Traumatic brain injury (TBI) is the leading cause of death and disability worldwide and has complicated underlying pathophysiology. Numerous TBI animal models have been developed over the past decade to effectively mimic the human TBI pathophysiology. These models are of mostly mammalian origin including rodents and non‐human primates. However, the mammalian models demanded higher costs and have lower throughput often limiting the progress in TBI research. Thus, this systematic review aims to discuss the potential benefits of non‐mammalian TBI models in terms of their face validity in resembling human TBI. Three databases were searched as follows: PubMed, Scopus, and Embase, for original articles relating to non‐mammalian TBI models, published between January 2010 and December 2019. A total of 29 articles were selected based on PRISMA model for critical appraisal. Zebrafish, both larvae and adult, was found to be the most utilized non‐mammalian TBI model in the current literature, followed by the fruit fly and roundworm. In conclusion, non‐mammalian TBI models have advantages over mammalian models especially for rapid, cost‐effective, and reproducible screening of effective treatment strategies and provide an opportunity to expedite the advancement of TBI research. Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (roundworm). Criteria that were discussed were mainly on type of animal model, the functional findings, pathophysiological outcomes, and the limitation of the respective models. In conclusion, this non‐mammalian animal model provides more simplistic approach to bridge the knowledge gaps within TBI research and may shorten the preclinical process for the final findings on the outcome prevention and cure.Traumatic brain injury (TBI) perpetuates lifelong and dynamic effects on health and well‐being. Most of the animal models previously developed to study the TBI pathophysiology were focused mainly on mammalian animal model. Despite of all the contributions that they offered, these mammalian animal models have some drawback such as huge sample size needed and very costly which resulted in lengthy process of preclinical development phase with hindered reproducibility option. Hence, this systematic review gathered all the previous non‐mammalian animal model used in TBI research from January 2010 to December 2019 via 3 search engines which were Embase, Scopus, and PubMed. PRISMA method was chosen to select the research article for the discussion. The result shown on 3 popular species comprised of  Preclinical TBI studies to date have heavily depended on the usage of mammalian models due to its close anatomical and physiological resemblance to humans. Rodent models, large mammalian models (pig and sheep) and non‐human primate models have successfully elucidated some of the cellular and molecular aspects of human TBI,Danio rerio), fruit fly (Drosophila melanogaster) and roundworm (Caenorhabditis elegans).Emerging TBI preclinical research reported on non‐mammalian animal models which includes zebrafish  original research articles within the specified publication date range, and (2) articles which provide sufficient information on the non‐mammalian animal TBI model utilized, enabling effective evaluation and comparison for this review. The following exclusion criteria were also applied; (1) non research publications or publication with incomplete information such as symposiums, conferences, editorials, book chapters, reviews, and systematic reviews, (2) duplicated articles, (3) non‐English articles, and (4) articles that were not related to non‐mammalian animal models used in traumatic brain injury research where they did not specifically mention the usage of non‐mammalian animal model in traumatic brain injury research. The study selection for this review was performed based on the Preferred Reporting Items for Systematic reviews and Meta‐Analyses guidelines (PRISMA).The quality of the articles included in this review was assessed using the SYstematic Review Centre for Laboratory animal Experimentation Risk of Bias (SYRCLE RoB tool)3st January 2010 – 31st December 2019), (b) 117 non‐original research articles, (c) 38 duplicates, and (d) 29 articles that were not related to non‐mammalian animal models of traumatic brain injury  56 articles not published within the specified date range (13.1Caenorhabditis worm (C. elegans) is one of the least used alternate choices of animal models in TBI research  in roundworms. The shock waves generated through this injury model shared similar properties as primary blast applied in larger animal models of TBI. One of the disadvantages of using this model is the risk of developing damaging cavitation produced by the shock wave.In brief, the shock wave was applied to the roundworms, contained in a medium, through the handpiece of the Swiss Dolor Clast therapeutic shock wave device Figure . This deC. elegans exhibited severe motor deficits and recovered within 10 min following the therapeutic shock wave injury. The recovery period shown by this less complex organism is the fastest as compared to the mammalian TBI models. This injury model has fulfilled two of the criteria for blast‐related neurotrauma models,Based on Table 3.1.2C. elegans's behavioral outcomesC. elegans at once,This model was developed intentionally to study and imitate the blast‐related mild TBI (br‐mTBI). Previous studies showed some irregularities in the C. elegans  device integrated with the worm‐on‐a‐chip microfluidic system and can be performed using two techniques; by introducing the SAW‐driven acoustic waves upright into a chamber fill up with s Figure  or by prC. elegans on the acoustic compressibility have been published in September 2018.C. elegans can be used in TBI research pertaining to learning and memory study.The exposure of the ultrasounds generated through this injury model reduces the worms mobility and causes morphological changes post‐injury.3.2Drosophila melanogaster has been utilized in numerous models of neurological disordersDrosophila models have around 75% genetic match with humans, hence this animal model is well suited for understanding the genetic changes within the central nervous system,3.2.1The high‐impact trauma (HIT) device was developed to study the more common closed head TBI’s underlying cellular and molecular mechanisms rather than penetrating TBI, in fruit flies.In brief, this model involves unanesthetized or anesthetized flies confined to the bottom part of a plastic vial. The end of the vial was fitted with a stationary cotton ball. The spring with the vial attached was deflected and released thereby rapidly contacting the polyurethane pad on the bench Figure . The mec24 after exposed to 2 h inter‐injury interval compared to only 5 min interval.24 with the time interval between the first with subsequent strike onto the fly had not been studied extensively in the mammalian model. Moreover, several studies using the HIT model in fruit flies have elucidated the importance of certain factors such as age, genetic susceptibility, and the effect of multiple TBI incidents on functional outcomes and mortality after TBI, all of which have been understudied in mammalian models of TBI.Traumatic brain injury outcomes generated through this injury model were similar to closed head TBI characteristics in humans3.2.2Mammalian blast TBI models are often time‐consuming, expensive, and difficult to generate in large numbers.This injury model displayed consistent and comparable outcomes to human mild blast TBI such as distinct motor dysfunction and high mortality rate, Table . Interes3.2.3Drosophila injury models which may have shown some inconsistencies in the outcomes measured due to the whole body exertion of impact,Unlike the other In brief, each fly was pushed into a P200 pipette using an aspirator and some air pressure. A specially designed strainer is then used to restrain the fly's head, thus ensuring that only the head is exposed outside the pipette. Next, using a micromanipulator, the head was positioned so that the back of the fly's head was in contact with the brass block attached to a spring‐type solenoid device Figure . The braIn fact, using this injury model not only can depicted the same phenotype seen in mammalian TBI models but the usage of mutants’ species in this project showed the importance of Drosophilia genetic tools in the investigation of the novel pathway underlying TBI. Different glial genes manage to be identified in this study but most of the genes were not well understood, and it is a good opportunity for this model to serve as first‐line screening for identifying in detail the other pathways that may modulate recovery. Besides that, this model may also help to understand the genomic response during TBI and the TBI recovery pathway, at a more effective rate than mammalian TBI models.3.2.4The omni bead ruptor TBI model was developed to investigate the long‐term effects of low impact or mild traumatic brain injury (mTBI).In contrast to the aforementioned HIT device,The selected study using this injury model, observed deficits in behavior and innate immune system and witnessed transformation in the profile of key autophagy markers, which were commonly described in the studies conducted in mammalian models of TBI.3.3Danio rerio) is one of the most common non‐mammalian animal models used in neuroscience research, even among TBI research as summarized in Table Zebrafish  is an excitotoxic metabolic agent that acts as an agonist at N‐methyl‐D‐aspartate (NMDA) receptors, therefore creating injury lesions when injected into the brain region as well as rapidly stimulates a neuroinflammatory response that promotes neuronal repair. Previously, this QA lesion injury model has commonly performed in rodent TBI models, but the extent of the neuronal repair was minimal,In brief, adult zebrafish were anesthetized and placed in a clay mold under a dissecting stereomicroscope. A 30‐gauge needle was filled up with 2.5 µl of 15 mM quinolinic acid and the needle was injected vertically through the skull into the right telencephalic hemisphere Figure  before iThe selected study in this review showed that lesion repair was strongly enhanced and more robust in the zebrafish telencephalon. Itthus proposed that QA‐induced brain lesioning model in zebrafish may provide an optimum tool to study neurodegeneration and neuronal damage replacing the less robust rodent QA lesion models. However, this model is still limited in terms of understanding the difference in molecular signals of QA lesioning compared to other excitotoxic agents, the long‐term behavioral outcomes associated with QA lesioning, and the comparison of regeneration mechanism between zebrafish and mammalian models.3.3.4When the stab lesion model's principles are applied to adult zebrafish with the injury site specified to the telencephalon area of zebrafish's brain, then the injury model is termed as the Telencephalon injury model. This model has been commonly used to study the neuroinflammatory response and other secondary injury pathological pathways post‐TBI. This model is unique as it is highly specific toward the telencephalon which has the capability of exhibiting strong regenerative properties after injury in zebrafish.Baumgart and his team reported that the injured wound was closed without any scarring and suggested that the reduction of oligodendrocyte progenitors (OPC) might be the reason underlying this regenerative phenomenon.In brief, a stab lesion was performed by inserting a needle into an anesthetized zebrafish through the nostril along the rostrocaudal body axis, passing the olfactory bulb until reaching the caudal area of telencephalon Figure . The val3.3.5High‐intensity focused ultrasound (HIFU) is the latest technology in