Despite breakthroughs in antiviral therapies, chronic viral hepatitis B and C are still the major causes of liver fibrosis and hepatocellular carcinoma (HCC). Importantly, even in patients with controlled infection or viral cure, the cancer risk cannot be fully eliminated, highlighting a persisting oncogenic pressure imposed by epigenetic imprinting and advanced liver disease. Reliable and minimally invasive biomarkers for early fibrosis and for residual HCC risk in HCV-cured patients are urgently needed. Chronic infection with HBV and/or HCV dysregulates oncogenic and profibrogenic signaling within the host, also displayed in the secretion of soluble factors to the blood. The study of virus-dysregulated signaling pathways may, therefore, contribute to the identification of reliable minimally invasive biomarkers for the detection of patients at early-stage liver disease potentially complementing existing noninvasive methods in clinics. With a focus on virus-induced signaling events, this review provides an overview of candidate blood biomarkers for liver disease and HCC risk associated with chronic viral hepatitis and epigenetic viral footprints. Chronic liver disease is a major health problem and globally associated with > 2 million deaths per year . The mosDespite tremendous advances in antiviral therapies, chronic viral hepatitis B and C are still the major etiology for chronic liver disease. Worldwide, an estimated 180 million people live with hepatitis B virus (HBV) and 75 million with hepatitis C virus (HCV), and for most, testing and treatment remain beyond reach . Both viThe most important measure to reduce HCC risk is to eliminate the underlying etiology. However, even though viral infection with HBV or HCV can be controlled or cured, the risk of developing HCC cannot be fully eradicated due to multiple reasons, especially in patients with already advanced liver disease. The mechanisms are not well understood, but evidence points towards epigenetic viral footprints that maintain dysregulated pro-oncogenic signal transduction. It is thus crucial to be able to identify patients with elevated HCC risk to stratify for a more frequent liver screening. Today, liver disease diagnosis and HCC risk assessment relies on a combination of imaging, blood markers and liver biopsies. While liver tissue allows a detailed transcriptomic HCC risk assessment linked to predictive transcriptomic signature ,20, liveAlthough HBV and HCV are hepatotropic, causing similar liver disease, they are very different viruses. HBV is an hepatotropic DNA virus of the Hepadnaviridae family, which specifically infects hepatocytes via the recently discovered functional receptor sodium taurocholate co-transporting polypeptide (NTCP)  and epidHCV is a single-stranded, positive-sense RNA virus of the Flaviviridae family that requires multiple host entry factors, including receptor tyrosine kinase signaling. After endocytosis, the RNA genome is translated into a viral polyprotein at the endoplasmic reticulum, leading to a massive reshaping of host membranes to a replication complex termed the membranous web. De novo virus assembles at lipid droplets, which are accumulated at the replication complex . HCVThe most important measure to treat liver disease and prevent HCC formation is the removal of the underlying etiology. The challenges to help patients with viral hepatitis are heterogenous: while an efficient preventive HBV vaccine is available, established chronic HBV infection can only be controlled but rarely eliminated due to a persistent chromosome-like viral DNA species and genome integrations . A “funcFor HCV, no vaccine is in reach, but the novel generation of antiviral therapies with direct acting antivirals (DAAs) can efficiently cure HCV infection . A new gThe majority of HCCs arise from liver cirrhosis, and thus the current HCC risk assessment is largely coupled to the staging of liver fibrosis. Despite the screening programs in cirrhotic patients, often the HCCs are diagnosed at an advanced stage. Less than 30–40% of them are eligible for a curative treatment using surgical approaches or radiological ablation . CurrentSeveral patient-derived transcriptomic signatures that associate with HCC risk and which are specific for certain etiologies or reflect a hepatic state of struggle in the liver independently from the underlying etiology have been identified in recent years . AlthouCurrent guidelines recommend the screening of HCC in at-risk patients using ultrasonography (US) of the liver every 6 months with or without serum alpha-fetoprotein (AFP) ,47, the Additional scoring systems have been predictive for HCC risk in patients with chronic liver disease. The GALAD score ,61 is deSignal transduction is an essential process involved in almost every step of cellular homeostasis. Signaling is tightly controlled, transmitting signals between cellular compartments and regulating gene transcriptional responses. Thus, the chronic dysregulation of signaling pathways is involved in the majority of diseases, including cancer . VirusesDespite differences in terms of structure and life cycle, HBV and HCV are believed to share common pathways which influence hepatic fibrosis and promote hepatocarcinogenesis ,72. It bGP73 has been suggested as a serum biomarker for liver cirrhosis in individuals with chronic HBV . MoreoveRecently, mac-2-binding protein glycan isomer (M2BPGi) has been shown to enhance the aggressiveness of HCC via the activation of the mTOR signaling pathway , harboriSignaling pathways play a pivotal role during viral hepatitis and liver fibrosis. Epidermal growth factor receptor (EGFR) signaling is required by HBV and HCV for entry ,105, whePersistent oxidative stress is an important factor in virus-induced liver fibrosis. Especially HCV infection, and its massive reorganization of cellular membranes to the replication complex, is a major cause of UPR and oxidative stress . HCV proHCV infection induces hypoxia in infected cells and stabilizes HIFs , which iGlypican-3 (GPC3) is an heparan sulfate proteoglycan that regulates cell morphology via the Hippo/YAP pathway. In a normal liver, the HCV entry factor CD81 interacts with GPC3 and inhibits the Hippo/YAP pathway. HCV E2 protein mimics the role of CD81 stimulating Hippo/YAP by engaging GPC3. In a chronically inflamed liver, HCV is thus likely to promote hepatic neoplasia by the growth of early CD81-negative neoplastic hepatocytes, which are resistant to HCV infection . Given iEpigenetic imprinting acts as a memory for environmental influences and disease . This haEpigenetic regulation of gene expression by HCV has been observed at the histone level. Such epigenetic footprints have been identified in cell lines and patients with chronic HCV infection and NASH ,142,143,HCV infection and liver disease largely impact miRNA expression , which iAlthough HBV and HCV cause both liver disease progression and HCC, the clinical challenges differ substantially. HBV infection can only be controlled but not eradicated because of a persisting chromosome-like cccDNA and genome integration . HCV infWhile HCV infection was rendered a curable disease due to efficient direct antiviral therapies, an important unmet medical need is to identify the fraction of patients with elevated HCC risk. In future, etiology-specific epigenetic markers, including histone modifications and miRNAs, will provide new perspectives for translation into correlating secreted biomarkers, which may be used for personalized approaches targeting specific groups of patients, e.g., biomarkers for HCC risk in HCV-cured patients.


Mild traumatic brain injury (TBI) often results in pathophysiological damage that can manifest as both acute and chronic neurological deficits. In an attempt to repair and reconnect disrupted circuits to compensate for loss of afferent and efferent connections, maladaptive circuitry is created and contributes to neurological deficits, including post-concussive symptoms. The TBI-induced pathology physically and metabolically changes the structure and function of neurons associated with behaviorally relevant circuit function. Complex neurological processing is governed, in part, by circuitry mediated by primary and modulatory neurotransmitter systems, where signaling is disrupted acutely and chronically after injury, and therefore serves as a primary target for treatment. Monitoring of neurotransmitter signaling in experimental models with technology empowered with improved temporal and spatial resolution is capable of recording in vivo extracellular neurotransmitter signaling in behaviorally relevant circuits. Here, we review preclinical evidence in TBI literature that implicates the role of neurotransmitter changes mediating circuit function that contributes to neurological deficits in the post-acute and chronic phases and methods developed for in vivo neurochemical monitoring. Coupling TBI models demonstrating chronic behavioral deficits with in vivo technologies capable of real-time monitoring of neurotransmitters provides an innovative approach to directly quantify and characterize neurotransmitter signaling as a universal consequence of TBI and the direct influence of pharmacological approaches on both behavior and signaling. Affecting over 2.5 million people across the United States, traumatic brain injury (TBI) is the leading cause of non-fatal injuries ,2. Over Damage after TBI can be classified as focal or diffuse injury. Focal brain injuries often produce overt injuries such as skull fracture, intracerebral and subdural hematomas, subarachnoid hemorrhage with significant elevations in intracranial pressure . DiffuseMild TBI cases 20–50%) develop persistent morbidities within one-month post-injury . Patient–50% deveAlthough diffuse white matter disconnections are a characteristic, multicentric outcome of the mechanical impact of TBI, the neurological impairment may reflect dysfunction rather than just neuronal damage. The initial injury and subsequent injury cascade influence the functional state of surviving neurons, astrocytes and microglia and compensatory alterations involving multiple neurotransmitter systems that lead to synaptic deficits that lead to deficits in circuit function. Insights into the neuronal circuits reveal anatomically unique changes in several neurotransmitter systems that modify neuronal dynamics, synaptic function, receptors and transporters ,30,31. OResearch into the pathology and pathophysiology of TBI has been rapidly advanced by the development of animal models. So far, the animal models replicate injury types, providing a better understanding of molecular and cellular mechanisms, neurological deficits along with possible rehabilitative/neuroprotective strategies. Rodent and non-human primate TBI models simplify the complex events observed in humans while consistently and robustly reproducing the primary and secondary injuries. In addition, TBI models have provided the opportunity to evaluate for translatable biomarkers that can track TBI progression in humans.For direct clinical relevance to diffuse TBI, several diffuse models of injury including fluid percussion, weight-drop, and blast injury models have been developed and modified to provide a preclinical platform recapitulating features of human TBI condition. The fluid percussion injury (FPI) imparts a fluid pressure pulse