDTs were available many febrile patients tested negative and hence other causes of fever were very likely to be considered. However, in the absence of mRDTs, health workers said several febrile patients were suspected to have malaria and were treated with anti-malarial drugs. They considered that reliance on clinical signs and symptoms only, is prone to lead to misdiagnosis and over-prescription of anti-malarial drugs.“Since we got the mRDT last year, I have never found more than eight patients with malaria in a day. However, when MRDTs are out of stock, we prescribe clinical malaria and cure them. Since we started using mRDT, malaria has really decreased though fever is still a big problem. Before mRDT was introduced to us, the situation was even worse.” Opinions of health workers towards management of persistent fevers following completion of anti-malarial dosage were quite divergent. While majority of health workers (10/14) reported opting for symptomatic diagnosis of non-malaria febrile illnesses and appropriate prescription of antibiotics following failed malaria treatment, however fewer health workers (4/14) reported to switch from first line anti-malarials (artemisinin-based combination therapy) to second line anti-malarial treatment (quinine).“If they come back with a persistent fever, we think they could not get cured, so we start the second line therapy such as quinine since initially we gave them Artmether Lumefantrine (ALU).” Proper management of non-malaria febrile illnesses is largely dependent on the capacity of the health facility to perform accurate diagnosis and treatment of these illnesses. Health workers in this study repeatedly reported lack of diagnostic facilities, shortage of trained health workers, and stock-out of medications as major barriers to proper management of non-malaria febrile illnesses. Our study revealed that from the 12 health facilities, only 2 had diagnostic facilities for a few febrile illnesses; the dispensary had Widal test for detection of typhoid and a microscope used in diagnosis of UTIs and mRDTs, while the health center only had a microscope. The remaining health facilities (10/12) had no diagnostic tests except mRDTs in few health facilities. Health workers explained that they experienced challenges in managing febrile patients without tools for laboratory investigation. Even though mRDT was named as the only diagnostic test which was used to rule out malaria from febrile patients, stock-out of mRDTs was mentioned as an ongoing problem. Health workers said that the supply of mRDTs from the government to the health facilities was normally done on a quarterly basis. However, all health workers repeatedly reported receiving inadequate mRDTs and there were frequent delays in supply of mRDTs. It was clearly stated by health workers that in most cases half way through a quarter, they experienced lack of mRDTs. Among 12 health facilities, only 4 had mRDTs available at the time of our visit. Health workers insisted need for diagnostic tools for malaria and non-malaria febrile illnesses.“We don't have a laboratory; so we are trying to exclude other diseases based on symptoms and physical examination. However, knowing that, this is urinary tract infection (UTI) or other infections that can cause fever is difficult; so you simply treat symptomatically.” “The first thing we need is a laboratory so that we can know the exact problem and control the misuse of anti-malarial drugs. Also, as I put it earlier, we don't have mRDTs, we simply offer anti-malarials and antibiotics, which is improper use of medicines.” During the interview with health workers, stock-out of medication was mentioned as a common problem. Drug shortages were reported in most (9/12) health facilities although a few (3/12) had a few boxes of basic drugs such as ALU, antibiotics  and pain killer (paracetamol).“Shortage of drugs is also a problem. You can place an order to the medical store department but the drugs will not be delivered in time and when delivered, they are usually are insufficient.”With regards to staffing, our study revealed significant shortage of health workers particularly in health facilities located in rural areas. Among visited health facilities, four  had more than two health workers at the level of clinical officers. The remaining eight health facilities each had only one clinical officer assisted by a nurse/midwife or medical attendant. Some of the interviewed health workers  explained that although they prescribe drugs to patients, they had inadequate skills to manage febrile patients. Moreover, health workers reported to work beyond normal working hours and thus attend more patients beyond the standard average number of patients per physician.“We have challenge of lack of manpower. We can say, members of staff are there, but in numbers, we are very few. You can be attending a child but a long queue of patients is still waiting for you. Sometimes you can be attending children in their ward; meanwhile other patients are waiting for you at the clinic.” (IDI-registered nurse)“I am alone, but I normally teach my colleagues how to manage patients in my absence. I'm offering training to a nurse midwife and medical attendant. The nurse midwife is able to manage patients but the medical attendant cannot. We do so because of the real situation as you can see we don't have enough health workers, but this doesn't mean that the medical attendant can perform it accurately. The midwife at least can perform better though not at a hundred percent.” In this study, we report that the awareness of the community on non-malaria febrile illnesses is low. Most of the study participants had little knowledge on non-malaria febrile illnesses and they considered most instances of fever to be synonymous with malaria. Some FGD participants had inappropriate conceptions about the causes of fever. Although the participants reported seeking treatment from health