TBI research which works by generating energy beams externally and focusing them directly on the desired injury site; the head in the case of TBI. A modified version of this model is the Pulsed HIFU (pHIFU), described as short pulses of HIFU that can induce clinically representative mechanical injury in the soft brain tissue.In brief, the anesthetized fish was placed in a holder by lying on its left side and were finely secured with surgical tape. The head of the zebrafish was positioned on top of a clear ultrasonic membrane opening below the holder Figure . The pulThis pHIFU injury model is a non‐invasive TBI model, that focuses on a specific target region, and has an automated behavioral assessment that can be performed through video tracking software. The selected study showed that caspase 3 activity was significantly altered post pHIFU which corresponded to the deficits in behavioral outcomes,3.3.6The weight drop model is a well‐established and clinically representative model of TBI in rodents.In brief, the zebrafish was fully anesthetized and was positioned with its dorsal side upward on a foam bed under the vertically erected plastic tubing. Then, the superior side of the head was properly positioned below the tube. A steel ball bearing with a mass of 0.0032 N was dropped through the plastic tube from a specified height, thereby impacting the cranium of the zebrafish at free‐fall speed and energy Figure . FinallyThe selected study also summarized in Table 3.3.7Intracerebral hemorrhage (ICH) injury accounts for about 10%–15% of strokes and about 40% of cases of disability worldwide.Intracerebral hemorrhage in larval zebrafish can show spontaneous brain‐specific bleeding without any invasive techniquesThe selected study showed cerebral bleeding in zebrafish larvae which led to an increase in neuronal cell death, an effect that can be similarly seen in humansThe major advantage of utilizing this zebrafish larval ICH model is the immediate intact in vivo imaging of ICH‐induced inflammatory process upon injury induction that enables cellular interactions and signaling within the brain to be observed spontaneously post ICH, a feature not possible with mammalian models of ICH. The major disadvantage of this injury model, on the other hand, is the lack of cranium in the zebrafish larvae limits this model from developing clinically representative intracranial pressure,3.3.8Glutamate excitotoxicity is a secondary injury hallmark of TBI. Therefore, the larval glutamate insult injury model is a highly specific model that investigates this secondary injury pathway and may only provide therapeutic solutions targeting this pathway, as it does not cover other aspects of TBI pathology. Nevertheless, this model may provide a clear, concise, and in‐depth understanding of TBI’s glutamate excitotoxicity injury mechanism and its subsequent outcome. Besides, this injury model is a non‐invasive model since the larvae can readily absorb glutamate acid and will not be injured mechanically. The secondary injury cascade following this injury model include axonal injury, cell death and synaptic dysfunction which depends on a dose‐severity relationship.In brief, early‐stage larvae were prepared in a 96‐well plate, one larva per wellThe selected study in Table 3.3.9This method induces a rapid neuroinflammatory and cellular death response similar to QA lesion but in the absence of excitotoxic agents, thus indicating this injury model to be purely mechanical. Moreover, unlike the brain mechanical lesion which requires the adult zebrafish brain to be exposed through surgical means, this larval stab lesion can be performed in the intact larvae without any incision or surgery. However, this model more closely resembles the penetrating TBI as seen in certain clinical TBI cases.o angle. This latter injury protocol was mainly used to investigate secondary neuronal cell death post‐injury,In brief, zebrafish larvae were anesthetized and placed with the abdomen facing downward onto an agarose plate. Then, by using a micromanipulator to handle the needle at a 45° angle, the needle was inserted into the hindbrain or any other intended regions, until the desired depth was reached Figure . The larThe study by Gan and his colleagues Table  showed t3.4C. elegans on regulation of naturally occurring axon regeneration following TBI have been reported.Some regenerations signaling pathways in vivo for The innate immune system is highly conserved between flies and humans.In the weight drop model of zebrafish, Gene Ontology (GO) discovery showed differentially expressed genes (DEG) identified in the zebrafish model after 3 and 21‐day post‐injury.Essential mature neuronal biomarkers such as MAP2a+b, parvalbumin, SV2, and metabotropic glutamate receptors 2 (mGLU2) were also identified in the zebrafish TBI model,The radial glial cell is known to have a strong ability to generate neurons.In mammals, brain injury stimulates cell proliferation and neurogenesis in the Subventricular zone (SVZ).Quinolinic Acid (QA) injury in zebrafish3.5Caenorhabditis elegans showed a reduction in mobility, paralysis, and morphological changes,Roundworm, fruit fly, and zebrafish been utilized as an animal model of traumatic brain injury are still new and not much research till recently. Most of the reported studies showed several biomarkers, and underlying mechanisms for the establishment of a disease model. As a TBI animal model, Drosophilia melanogaster can be the best candidate for the first screening test to test the important pathways that modulate recovery. Drosophila model showed a strong upregulated immune response shortly after TBI. Immune and stress response make up 157 out of 512 different glial genes.On the other hand, Danio rerio was reported to offer a wide range of opportunities to study inflammatory response and successful regeneration programs in the Central Nervous System.Apart from that, the wound healing related pathway process reported with the interaction between inflammation, neurogenesis, and angiogenesis has been characterized in this study.Not to forget, the unique Danio rerio larvae, with undeveloped blood‐brain barrierAll the non‐mammalian model has a great potential to expedite the stream of drug discovery for the TBI treatment. Ongoing research of TBI with a non‐mammalian model is often compared with the current established mammalian model. Both models are essential in TBI research, where non‐mammalian helps in screening for the important gene expression involved, biomarkers, and mechanisms for TBI research due to its simple nervous system and highly reproducible. On the other hand, the mammalian TBI model is more advanced in its robust research background focusing on the mechanism and important biomarkers.4In summary, we believe this is the first review that summarizes and evaluates non‐mammalian models of TBI while critically comparing them with mammalian models of TBI, in hopes to elucidate a better, cheaper, faster, and more efficient alternative TBI model that may further advance current TBI research. Among the three species of non‐mammalian TBI model retrieved in this review, comprised of zebrafish, roundworm, and fruit fly, zebrafish is the one most frequent model used in showcasing a variety of injury conditions to mimic human TBI. Moreover, since both the larvae and adult zebrafish can be effectively sampled to inflict clinically representative TBI, whether penetrating or closed head injury, an endless possibility of TBI investigations such as aging effects, intervention studies, and time progression outcomes, can be performed with hassle‐free. On the other hand, the roundworm and fruit fly are mostly ideal for high‐throughput TBI treatment screening studies. They may be limited in terms of pathophysiological resemblance to human TBI, despite the similarities in some of the functional outcomes displayed post‐injury when compared to mammalian TBI models and human TBI. Taken together, the non‐mammalian TBI models provide a more simplistic approach to bridge the knowledge gaps within TBI research and may shorten the road toward TBI outcome prevention and cure, by surpassing the limitations of mammalian models of TBI. Apart from that, these animal models could be used together with the mammalian model and help on the trial basis of research before moving to an in‐depth investigation to find the novel mechanism and therapeutic approaches against TBI.All authors declare that they have no conflict of interest.NAZ performed literature search, analyzed, and wrote the manuscript. AA contributed to quality analysis, writing, and editing. MFS conceptualized the idea, designed, and supervised the work. AA, IA, SAZA, IO and MFS provided their critical comments and were involved in editing and proofreading. All authors gave their final approval prior to the submission of this manuscript.Table S1Click here for additional data file.


Hepatitis C virus (HCV) infects 180 million people worldwide and over 4 million people in the United States. HCV infection is a major cause of chronic liver disease and is recognized as a risk factor for clinical cardiovascular disease (CVD). Many studies have shown increased prevalence of cardiac and inflammatory biomarkers in patients with chronic HCV infection (CHC), and though these markers may be used to risk stratify people for cardiac disease in the general population their role in the HCV population is unknown. Patients with CHC have elevated cardiac and inflammatory biomarkers compared to noninfected controls which may play a role in CVD risk stratification. We undertook a systematic review of inflammatory and cardiac biomarkers in people with HCV infection with a focus on the effect of CHC on serum levels of these markers and their utility as predictors of CVD in this population. Medline, EMBASE, and Cochrane databases were searched for relevant articles until June 2019. A total of 2430 results were reviewed with 115 studies included. Our review revealed that HCV infection significantly alters serum levels of markers of inflammation, endothelial function, and cardiac dysfunction prior to HCV treatment, and some of which may change in response to HCV therapy. Current risk stratification tools for development of CVD in the general population may not account for the increased inflammatory markers that appear to be elevated among HCV‐infected patients contributing to increased CVD risk. ALDalcoholic liver diseasesARTantiretroviral therapyASC HCVasymptomatic HCVBMIbody mass indexBNPbrain natriuretic peptideCADcoronary artery diseaseCFRcoronary flow reserveCHCchronic hepatitis CCHFcongestive Heart FailureCLDchronic liver diseaseCRFchronic renal failureCRPC‐reactive proteincTn‐Icardiac troponin IcTn‐Tcardiac troponin TCVAcerebrovascular accidentCVDcardiovascular diseaseDAAsdirectly acting antiviral agentsDMdiabetes mellitusESRDend‐stage renal diseaseGIBgastrointestinal bleedinggpglycoproteinHAIhepatic activity indexHAVhepatitis A virusHBVhepatitis B virusHCVhepatitis C virusHDhemodialysisHDVhepatitis D virusHIVhuman immunodeficiency virushsCRPhigh‐sensitivity CRPHTNhypertensionIDUinjection drug useILinterleukinINF‐ αinterferon alphaLPSlipoprotein polysaccharideNRnonrespondersNT‐proBNPN‐terminal pro b‐type natriuretic peptideOIsopportunistic infectionssCDsoluble cellular differentiationsE‐selectinsoluble E‐selectinsICAM‐1soluble intercellular adhesion molecule‐1SLEsystemic lupus erythematosussP‐selectinsoluble P‐selectinsTWEAKsoluble Tumor necrosis factor like weak inducer of apoptosissVCAM‐1soluble vascular cell adhesion molecule‐1SVRsustained virologic responseTNF‐ alpha (or TNF‐α)tumor necrosis factor‐alphaTNFRTNF‐α receptorsTnItroponin ITnTtroponin TTWEAKTNF like weak inducer of apoptosis,VLviral load.1Hepatitis C virus (HCV) is a single stranded RNA virus belonging to the Flaviviridae family. HCV has a global prevalence of 2.5% and infects 180 million people worldwide.Chronic viral diseases, in particular human immunodeficiency virus (HIV), have been strongly linked to the development of clinical cardiac diseases.2We conducted a search based on PRISMA guidelinesCardiac biomarkers were categorized into three main groups: biomarkers of inflammation, biomarkers of endothelial function, and biomarkers of cardiac dysfunction.3The search performed in June 2019 yielded 1255 results on Medline, 1100 results on EMBASE, and 75 results on Cochrane giving a total of 2430 results. After duplicates were removed, 2394 references remained for review. Twenty‐four additional articles were added after performing ancestry and bibliography searches of all relevant articles, meta‐analyses, and systematic reviews. On review of titles and abstracts, 2156 were removed as they were found to be not relevant to our review. Of the remaining 262, 147 were removed because they did not meet eligibility criteria: 66 lacked a seronegative control group, 39 did not examine biomarkers of interest, 27 described only expression of and not serum levels of biomarkers, eight had relevant data missing, four were in a pediatric population, and three described levels of biomarkers following stimulation. Ultimately, 115 studies were included in our review .3.1Biomarkers of inflammation are commonly used to assess CVD risk in the general population. Fifty‐six studies evaluating the effect of HCV infection on inflammatory biomarkers were reviewed Table  BiomarkeIL‐10 is an anti‐inflammatory cytokine that regulates the production of proinflammatory cytokinesTNF‐α is a proinflammatory cytokine secreted by activated monocytes and macrophages in response to various infections. TNF‐α stimulates the release of acute phase proteins in the liver leading to lymphocyte and endothelial activation,C‐reactive protein (CRP) is an acute phase protein produced predominantly by hepatocytes, and influenced by IL‐6 and TNF‐α.Furthermore, monocyte/macrophage activation markers of immune activation such as soluble CD163 (sCD163)Numerous studies have demonstrated increased expression of and serum levels of IL‐6,In a cohort of 981 patients with coronary heart disease enrolled in the Heart and Soul study, HCV seropositivity was associated with changes in levels of CRP, TNF‐α, IL‐6 increased Framingham risk scores,However, other studies have reported different findings. These studies were smaller in size,Studies have been mixed about the association between CRP levels and CHC infection. Some studies have demonstrated decreased CRP levels in CHC infection, which was postulated to be due to decreased production of CRP.In contrast, other studies have shown elevated CRP levels in CHC infection.Among the reported studies, few have linked altered levels of biomarkers with subclinical and clinical CVD in CHC. Tsui et al demonstrated increased levels of CRP, TNF‐α, IL‐6, and hospitalizations due to clinical cardiac failure among HCV positive patients compared to controls.Finally, many studies investigated the effect of HCV therapy on inflammatory markers and the majority demonstrated that HCV therapy significantly altered levels of inflammatory levels and/or predicted treatment success.Taken the together, the preponderance of data shows a clear modulation of the immune system, an imbalance of pro‐and anti‐inflammatory biomarkers with a shift towards a proinflammatory state, and increased serum inflammatory levels among CHC‐infected patients. However, possibly due to blunted hepatocyte response and decreased production of CRP, CHC patients may score lower on CVD risk assessment models which rely heavily on CRP values.3.2One of the early sentinel events in the development of atherosclerosis is endothelial dysfunction which results in an increased interaction between circulating leukocytes and the endothelium.Elevated levels of these markers have been correlated with increased risk of cardiovascular mortality in numerous studies in the general population.Six studies investigated the serum levels of markers of endothelial function among HCV patients Table  ElevatedIn a study of 60 patients with CHC infection and age‐matched controls, higher levels of hsCRP, sICAM‐1, sVCAM‐1, and s‐E‐selectin were found among CHC patients compared to controls and increased biomarker levels correlated with carotid intima thickness.3.3Brain natriuretic peptide (BNP) and N‐terminal proBNP (NT‐proBNP) are natriuretic hormones that are primarily released by the ventricles of the heart. Plasma BNP provides prognostic information in patients with chronic heart failure and in those with asymptomatic or symptomatic left ventricular dysfunction.Troponin T (TnT) and Troponin I (TnI) are cardiac proteins which regulate the calcium mediated interactions between actin and myosinNine studies evaluated serum levels of these biomarkers in CHC‐infected patients. Table  Among CHChe et al found higher levels of NT‐ proBNP levels and a greater proportion of patients with impaired diastolic filling among HCV‐infected patients compared to controls, and the NT‐proBNP levels correlated with impaired diastolic filling.The data above linking HCV infection with elevated biomarker levels and echocardiographic evidence of diastolic dysfunction suggest that HCV infection influences cardiac function asymptomatically and that testing with NT‐proBNP may have a role in identifying patients with low‐grade cardiac dysfunction and increased CVD mortality risk. There were no studies evaluating the change of these markers with HCV therapy.44.1HIV infection has been strongly associated with increased risk of CVD through immune activation, exposure to antiretroviral therapy (ART) and disproportionately increased traditional risk factors for CVD among the HIV population.Sixteen studies have evaluated the effect of both HIV mono‐infection and HIV/HCV co‐infection on inflammatory biomarkers. Table  The majoStudies of markers of endothelial function in HIV/HCV co‐infected patients have reported similar results to studies in HCV mono‐infected patients Table  Studies Therefore, the totality of the data suggests that while HIV mono‐infection has a significant influence on serum cardiovascular biomarkers, HCV co‐infection may amplify this effect and further increase CVD risk. In addition, it may be possible to identify this increased risk in the HIV/HCV co‐infected population through measurement of select inflammatory and endothelial biomarkers.4.2The additive effect of comorbidities such as diabetes mellitusTNF‐α and IL‐6 have been elevated in ESRD patients with and without HCV compared to healthy controls.The immune activation associated with CHC infection can lead to the development of immune related complications, which may further influence the levels of inflammatory markers associated with CVD. Increased levels of IL‐6, IL‐10, TNF‐α, and sTNFR in HCV‐infected patients with complications such as cryoglobulinemia, Sjogren syndrome, lymphoproliferative diseases, hemophilia, and HCV related arthritis have been reported compared to CHC‐infected patients without such complications or to healthy controls.Therefore, special consideration may be needed when interpreting serum inflammatory markers in HCV populations who have DM, ESRD, and/or concomitant immune complications. In these populations with HCV infection and inflammatory conditions, there we no studies evaluating the change of these markers with HCV treatment, and none evaluated the association of these markers to subclinical and/or clinical CVD.5We found that the preponderance of data suggested that HCV infection significantly alters serum levels of markers of inflammation, endothelial function, and cardiac dysfunction prior to HCV treatment, and some of which may change in response to HCV therapy. The majority of studies demonstrate an imbalance of pro‐and anti‐inflammatory biomarkers with a shift towards a proinflammatory state among CHC‐infected patients.Clinical presentations of CVD are preceded by an asymptomatic period, which can be long and insidious, but during this period biomarkers can be critical in identifying pathological developments that may lead to clinical cardiac events. Biomarkers are useful tools to risk stratify patients for development of disease and to monitor disease outcomes in response to therapies. Cardiac biomarkers used for these purposes in the general population may be useful in the HCV population too. Since the majority of studies consistently found increased levels of the pro‐inflammatory markers IL‐6 and TNF‐α and markers of endothelial function and cardiac dysfunction among HCV‐infected compared to uninfected patients, incorporating these biomarkers into risk stratification tools may improve the ability to discern an individual's risk for developing CVD among HCV‐infected patients who were determined to be at intermediate risk based on standard CVD assessment tools using traditional markers such as CRP only.Traditional risk stratification tools for CVD have been established and widely adopted in routine practice. Risk scoring tools such as the atherosclerotic CVD (ASCVD) risk score incorporate traditional risk factors such as smoking, hypertension and DM, and though these risk factors certainly contribute to an individual's CVD risk they do not fully account for the increased atherosclerosis and CVD events among certain high‐risk groups such as the HCV‐infected patient population.CRP is an established cardiac biomarker for increased CVD risk and has been included in traditional risk assessment algorithms. Treatment strategies based on hsCRP levels have resulted in reduced CVD events,Another advantage of some nontraditional biomarkers is their relationship to CHC disease stage,Ours is one of the first comprehensive reviews of the literature reporting on the associations between HCV and a wide array of nontraditional potential CVD biomarkers such as endothelial markers. Strengths of our review included an extensive, systematic review of the topic and inclusion of various study designs, thereby allowing us to include many studies with pertinent, valuable findings. Furthermore, it is the first review to examine the possible additive influence of common comorbidities such as HIV co‐infection, DM, and ESRD on CHC and their effects on serum cardiac biomarkers. In addition, our review reported on the effect of antiviral therapy on these cardiac markers, which is of particular interest in the DAA era. Only one other similar systemic review by Osibogun et al included HIV/HCV co‐infected patients but it included 28 studies only.In conclusion, inflammatory pathways are fundamental to the pathogenesis of atherosclerosis and the development of cardiac events, and CHC infection has been shown to heighten the state of inflammation in these patients. Current risk stratification tools for development of CVD in the general population do not account for many of the inflammatory markers that appear to be elevated among HCV‐infected patients, and therefore there is concern that the CVD risk in these patients may be underestimated. Given the burden of HCV infection both nationally and internationally, there is an urgent need to evaluate additional cardiac biomarkers that may potentially identify or better discriminate CVD risk among HCV‐infected patients more accurately than those in the current standard risk assessment tools. Further prospective studies are needed to confirm the predictive value of these biomarkers of interest in this patient population.The authors declare no potential conflict of interests.Appendix S1. Supporting Information.Click here for additional data file.FIGURE S1 Prisma flow diagramClick here for additional data file.