facilities, visiting traditional healers and self-medication were also practiced. Interviewed health workers reported using mRDTs to distinguish non-malaria causes of fever from malaria. However, mRDT negative patients were diagnosed based on symptoms and clinical signs of the patient. In the absence of mRDTs health workers reported giving anti-malarial drugs to many febrile patients as compared to when mRDTs were available. The lack of diagnostic facilities for non-malarial febrile illnesses, stock-out of mRDTs and shortages of drugs were repeatedly reported by all health workers. Therefore, this study revealed inadequate capacity of health facilities to manage febrile illnesses particularly at primary health facilities .Fever is a common medical problem in children which may prompt parents to seek medical care. Our results have shown that some participants could not explain the meaning of the term fever and the majority described fever as an illness condition or associated it with symptoms/signs of the disease. In addition, other participants mentioned change of weather and exposure to sunlight as causes of fever in children. The present findings are consistent with the study from Uganda where the study participants used the term fever to describe an illness condition We have indicated minimal awareness of community members on non-malaria febrile illnesses. However, a few participants particularly parents aged more than 30 years were more knowledgeable about non-malaria febrile illnesses as compared to younger parents. Likewise, all interviewed health workers admitted that the communities lack knowledge or had little knowledge on these illnesses. This finding is consistent with results from other studies in Northwestern Ethiopia and Zimbabwe which reported very low awareness of the community regarding non-malaria febrile illnesses With regards to treatment seeking for febrile illnesses, although some FGD participants reported seeking treatment primarily from health facilities, however, other participants sought treatment from traditional healers. These findings however agree with previous studies in Tanzania where a large percentage of members of the community sought treatment from health care facilities and only a few opted for traditional health care Self-medication without medical prescription was found to be a common practice among many FGD participants. Self-medication with anti-malarial drugs has widely been practiced in several countries including Tanzania The current study has shown that health workers used mRDTs to distinguish non-malaria causes of fever from malaria. However, for the diagnosis of mRDT negative children, health workers reported to rely on symptoms and clinical signs to distinguish non-malaria febrile illnesses. In Tanzania, there has been a roll out of mRDT that was introduced at all levels of health care for parasitological confirmation of malaria prior to treatment The proper management of non-malarial febrile illnesses depends highly on the availability of diagnostic facilities, professional health workers, medications, transport and communication infrastructures. However, complaints of lack of diagnostic facilities, shortage of health workers, and stock-out of medications were raised by majority of health workers. We found that most health facilities had no diagnostic tests for febrile illnesses. Some non-malaria febrile illnesses are potentially life-threatening and hence failure to diagnose and treat them can result into prolonged and worsening illness with repeated visits to health facilities FGD participants were selected from different divisions, wards and hamlets, hence they were from diverse demographic backgrounds and their views were a representation of the general population in the district. Interviewed health workers were selected from different health care levels, dispensary to hospital levels. This was purposively done to grasp a wide scope of attitudes and practices by health care staff from the few health facilities which were visited. During focused group discussion with communities more discussions were done with women as compared to men, but this was purposively done because women are responsible for child care in the family hence their views were expected to give a balanced representation of the household health. However, the results of this study showed that both men and women had similar level of knowledge when it comes to perceptions of non-malaria febrile illnesses. It is also possible that some information was lost during the translation of the transcripts before analysis.This study has demonstrated that the awareness and level of knowledge of communities on non-malaria febrile illnesses was low. Knowledge from this and other similar studies will provide insights into better and practicable methods for improving the management of febrile patients. The wrong perception among communities, whereas fever is understood as being synonymous with malaria, as encountered in this study pose a challenge to the health sector and thus we emphasizes the need of creating public awareness regarding causes of fever other than malaria. Community misconceptions on fever and its causes must be addressed since such beliefs often distract or delay treatment seeking from health care facilities. It is also crucial that relevant authorities intervene against existing habits of self-medication and seeking treatment from traditional healers. Appropriate treatment of febrile patients will require inputs geared towards strengthening of diagnostic facilities, drugs availability and optimal staffing of health facilities. Therefore, it is advisable that the government and other stakeholders should take appropriate measures to improve health care services delivery.Checklist S1STROBE checklist.(DOC)Click here for additional data file.Supporting Information S1Topic guide for interviews and discussions.(DOC)Click here for additional data file.