Femoral avulsion fracture of the posterior cruciate ligament (PCL) in association with meniscus tear is extremely rare in children, and similar cases are not available in the existing literature.In this paper, we present a case of a 9‐year‐old boy treated by an arthroscopic repair technique using two transfemoral tunnels sparing the epiphyseal plate for 8‐shaped suture fixation of femoral avulsion fracture of the PCL and using the Fastfix repair system for a meniscus tear. At 3‐month postoperative, the boy showed satisfactory recovery with a full range of motion of the right knee and normal muscular strength, and no complications were observed after operation. The patient had been followed up for 24 months and his final assessment demonstrated stable painless knee with excellent functional outcome. In view of this, we suggest that the “figure‐of‐eight” suture technique using two transfemoral tunnels sparing the epiphyseal plate can be considered a new choice for the treatment of the PCL femoral attachment avulsion, especially in skeletally immature patients. In addition, six similar cases were found in a comprehensive literature review targeting femoral avulsion fracture of the PCL. According to the relevant findings and cases studies, we proposed a new classification named “Xiangya” which might facilitate future clinical decision making. In general, posterior cruciate ligament (PCL) injuries can be classified into several patterns according to the anatomic lesion, including mid‐substance failure, tibial avulsion, femoral peel‐off, and femoral avulsion. Injuries to the PCL usually involve ligament substance or tibial insertion and, less commonly, avulsion from the femoral insertionSpecifically, this study was performed with the following purposes: (i) to provide a new technique for the treatment of PCL femoral attachment avulsion especially in skeletally immature patients; and (ii) to explore a new classification of PCL femoral avulsion fracture to facilitate clinicians in making more appropriate surgery decisions for patients with PCL femoral attachment avulsion.Our targeted case corresponds to a boy with PCL avulsion from the femoral insertion in association with a meniscus tear who received treatment in our hospital. Written informed consent had been obtained from the patient prior to the research alongside all the related clinical reports with detailed clinical diagnosis and treatment data. PubMed and Science Direct databases were searched using the following key words: “femoral avulsion fracture” and “posterior cruciate ligament”. Due to the low‐incidence, large‐sample summative studies, and with randomized controlled trials still lacking, all the relevant publications that exist as case reports. Therefore, the included literature is mainly based on detailed records of the patients' specific conditions, treatment processes, and follow‐up articles. For this reason, the patients' basic characteristics and treatment plans were analyzed.A 9‐year‐old boy injured his right knee in a fall while riding a motorcycle. He complained of pain and swelling, and was admitted to our hospital 7 days after the incident. Detailed physical examination was difficult to carry out due to the pain. Plain radiography did not reveal femoral avulsion fracture of the PCL. X‐ray images of the right knee showed a displaced femoral avulsion fracture of the PCL Fig. A. On theAnesthesia and Position. General anesthesia was executed in the operation with the patient being placed in a dorsal position. His right knee showed a positive result of posterior drawer test under general anesthesia, and >1 cm posterior sagging was observed in comparison with the left knee.A thigh tourniquet was typically used to control bleeding and improve visualization. Routine anterolateral and anteromedial portals were created for intraarticular surgery. The arthroscopic view was obtained from an anterolateral portal. Hematoma and large blood clots in the joint were carefully removed to expose the femoral avulsion fracture Fig. A of the Meniscus tear was identified and probed with a small metal hook Fig. B. There The radial tear in the body of medial meniscus was repaired with the Fastfix meniscus repair device before reduction of the femoral avulsion fracture Fig. C. Then, After the surgery, the patient was required to wear a plaster cast for 2 weeks to heal the fracture Fig. F, and fuThe operation lasted for about 90 min with a total bleeding loss of about 15 ml. It was found that the osteochondral fracture was stable in full range of motion (ROM) after being fixed, the articular surface had recovered its smoothness under arthroscopy, and the PCL reattachment held tension on testing Fig. E. A postAt 1 month postoperative, the pain and claudication symptoms had completely disappeared. Flexion of the knee was approximately 110° at 1 month Fig. A, 125° aForty‐six similar cases were found from the literature search and six cases involving femoral attachment avulsion fractures in children were retained (an overview was given in Table The PCL is approximately twice as strong as the anterior cruciate ligament (ACL) and represents the strongest ligament of the kneeUp to the present, practical experience in the treatment of femoral avulsion fractures of the PCL for children is limited due to the low incidence of such injuries. Although some similar cases were reported in the literature, a “gold standard” is not yet availableAfter a comprehensive search, six cases in adolescents were found in the literature Table , includiThere was no consensus about whether to use a cast or brace after surgery. According to Table The case presented in our study featured somewhat unusual characteristics. First, the avulsion occurred at the femoral origin of the PCL. Second, the fragment could be fixed regardless of its shape or size by using two transfemoral tunnels sparing the epiphyseal plate for 8‐shaped suture fixation. This new technique overcame the disadvantage of screws which could only fix large fracture fragments. Third, our “figure‐of‐eight” suture technique can be regarded as an improvement on previous techniques by providing perfect stability to the knee to allow earlier functional exercises. Fourth, growth arrest or angular deformity related to physeal injury must be taken into consideration during the operation. Our technique avoided distal femoral physis when placing the medial femoral condylar drill holes, and the fragment was fixed without disrupting future growth potential.The advantages of this technique include the ability to diagnose and treat concomitant injuries, anatomic reduction of the fragment and stabilization in a limited surgical time, as well as early rehabilitation with full weight bearing. Last but not least, there is no need for device removal. Although controversy still exists, we believe that the physeal‐sparing technique can address the concerns about growth disturbance to a large extent. To the best of our knowledge, this is the first study that investigates the arthroscopic treatment of displaced femoral avulsion fracture of the PCL by using two transfemoral tunnels sparing the epiphyseal plate for 8‐shaped suture fixation. Of course, we have also noticed some deficiencies in the present study. It had been only 24 months since the operation, and follow‐up is still in progress.At present, no relevant reports have been found on the classification of PCL femoral avulsion fracture. Therefore, we proposed a new classification of PCL femoral avulsion fracture named “Xiangya”, which depended mainly on the size and displacement degree of the fracture fragment. We hoped this classification would provide some reference for future clinical decision making and treatment of PCL femoral attachment avulsion fractures Fig. . SpecifiThe present study involves several limitations. Firstly, we only focused on one case, and there was the lack of a randomized controlled study, so more cases are needed to further validate the effectiveness of this new technique. Secondly, our case's follow‐up period was relatively short. It is necessary to execute a longer term follow‐up to evaluate growth disturbances. Nevertheless, the follow‐up for our case is 24 months, which is deemed sufficiently robust to report on the clinical outcomes. Thirdly, the practicability of this new “Xiangya” classification needs to be tested by further clinical practice.In conclusion, our “figure‐of‐eight” suture technique using two transfemoral tunnels sparing the epiphyseal plate can be a new choice of treatment for the PCL femoral attachment avulsion, especially in skeletally immature patients.


Bos taurus reference genome assembly (ARS‐UCD1.2), it is the opportune time to upgrade the bovine gene set by seeking input from researchers. Furthermore, advances in graphical genome annotation tools now make it possible for researchers to leverage sequence data generated with the latest technologies to collaboratively curate genes. For many years the Bovine Genome Database (BGD) has provided tools such as the apollo genome annotation editor to support manual bovine gene curation. The goal of this paper is to explain the reasoning behind the decisions made in the manual gene curation process while providing examples using the existing BGD tools. We will describe the sources of gene annotation evidence provided at the BGD, including RNA‐seq and Iso‐Seq data. We will also explain how to interpret various data visualizations when curating gene models, and will demonstrate the value of manual gene annotation. The process described here can be applied to manual gene curation for other species with similar tools. With a better understanding of manual gene annotation, researchers will be encouraged to edit gene models and contribute to the enhancement of livestock gene sets.With the availability of a new highly contiguous  Bos taurus) genome has been used to identify genes associated with such complex traits . The Ensembl gene set (Ensembl95 Release) was downloaded from Ensembl (http://ftp.ensembl.org/pub/release‐95/gff3/bos_taurus/).The bovine ARS‐UCD1.2 genome assembly and RefSeq gene set were downloaded from NCBI  RNA‐seq, paired‐end (PE) RNA‐seq and Iso‐Seq data, predominantly from tissues isolated from the reference genome individual (Line 1 Hereford Dominette 01449) and closely related individuals Table . Sampleset al. : PE RNA‐fastq‐mcf (https://code.google.com/p/ea‐utils/wiki/FastqMcf), quality trimmed using dynamictrim . The trimmed reads were aligned to the unmasked ARS‐UCD1.2 genome assembly with Hisat2  and in Dunn et al.  allows the Faceted Track Selector to be opened, which provides over 800 tracks organized into categories, with a filterable and searchable table and flexible search functions. Once logged in to apollo, tracks can be selected either with the Tracks Tab in the Information Panel or the Faceted Track Selector. The Select Tracks tab is available by clicking the icon that resembles a list, under the icon used to open or close the Information Panel (described above). On the left of the Faceted Track Selector is a list of tracks organized according to track type, and in reference to gene expression tracks, Organ System and Brenda Tissue Ontology. Highlighting one or more Data Type, Organ System or Brenda Tissue Ontology in the left panel filters the tracks, so that only those tracks are shown in the table on the right. The table, with additional details about the RNA‐seq tracks, is searchable by entering text in the ‘Contains text’ search box above the table. Select tracks for viewing by clicking the boxes on the left side of the table. Clicking ‘Back to Browser’ brings the browser area with selected tracks into view.The Select Tracks tab that appears in the upper left of a The types of tracks most often used in manual gene annotation are computed gene prediction and RNA expression tracks. The gene prediction tracks in the BGD represent RefSeq and Ensembl transcripts, including protein‐coding, long non‐coding and other RNA. Gene prediction tracks appear as histograms depicting gene density when zoomed out, and allow the visualization of predicted introns and exons when zoomed in.gmap tracks provide the most reliable information, because the transcripts have not been further assembled, which could introduce errors. On the other hand, some large genes, such as titin (TTN) are prone to produce fragments even with Iso‐Seq, and assembly with PASA can help reconstruct full‐length isoforms, as well as reduce redundancy.The BGD provides three kinds of RNA expression data generated from the tissues of Dominette and her close relatives: SE Ilumina RNA‐seq, PE RNA‐seq and Iso‐Seq. Each type of expression data is provided in several visualizations. Iso‐Seq should be the first expression data type investigated, because an Iso‐Seq sequence usually includes the entire transcript, without the need for assembly. In addition to identifying new transcript isoforms, Iso‐Seq can provide evidence to support the merging of gene predictions and to create novel isoform and gene annotations. A disadvantage of the bovine Iso‐Seq data compared with the bovine RNA‐seq data is that there are fewer Iso‐Seq sequenced tissue transcriptomes. Furthermore, the depth of sequencing tends to be lower for Iso‐Seq than RNA‐seq and isoforms found in RNA‐seq data may not be present in the Iso‐Seq data. Of the Iso‐Seq tracks, ‘Iso‐Seq Combined PASA Assembly’ is the track of choice to determine if there is any Iso‐Seq evidence for gene or isoform structure Fig. . The iden = 90 datasets) or PE (n = 40 datasets) that are formatted in six different track types. StringTie tracks show transcript models inferred by assembling RNA‐seq reads, providing the advantages of reduced data redundancy and ease of visualizing the possibility of new isoforms. Whereas predicted transcripts assembled from short RNA‐seq reads are considered to be less reliable than full‐length Iso‐Seq transcripts, the higher RNA‐seq sequencing depth leads to the StringTie tracks having the advantage of higher overall gene representation. Similar to gene predictions and Iso‐Seq, StringTie tracks allow the visualization of predicted introns and exons when zoomed in, and when zoomed out visualizations appear as histograms depicting transcript density.The bovine RNA‐seq tracks include SE . RNA‐seq junction tracks are available as arcs or as flat tracks, and can quickly reveal whether an RNA‐seq dataset would be useful in resolving a split/merge issue. The arc version is a visualization that collapses junctions from individual reads for easiest viewing when zoomed out to see an entire gene or the region between genes Fig. . The thiBAM tracks show alignments of individual RNA‐seq reads, and are available in two forms, dense and draggable, both of which are useful in predicting intron/exon boundaries and require sufficient zoom level to avoid an error message. The dense track Fig.  is the let al. et al. A challenge with the availability of RNA‐seq tracks from 130 experiments is selecting the experiment with the best information for the gene of interest. A solution is to query BovineMine  or tblastn (for protein queries) and then viewing alignments of blast high‐scoring segment pairs (HSPs) in jbrowse. If logged into apollo, individual blast HSPs can be dragged to the Editing Area to start a new annotation. The blast server is especially useful for identifying the locations of bovine genes based on their homologs, for example, from model organisms such as human or mouse.The BGD Here we describe in general terms how to annotate a gene using BGD tools in order to convey what the annotation process entails. Detailed step‐by‐step examples are provided in Appendices blast search with a bovine cDNA or protein homolog sequence. After navigating to the genomic region containing the gene of interest, select tracks to display the gene model and isoforms using the Faceted Track Selector. First inspect the RefSeq and Ensembl tracks for differences across the gene sets. If there is no major disagreement, such as when one gene set indicates two genes and the other indicates one (a split/merge issue), the annotation can be initiated using the RefSeq and Ensembl transcripts. The goal is to annotate an entire gene, if possible, which entails annotating all of the transcript isoforms for which there is evidence. To begin, select a unique set of isoforms from the RefSeq and Ensembl tracks, but do not select isoforms with major incongruences compared with others without further inspection. Drag each selected isoform to the Editing Area after right‐clicking an intron. Sometimes two isoforms are identical across genes sets, in which case select only one. Sometimes an isoform in one gene set will appear to be a fragment of an isoform found in the other, in which case, select the longer isoform. There is no need to be overly concerned about the choice of isoforms at this point, because each isoform can be easily deleted from the Editing Area.Prior to starting annotation, identify a gene or genomic region of interest, for example, by searching with a RefSeq or Ensembl transcript id, or by performing a gmap tracks for each tissue  or cDNA sequence; ‘Show History’ to see modifications made; ‘Undo/Redo’ to undo or redo the previous edits; and ‘Edit Information’ to add more information about the annotation. orm Fig. . Modificinterpro database can provide additional evidence of protein sequence integrity (Mitchell et al. After edits to the annotation are complete, the next step is to obtain the sequence and search a well‐curated protein database such as UniProt/SwissProt (UniProt Consortium et al. After completing the annotation process, you may have determined that the original predicted isoforms needed no modification and that there were no new isoforms; nevertheless, the gene annotation is important because it indicates that a predicted gene has been reviewed and confirmed. Whether or not you modified the annotation, you can provide additional information about the gene using the Information Editor, available by right‐clicking the annotation. The form provides text entry boxes for gene symbol, description, database cross‐references, GO, PubMed identifiers and comments. For gene symbols, you should use standard nomenclature established by the Vertebrate Gene Nomenclature Committee (Braschi Throughout the annotation process, the server automatically saves annotation changes and the history of changes at each step. Both the annotation and change histories are available to all logged in users. Any user can make edits to a previous annotation or delete the annotation from the editing area, but you should never delete another’s work as the history will also be deleted. The annotation owner, visible by hovering over the annotation, should be contacted if changes need to be made to the annotation.apollo tool will be immediately available to the bovine research community, and will periodically be submitted to NCBI for inclusion in future bovine genome annotation updates. Whether the objective is overall gene set improvement or examining specific genes of interest, we encourage the research community to use the freely accessible annotation tools at the BGD and we welcome suggestions to improve the utility of the resource.With the release of a new highly contiguous bovine reference genome assembly, a collaborative effort to manually annotate genes would greatly enhance the bovine community’s genomics resources. Gene models curated using the BGD Table S1 RNA‐seq sample metadata.Click here for additional data file.Table S2 Two‐letter codes for Iso‐seqpasa combined assembly track.Click here for additional data file.Appendix S1 Annotation example 1.Click here for additional data file.Appendix S2 Annotation example 2.Click here for additional data file.


The guidelines further state that CBTp “can be started either during the acute phase or later, including in inpatient settings” (guidelines 1.4.4.1). There is an additional recommendation that when CBTp is started within inpatient settings “the full course should be continued after discharge without unnecessary interruption” (guideline 1.4.4.5). However, it is not clear whether these guidelines are a good fit for how care pathways are currently configured in NHS mental health trusts. Trusts may not be able to offer continuity of care between inpatient and community settings for various reasons. For example, inpatient and community psychology services may be separate from each other, or there may be no CBTp provision at all within inpatient services. Although previous audits have found generally low levels of the provision of CBTp for service users with psychosis within NHS mental health trusts,Guidelines from the United Kingdom's National Institute for Health and Care Excellence (NICE) for psychosis and schizophrenia  for service users with psychosis, to continue a full course of CBT which has been started within an inpatient setting, without interruption after discharge, anywhere in the Trust?Survey Question: Is it currently Ethical approval for the study was given by the University of Bath Psychology Research Ethics Committee (Ref: 19‐319). The survey question below was distributed was via email to Heads of Psychological Therapies  at NHS mental health trusts in England. Healthcare is devolved in the other nations of the United Kingdom, hence our focus on England only.The email made it clear that participation was voluntary, and we were not asking for any data on numbers of service users who have received psychological therapies, as our question related only to care pathway configuration  and received 26 responses . In total, we received responses from 48% of all NHS mental health trusts in England (26/54). We received at least one response from all major geographical regions of England . Additionally, some trusts said it would only be possible for service users meeting criteria for early intervention (EI) services (n = 5). Some trusts gave additional information about how service configuration supported continuity of therapy provision. This was achieved in one of three main ways; (a) psychology staff had split inpatient/community posts (n = 3), (b) community staff could in‐reach to inpatient settings (n = 3), (c) inpatient staff could follow patients into the community to continue therapy which had started during an inpatient admission (n = 1).Approximately half of all trusts responded “yes” to the survey question . However, out of these who responded yes it was possible, some commented that it was not common practice due to limited staff resources (n = 9). Additional reasons given included CBTp not being offered at all within inpatient settings (n = 4), long waiting lists for therapy in the community (n = 2), and CBTp being mainly available in early intervention (EI) services (n = 1).For the 13 trusts who responded “no” to the survey question, the main reason given for this was that there was a separation between inpatient and community services, with no staff working across these settings in an integrated way  have been explained.Data S1. Supporting Information.Click here for additional data file.


As a user‐controlled HIV prevention method, oral pre‐exposure prophylaxis (PrEP) holds particular promise for adolescent girls and young women (AGYW). HIV prevention cascades, critical frameworks for the design and evaluation of PrEP programmes, outline the priorities of identifying individuals at greatest HIV risk and motivating them to initiate PrEP through perceived HIV risk. To inform future iterations of these cascades and PrEP delivery for AGYW, the objective of this study was to understand the level of interest in PrEP among AGYW at highest HIV risk, and the potential role of perceived risk in motivating PrEP interest.Using data from a cohort study of HIV‐negative AGYW in Lilongwe, Malawi (February 2016 to August 2017), we assessed the relationship between epidemiologic HIV risk  and PrEP interest, and the extent to which perceived risk explains the relationship between HIV risk and PrEP interest. We further aimed to operationalize the pre‐initiation steps of the HIV prevention cascade in the study population.In total, 825 AGYW were included in analyses, of which 43% met the criterion for high epidemiologic HIV risk. While epidemiologic risk scores were positively associated with PrEP interest, high numbers of AGYW both above and below the high‐risk cutoff were very interested in PrEP (68% vs. 63%). Perceived risk partially explained the relationship between HIV risk and PrEP interest; greater epidemiologic HIV risk was associated with high perceived risk, which was in turn associated with PrEP interest. Many more high‐risk AGYW were interested in PrEP (68%) than expressed a high level of perceived HIV risk (26%).These results highlight key relationships between epidemiologic HIV risk, risk perception and interest in PrEP. While risk perception did partially explain the relationship between epidemiologic risk and PrEP interest, there may be other important motivational mechanisms that are not captured in many HIV prevention cascades. The high number of participants with risk scores below the high‐risk cutoff who both expressed high perceived risk and interest in PrEP suggests that demand for PrEP among AGYW may not be well aligned with epidemiologic risk. In Mhow AGYW at greatest risk may become motivated to consider PrEP.Following World Health Organization (WHO) guidelines recommending offering PrEP to at‐risk populations, including AGYW in high burden settings , many coHIV prevention cascades provide valuable frameworks and metrics by which to evaluate the impact of prevention programmes, and to identify important intermediary endpoints to guide the development of effective prevention programmes . The majTo address this gap, using data from an observational cohort study with AGYW in Lilongwe, Malawi we aim to answer two primary questions: (1) Are those AGYW at highest HIV risk actually the most likely to (a) perceive themselves to be at risk and (b) express interest in PrEP use? and (2) To what extent does perceived risk explain the relationship between HIV risk and PrEP interest? We further aim to operationalize the pre‐initiation stages of the prevention cascade in the study population. The answers to these questions will build understanding of likely outcomes for key precursors to PrEP interest and uptake in the early days of rollout to AGYW.22.1The Girl Power‐Malawi study was conducted at four health centres in Lilongwe, Malawi from February 2016 to August 2017 and assessed four service delivery models for AGYW . All cli2.2Two‐hundred and fifty AGYW were recruited from the catchment areas surrounding each of the four study clinics  through community outreach, participant referral, and self‐referral. AGYW were eligible to participate if they were 15 to 24 years old, from the catchment area, and willing to provide locator information . AGYW who were sexually active in the past six months were purposively recruited; study staff informally discussed romantic relationships and sexual activity with AGYW and invited those with current or past sexual activity for screening. Eligible and consenting participants were enrolled and followed for one year. All participants were asked to complete a behavioural survey at baseline, six months, and one year assessing socioeconomic, behavioural, biomedical and partnership characteristics; interest in using PrEP; and HIV risk perception. Surveys were administered in Chichewa by young female research officers using Open Data Kit software . Phone a2.3Girl Power‐Malawi received approval from the National Health Science Research Committee in Malawi and the University of North Carolina Institutional Review Board. Voluntary written informed consent was obtained from participants 18 to 24 years old. Assent and permission by a parent, guardian or authorized representative were obtained for adolescents 15 to 17 years old. In cases of limited literacy, an impartial witness was present.2.42.4.1Indicators of HIV risk used were previously identified as those associated with HIV incidence in the Girl Power‐Malawi cohort . All nin2.4.2AGYW rated their perceived lifetime chance of acquiring HIV as: “no chance,” “small chance” or “high chance.” In bivariate analyses, this was dichotomized as “high chance” (1) versus other responses (0).2.4.3AGYW were rated their potential interest in using PrEP after receiving this explanation: “PrEP is a medicine that can be used to prevent HIV for people who are HIV‐negative. To be protected with PrEP, a pill is taken every day. These pills contain some of the same medicine used to treat people who already have HIV. PrEP is not currently available in Malawi.” Participants rated their interest in trying PrEP if it were available at the study clinic . For multivariate analyses, this was dichotomized as “very interested” (1) versus other responses (0).Control variables included highest level of education (grade level) and household economic status evaluated through an adapted Filmer Pritchett Wealth Index including 13 household assets . A compo2.5All analyses were performed in SAS v 9.4. We tested the following hypotheses: 1) Women with greater HIV risk will be more likely to a) be very interested in PrEP and b) perceive high HIV risk; 2) Women with greater perceived risk will be more likely to be very interested in PrEP; (3) Perceived risk will mediate the relationship between HIV risk and PrEP interest per hypotheses 1&2. We described the frequencies and percentages of categorical variables, and the median and inter‐quartile range of continuous variables  perceived no lifetime risk of HIV infection, while 15% and 18% perceived a small or high risk respectively. The majority (66%) were very interested in PrEP while 22% were somewhat interested, and 13% reported no interest.3.2lower odds of PrEP interest than those younger than 20 years. The other risk factors were not associated with PrEP interest.The number of reported risk factors was associated with PrEP interest Table ; each ad3.3Both level of perceived risk and reporting “high” perceived HIV risk were associated with PrEP interest Table . AGYW re3.4The indirect effect of epidemiologic HIV risk on PrEP interest through the mediator of perceived HIV risk was significant for both the binary indicator of high HIV risk and HIV risk score . Approximately two‐thirds of both high‐risk and lower‐risk women were very interested in PrEP. This generally high level of PrEP interest has been found in other populations of AGYW and older women in South Africa , 32, 33 4.2Although women with higher epidemiologic risk were more likely to report high perceived risk, 74% of those with high‐risk scores did not have high perceived risk. These results echo findings from FEM‐PrEP that half of women sero‐converting while taking PrEP had reported no perceived chance of HIV acquisition. This was attributed by the investigators to overestimation of protective behaviours and protective reasoning  .4.3The mediation results indicated that perceived risk partially explained the relationship between epidemiologic risk and PrEP interest, thus there is reason to infer that perceived risk may be a motivating mechanism for PrEP interest in higher‐risk women. Future randomized studies should seek to understand if interventions promoting accurate risk perception could promote PrEP initiation . Yet, peMore research is needed to understand why many women who perceive no lifetime HIV risk would be very interested in PrEP. Previous evidence suggests that AGYW’s PrEP decision‐making can be driven by emotion and motivated reasoning about partner risk . Our qua4.4Unlike HIV treatment cascades, our operationalized pre‐initiation PrEP cascade Figure  was non‐Our results highlight one further question about the application of the cascade framework for PrEP. Over 60% of participants with low/moderate‐risk scores were very interest in PrEP, and 12% of women with these scores reported high perceived risk. Should these women be excluded from the PrEP “target population” as defined by cascades? Women may be reticent to report risk behaviours , 42, or 5The results of this study should be interpreted with key limitations in mind. First, participants’ reports of PrEP interest, sexual behaviours and perceptions of risk were likely susceptible to social desirability bias. To mitigate this issue, participants were assured that their responses were confidential and that there were no right or wrong answers. We also used the highest threshold (“very”) for PrEP interest to increase the theoretical specificity of this indicator. Second, few participants had previous knowledge of PrEP and PrEP was not available in the study setting at measurement, thus their reported interest in PrEP was hypothetical and based on limited consideration. Third, perceived HIV risk was measured with a single item, and thus may not capture all dimensions of perceived risk. Fourth, due to low cell counts for the associations between STI symptoms and PrEP interest, and between being separated/divorced/widowed and PrEP interest, tests may have been underpowered. Fifth, causal interpretations of all measures of association presented should be made with caution as they represent cross‐sectional associations; qualitative studies are needed to better understand the causality of the relationships studied. Finally, sexually active AGYW were purposively recruited for participation from urban and periurban settings in Lilongwe. The findings of this study are primarily generalizeable to sexually active AGYW living in similar urban and periurban settings in the region.6Our results bring to light relationships between epidemiologic risk, risk perception and PrEP interest and indicate directions for future research to inform effective HIV prevention programmes. A better understanding of mechanisms motivating PrEP interest beyond perceived risk is needed to inform delivery of PrEP among AGYW in high burden settings. Our results suggest that early demand for PrEP may not be well aligned with epidemiologic risk in this population; more research is needed to understand the implications of expanding or retaining current target population and PrEP eligibility definitions which may exclude many interested and motivated potential users not meeting a cutoff for epidemiologic risk.The authors have no competing interests to declare.LH conducted the analysis and wrote the manuscript under the guidance of NR. NR conceptualized and led the Girl Power study. MH contributed to the overall study conceptualization and HIV‐related analyses. LB and AP Contributed to the overall study conceptualization and design. BM contributed to study design and study implementation. MC contributed to data analysis. All authors contributed to the writing of the manuscript and approved the final manuscript for publication.


RNA interference is a biological process whereby small RNAs inhibit gene expression through neutralizing targeted mRNA molecules. This process is conserved in eukaryotes. Here, recent work regarding the mechanisms of how small RNAs move within and between organisms is examined. Small RNAs can move locally and systemically in plants through plasmodesmata and phloem, respectively. In fungi, transportation of small RNAs may also be achieved by septal pores and vesicles. Recent evidence also supports bidirectional cross‐kingdom communication of small RNAs between host plants and adapted fungal pathogens to affect the outcome of infection. We discuss several mechanisms for small RNA trafficking and describe evidence for transport through naked form, combined with RNA‐binding proteins or enclosed by vesicles. We summarize the evidence and possible mechanisms that small RNAs move within and between plants and fungi in naked form, combined with RNA‐binding proteins or enclosed in vesicles. AGOArgonauteCCcompanion cellCWcell wallCYPcytochrome P450 lanosterol C‐14 α‐demethylaseDCLdicer‐likeDMdesmotubuledsRNAdouble‐stranded RNAEHMxextrahaustorial matrixERendoplasmic reticulumEVextracellular vesicleFCWfungal cell wallFIGSfilamentous organism‐induced gene silencingFPMfungal plasma membraneGGolgiGFPgreen fluorescent proteinHIGShost‐induced gene silencingmiRNAmicroRNAMPmovement proteinMVBmultiple vesicle bodyNnucleusPCMplant plasma membranePCWplant cell wallPDplasmodesmatapiRNAPIWI‐interacting RNAPMplasma membraneRBPRNA‐binding proteinsRDRPRNA‐dependent RNA polymeraseRNAiRNA interferenceRNPCribonucleoprotein complexSCsource cellSEsieve tube elementsiRNAsmall interfering RNASPsieve tube plate or septal pore1Escherichia coli in 1984  of miR393  to meiotically active cells such as anthers and flowers  or externally (apoplast). In either case, evidence exists that small RNAs can be transported either in naked form or encased in vesicles  to the Golgi apparatus and then loading to a complex network of vesicles. Small RNAs can be sorted to transporting vesicles fusing with the plasma membrane and then released by exocytosis , a plasma membrane‐lined pore acting as an intercellular channel that connects the plant cytoplasm of connected cells Figure a. There et al., et al., The PD size exclusion limit is 30–50 kDa and may dictate which forms of small RNAs can move through the PD. Naked small RNAs are around 15 kDa, thus their free diffusion through the PD should not be limited  phloem binds single‐stranded small RNAs moving from cell to cell through the PD , which are at the core of ribonucleoprotein complexes (RNPCs), are important for RNA movement  and an isotrichodermin C‐15 hydroxylase (HiC‐15). Deletion of those two genes in the fungus inhibited microsclerotia formation or hyphae growth, respectively, and down‐regulation of Clp‐1 and HiC‐15 through small RNAs from the host plant interfered with fungal pathogenicity , a novel RNA‐based technology for the efficient control of fungal pathogens and other pests  of how they move remains to be fully determined.4.3et al., B. cinerea DCL1 and DCL2 genes were directly sprayed to Arabidopsis and tomato, treated plants gained resistance to grey mould disease, suggesting the naked exogenous small RNAs were assimilated into the pathogen and interfered with fungal virulence .For both plants and fungi, vesicles containing small RNAs can be generated inside cells as well as secreted to the extracellular environment. Evidence suggests that such vesicles can also be taken up by fungi  were abundant in haustoria and putative exosome vesicles were detected in the extracellular space and extrahaustorial matrix (EHMx), suggesting the existence of an exosome‐mediated secretion pathway for the interaction area between plants and fungi. These and numerous other examples support a role of vesicles in plant–fungal pathogen interactions (Chowdhury, Studies of mammals such as mice also suggested that small RNAs can be transferred between different species mediated by EVs (Knip et al., et al., et al., et al., Direct evidence for the role of vesicles in cross‐kingdom communication could be obtained through the isolation of vesicles derived from HIGS transgenic plants followed by evaluation of the presence of target small RNAs inside vesicles. Such vesicles could then be co‐cultured with fungi to confirm the ability to confer RNA silencing. Using fluorescence or radioactively labelled small RNAs would facilitate monitoring of small RNA movement. Chemical inhibitors such as Brefeldin A (Nebenfuhr The possible mechanisms of small RNA absorption remain unknown. Secretion and absorption may involve cell membrane proteins. Currently, there are limited reports regarding protein channels for small RNA movement in plants. In addition, how specific small RNAs are sorted for secretion and absorption remain enigmatic. Though much work needs to be done, studies of small RNA communication will probably provide applications for enhancing sustainable agriculture. Gene silencing of pathogen genes by HIGS or the direct application of dsRNA, for instance, is a highly promising strategy to provide resistance to plant disease.5The RNAi system is largely conserved among eukaryotes. It plays important biological roles, including in disease processes. Both plants and fungi can generate small RNAs and induce gene silencing. In plants small RNAs can be transported both short and long distances. For the latter, small RNAs are transferred from cell to cell through plasmodesmata and the plant phloem. Small RNAs can be transferred nakedly, bound by proteins, or packed into vesicles. In fungi small RNAs can be transferred through septa pores as well as secreted and absorbed in vesicles. EVs are present at the interface of plant–fungus interactions. However, compelling data are still needed to illuminate the underlying mechanisms by which small RNA communication occurs across plants and fungal cells. The knowledge of how small RNAs are sorted and transferred to target cells is also unclear. Answers to these questions and others related to cross‐kingdom communication will not only enrich our understanding of plant disease processes but also aid in the development of powerful new tools for disease control.


Adoption of emergency department (ED) initiation of buprenorphine (BUP) for opioid use disorder (OUD) into routine emergency care has been slow, partly due to clinicians’ unfamiliarity with this practice and perceptions that it is complicated and time‐consuming. To address these barriers and guide emergency clinicians through the process of BUP initiation, we implemented a user‐centered computerized clinical decision support system (CDS). This study was conducted to assess the feasibility of implementation and to evaluate the preliminary efficacy of the intervention to increase the rate of ED‐initiated BUP.An interrupted time series study was conducted in an urban, academic ED from April 2018 to February 2019 (preimplementation phase), March 2019 to August 2019 (implementation phase), and September 2019 to December 2019 (maintenance phase) to study the effect of the intervention on adult ED patients identified by a validated electronic health record (EHR)‐based computable phenotype consisting of structured data consistent with potential cases of OUD who would benefit from BUP treatment. The intervention offers flexible CDS for identification of OUD, assessment of opioid withdrawal, and motivation of readiness to start treatment and automates EHR activities related to ED initiation of BUP . The primary outcome was the rate of ED‐initiated BUP. Secondary outcomes were launch of the intervention, prescription for naloxone at ED discharge, and referral for ongoing addiction treatment.Of the 141,041 unique patients presenting to the ED over the preimplementation and implementation phases , 906 (574 preimplementation and 332 implementation) met OUD phenotype and inclusion criteria. The rate of BUP initiation increased from 3.5% (20/574) in the preimplementation phase to 6.6% (22/332) in the implementation phase (p = 0.03). After the temporal trend of the number of physician's with X‐waiver training and other covariates were adjusted for, the relative risk of BUP initiation rate was 2.73 . Similarly, the number of unique attendings who initiated BUP increased modestly 7/53 (13.0%) to 13/57  after offering just‐in‐time training during the implementation period. The rate of naloxone prescribed at discharge also increased . The intervention received a system usability scale score of 82.0 (95% CI = 76.7 to 87.2).Implementation of user‐centered CDS at a single ED was feasible, acceptable, and associated with increased rates of ED‐initiated BUP and naloxone prescribing in patients with OUD and a doubling of the number of unique physicians adopting the practice. We have implemented this intervention across several health systems in an ongoing trial to assess its effectiveness, scalability, and generalizability. An estimated 2.1 million people nationally suffer from opioid use disorder (OUD), contributing to nearly 50,000 overdose deaths each year.Opioid agonist medications, such as buprenorphine/naloxone (BUP) and methadone, are the current standard of treatment for OUD and have been shown to reduce withdrawal symptoms, craving, relapse, overdose, and mortality .Numerous patient‐side barriers currently limit the adoption of BUP initiation, including confusion and cultural stigma surrounding medication therapy for OUD and patient perceptions that such treatment is harmful, inferior to detoxification, and even incompatible with being truly “drug‐free.”To address these barriers to implementation and to simplify the practice of ED‐initiated BUP, we developed a user‐centered CDS called EMBED (EMergency department‐initiated BuprenorphinE for opioid use Disorder).A single‐site time series study evaluating the preliminary efficacy of the EMBED intervention was conducted during April 2018 to December 2019 in an urban, academic Level I trauma center ED with 103,000 annual patient visits. The time series was divided into three phases for analysis: 1) preimplementation phase (April 2018–February 2019), 2) implementation phase (March 2019–August 2019), and 3) maintenance phase (September 2019–December 2019). The phased rollout of the CDS intervention began with a soft go‐live in mid‐January 2019. Users were then made aware of the CDS's availability when full functionality was achieved in early March 2019. Provider feedback on the CDS was collected in the implementation phase to assist with planning the subsequent trial. The study protocol was reviewed and approved by our institutional review board (Protocol 2000022749).Eligible patients were adult ED patients meeting the criteria of a computable phenotype derived from electronic health record (EHR) data that was developed to capture ED patients likely to have OUD and not actively on medication for OUD .The study intervention included an integrated Web application for decision support and automation of EHR workflow that streamlines the practice of initiating BUP in the ED Figure . Full dehttp://onlinelibrary.wiley.com/doi/10.1111/acem.14002/full). A convenience sample of physicians completing the tutorial was utilized to gather qualitative feedback via semistructured interview questions. A formal qualitative analysis was not performed, but feedback on the CDS obtained through these interviews was synthesized and categorized according to common, recurring themes as displayed in Data Supplement To enhance use, midway through the implementation period, 5‐minute one‐on‐one tutorials were performed by author WCH to provide just‐in‐time training as an additional academic detailing component of the intervention. Compared to CDS alone, academic detailing combined with CDS has been shown to increase use when introducing a new CDS.The primary outcome of this study was BUP initiation rate in the ED, defined as whether or not an eligible patient was administered BUP in the ED and/or prescribed BUP on discharge from the ED. Secondary outcomes to evaluate preliminary efficacy of the intervention's implementation included the attending physician adoption rate of the practice of ED initiation of BUP at least once in the study phase as well as the following patient‐level rates in the cascade of care for treatment among eligible patients:reach of the intervention—the proportion of the target population that participated in the intervention—was the proportion of unique attendings who ever launched the intervention. Effectiveness was assessed based on the rate of BUP initiation in the ED during the implementation period (the primary outcome). Adoption was based on the proportion of unique attendings who initiated BUP, and Implementation—the extent to which the intervention is implemented as intended—was the proportion of phenotype‐positive patients for whom the intervention was launched. Maintenance was the rate of BUP initiation in the ED after training ended (September–December 2019).The RE‐AIM  framework was also used to evaluate the success of the intervention.Quantitative outcome data from the three study periods were extracted from the study site's EHR database using structured query language (SQL). The SQL query  performed by author WCH in the ED. The interview consisted of three parts: 1) system usability scale (SUS), 2) net promoter score (NPS), and 3) three open‐ended questions focusing on barriers to implementation and ways to address them (Data Supplement Patient characteristics were summarized as means and standard deviations (SDs) or frequencies and percentages as appropriate for the preimplementation and implementation periods. For patients with multiple visits, only the first visit was used to analyze patient‐level outcomes. However, analysis of physician‐level outcomes  included multiple visits made by a single patient. T‐tests and chi‐square or Fisher's exact tests were used to make unadjusted comparisons of demographics, primary outcome rates of BUP initiation and secondary patient outcomes between periods. McNemar's test was used to compare the number of unique attendings who initiated BUP who were present during both the preimplementation and the implementation phases, while the generalized estimating equation method was used as a supportive analysis of all attendings . Following unadjusted analysis, a multivariate logistic regression model was used to adjust for age, race, sex, the number of waivered physicians, naloxone prescription within the past 24 months, and OUD diagnosis on problems list. To further contrast BUP initiation between study phases while “detrending” the time effect, Poisson regression was utilized for the interrupted time series adjusted analyses. For this analysis, an offset was used for the monthly volume of patients presenting to the ED with OUD. Relative risk and 95% confidence interval (CI) are reported, with values above 1 corresponding to greater relative rates of ED BUP initiation. Physician X‐waiver status was used as a covariate in this analysis. All analyses were performed using SAS 9.4. Statistical significance was set as p < 0.05, two‐sided.Of the 141,014 total ED visits during the two study phases used for analysis of main outcomes, 906 (574 preimplementation and 332 implementation) met inclusion criteria for analysis. Of these 906 OUD phenotype–positive visits, 98 patients in total (11%) had more than one ED visit, including a maximum of four visits (of which four patients had). Across these two phases, patients had a mean age of 39.9 years, 31.2% were female, 71.7% were white, and 73.4% had Medicaid insurance  was 3.5% 20/574) in the preimplementation phase and 6.6% (22/332) in the implementation phase  who initiated BUP did not change significantly from 14/58 (24.1%) in the preimplementation phase to 16/58 (27.6%) in the implementation phase (p = 0.65). After just‐in‐time training was added for the second half of the implementation phase, the number of unique attendings who initiated BUP increased from 7/53 (13.0%) to 13/57 . The addition of just‐in‐time training was also associated with an increase in the proportion of unique attendings who launched the intervention in the implementation period .Evaluation of the intervention's implementation using the RE‐AIM framework Table  shows thAs shown in Data Supplement In this interrupted time series evaluating the preliminary efficacy of the EMBED intervention at a single site, implementation of a user‐centered CDS with a brief, just‐in‐time training was associated with close to a doubling in the BUP initiation rate in the ED for patients with OUD and receiving a prescription for naloxone at discharge. After adjusting for the temporal trend of physician waiver training, the increased rate of BUP initiation was no longer statistically significant. The primary outcome was lower in the maintenance phase compared to the implementation phase Figure . Given tDespite the ED's potential to initiate treatment for a large number of patients with OUD, a cohort study conducted in Massachusetts indicates that this potential has yet to be realized. Among the 17,000 patients who had an ED visit for nonfatal opioid overdose between 2012 and 2014, only one in three received a MOUD in the 12‐month period following their overdose. Compared to no MOUD treatment, both methadone and BUP were associated with decreased all‐cause mortality and opioid‐related mortality.A number of barriers currently limit the rate of BUP initiation in the ED. One such barrier directly addressed by the EMBED intervention is how the majority of emergency physicians feel unprepared to provide OUD care; a 2019 survey of physicians from two urban, academic EDs found that fewer than half of respondents felt prepared in several components of OUD emergency care.Although 93% of emergency medicine faculty had an X‐waiver by the end of the preimplementation phase, the proportion of unique attendings who had adopted the practice of ED initiation of BUP was low (19.2%). In the implementation phase, the proportion of X‐waivered physicians increased slightly to 97%. However, during the implementation phase, we observed a close to a doubling in the proportion of unique attendings who had adopted the practice of ED initiation of BUP . Although this increase was not statistically significant, it seems more clinically significant and suggestive that the barrier of X‐waivered physicians actually adopting the practice of ED‐initiated BUP may require a user‐centered CDS with just‐in‐time training. Similarly, although not statistically significant, the proportion of unique attendings who initiated BUP after we began just‐in‐time training nearly doubled. Taking all of these findings together, we hypothesize that all three  may be necessary and complementary to increase adoption of ED‐initiated BUP.Creation of a CDS with these capabilities is the result of our choice to employ a user‐centered design process which involved identifying users' needs and incorporating their feedback in each phase of iterative prototype development.The decision to incorporate training as part of the intervention turned out to be one of the study's strengths. Although not statistically significant, increased rates of the primary outcome with one‐on‐one training component suggests that this feature of the intervention is necessary to increase user recognition of its presence and value. The training component also leverages the science of “diffusion of innovations,” as a way to promote intervention adoption via communication and sharing by local champions and among colleagues within a social system.Planning for the upcoming trial is also supported by the collection of qualitative data that resulted in a richer data set and a better understanding of barriers to adoption and possible solutions. Some of these findings include the need to increase awareness of the intervention, for the CDS to display calculated scores on the home screen, and to increase clarity of the referral process.There are many barriers to adoption of ED initiation of BUP into routine emergency care. For example, the EMBED intervention does not address negative attitudes toward addiction, the inconvenience of obtaining an X‐waiver to prescribe BUP, the limited number of providers with an X‐waiver, and access and availability to MOUD in the community. This study was also conducted in a single ED with a high rate of X‐waivered physicians compared to nonacademic or rural EDs. Because physicians without an X‐waiver are limited in their ability to prescribe BUP for home induction, this could have had an impact on the primary outcome of rates of ED BUP initiation. Another weakness of our study's design is that it was neither randomized nor controlled, so causality between the intervention and outcomes could not be established. Given the urgency of the opioid crisis, additional temporal trends could have occurred that were not adjusted and remain as confounders. The increase in X‐waivered physicians, although identified and adjusted during analysis, is an example of one trend which alone could have impacted study results if overlooked. Finally, in an effort to avoid unintended consequences from a hard‐stop alert that triggered the CDS, not all attending physicians utilized the intervention during the implementation phase.Future research could explore alternative approaches to triggering the intervention in a nonobstructive manner and whether lack of a hard‐stop alert represents another barrier to physician adoption. Additional investigative efforts are necessary to explore the extent to which findings of the study are generalizable across different patient populations, health care systems, and EHR platforms. Similarly, the scalability of EMBED as a solution to increasing adoption of BUP initiation in the ED will require further investigation, particularly across different health care systems using different EHRs. The upcoming pragmatic trial to be launched in 20 EDs across five health care systems using different EHR vendors may provide answers to some of these remaining questions.This interrupted time series study demonstrates that implementation of the EMBED intervention in a single ED is feasible, acceptable, and estimated to be efficacious at increasing rates of buprenorphine initiation for the treatment of opioid use disorder in the setting of a temporal trend of increased physician X‐waiver training. The intervention was associated with a doubling of the buprenorphine initiation rate, emphasizing the importance of user‐centered health IT to change practice around evidence‐based medicine that may be slowly adopted. Our findings suggest that we should proceed with the larger effectiveness trial to explore whether these preliminary findings are significant and generalizable. Knowledge gained from the study will continue to inform the trial, particularly the finding of how one‐on‐one training is a necessary part of the intervention to increase and sustain adoption of clinical decision support.Data Supplement S1. Supplemental material.Click here for additional data file.Data Supplement S2. Supplemental material.Click here for additional data file.


Dear editor,Since the middle of December 2019, human‐to‐human transmission of coronavirus disease (COVID‐19) has occurred among close contacts.1PTSD is defined as a stress‐related disorder with subsequent autoimmune disease that may arise after exposure to a serious traumatic event or injury.+ Th1 subsets release Th1 cytokines that activate the inflammatory cellular immune response. The response involves IL12 and IFN‐G, which is strongly suppressed by IL10. This action helps to shift the cellular immune response from anti‐inflammatory process of Th1 to Th2 via adrenergic agonists as a result of stress. Moreover, the immunosuppressive effect is specific to the inflammatory cellular immune system. A shift from Th1 to Th2 cellular is strongly enhanced through the suppression of IL12, which is a major Th1 agonist within humoral immunity. However, the shift occurs proportionately rather than quantitatively. These effects above are observed over both the short and long term in PTSD. This decreased reaction of the immune system is also observed due to senescence with the chronic down‐regulation of cortisol receptors sites. The down‐regulation of cortisol receptors may reduce the capacity of lymphocytes to respond to anti‐inflammatory signals and allow other cytokine‐mediated processes to dominate in patients with PTSD.A study showed that chronic stress elicits the simultaneous suppression and enhancement of the immune response via alteration of the cytokine expression pattern.2Many clinical observations have shown that elderly patients, those with an underlying chronic disease and treated with immunosuppressants, or patients otherwise in an immunosuppressed state could suffer from a decreased immune response and greater susceptibility to life‐threatening virus infections. Such infections show rapid national and international spread, such as in the case of SARS‐CoV‐2, which is currently posing a global health emergency.Another clue has been showed that pregnancy is an independent risk factor to develop severe virus pneumonia under immune tolerance. The pregnancy bias toward Th2 system dominance which left pregnant woman vulnerable to viral infections might bring a challenge for the prevention of SARS‐CoV‐2 infection.In addition, it has been showed that immunosuppression (secondary to disease or treatment) is by far the most identified risk factor to develop severe viral pneumonia by different respiratory virus families.3During such a stressful period, psychological services and crisis interventions are needed at an early stage in almost all groups to reduce PTSD and in order to alleviate the current acute stress responses of individuals and patients and reduce the incidence of PTSD to prevent immunosuppression, thus breaking the vicious circle. Evidence‐based medicine is of great importance to conduct population‐based psychiatric surveys on the symptomatology of PTSD.The authors have no conflicts of interest.The work was supported by the National Key Research and Development Program of China (2016YFC1307100), the National Natural Science Foundation of China , the Innovative Research Team of High‐level Local Universities in Shanghai, and the Key Program of Nantong University Clinical Medicine Special Project (2019JZ021).