to the intact dura through a craniotomy which can be located laterally or centrally over the brain (for a diffuse or mixed pathology). The pressure pulse is a variable that can be controlled to alter injury severity. Midline  FPI is predominantly used to produce DAI, petechial hemorrhage, and white matter damage in both hemispheres . In releBehavioral alterations have been observed in most of the TBI animal models and characterization of behavioral assays is essential for understanding neural systems influencing the behavioral phenotype. As a valuable read-out to probe detailed mechanisms of functions and dysfunctions, various behavioral paradigms help characterize the behavioral repertoire similar to the acute and chronic sequelae that TBI survivors suffer. The assays provide for the sensitive, objective, and quantifiable behavioral measures with the use of video tracking, automated recordings, and detection technologies to record and analyze the behaviors. The assays include neurobehavioral assessment , exploraAnimal models have extended clinical findings to show impaired neuroplasticity after diffuse TBI . Neural Dysregulation of the neurotransmitter systems has been associated with several neurodegenerative diseases as well as neuropsychiatric disorders. Alterations in neurotransmitters after TBI leads to compensatory alterations in neurotransmitter receptors and related signaling pathways, important for circuit-level information transmission. The function of brain circuits is governed, in part, by several neurotransmitter systems that regulate neuronal information flow through exocytosis, uptake and recycling events.Glutamate is the primary excitatory neurotransmitter and more commonly implicated in TBI neuronal susceptibility to excitotoxic injury . ReleaseSeveral studies demonstrate significant glutamate dysregulation as an instigator of acute pathophysiology in days following TBI. Clinical microdialysis studies demonstrate increased extracellular glutamate levels as early as 24 h post-injury and up to 9 days post-injury ,61,62. FA and GABAB receptors located on the axonal terminals GABAergic transmission is terminated by clearance from the extracellular space by GABA transporters (GATs) on surrounding neurons and glia. GABA receptors regulate information processing with high physiological relevance through the maintenance of phasic and tonic inhibition, and modulation of neuronal excitability  choline infusion measurements revealed that TBI alters ACh turnover in the rat midbrain at 12 min and 4 h after moderate FPI, indicating enhanced cholinergic transmission [3H] choline uptake thus suggesting that the cholinergic dysfunction is mainly associated with lower ability of cholinergic neurons to clear extracellular choline [Animal studies have shown acute effects of FPI on increased hippocampal ACh levels . Phosphosmission . Cognitismission ,118. Expsmission . It has  choline . In vivo choline ,122. At  choline . Other f choline . There i choline  and redu choline .Serotonin  is involved in the regulation of mood, where dysregulation of 5-HT neurotransmission plays a crucial role in neuropsychiatric disorders in the general population and TBI patients . ModeratThe chronic consequences of TBI on psychiatric health have been identified not only after severe injury but also in several cases classified as mild to moderate. Psychiatric problems can be a major clinical problem as it potentially interferes with rehabilitation and overall recovery. The 5-HT system has wide distribution throughout the brain regulating various behavioral processes . Over onThere is growing evidence that changes in neurotransmission associated with TBI can instigate, accelerate, or exacerbate symptoms associated with chronic traumatic encephalopathy, epilepsy, PD, AD (and other forms of dementia), amyotrophic lateral sclerosis, HD, PTSD, ADHD, anxiety disorders, and depression disorders ,135. ModStudies evaluating the extracellular dynamics of neurotransmitters can provide critical information regarding neuronal communication that is disrupted after TBI. Insight may be gained into aspects of functional neuronal communication by evaluating region-dependent pre-synaptic release, extracellular clearance, basal concentrations in anesthetized animals and behaviorally relevant signaling in freely-moving animals. Several techniques have been developed and applied for in vivo measurement of neurotransmission in various brain disorders. The functional role for neurotransmitter release events in the regulation of homeostasis, as well as influence on behaviors, have been previously reported. The use of cell-based fluorescent sensors , microdi−1). The microelectrode measures the current as cyclic voltammogram as a characteristic oxidation and reduction peaks by subtracting the large background current. Other biosensors use enzyme-based microelectrodes with amperometric detection that provides the advantage of better resolution and minimal invasion/tissue damage [Electrochemical detection with biosensors allows for real-time observation and quantification of neurotransmitter events. Biosensors often use a biological element (enzyme) to bind a specific analyte of interest and through the application of current, can detect and quantify the extracellular levels and kinetics of release and clearance at 2–100 Hz. The size of the sensors has also been decreased and in some instances, consists of several microsensors on a single electrode. Application of various exclusion layers to the microsensors during the fabrication process allows for increased selectivity, to avoid recording endogenous electroactive molecules that can prevent the detection of the analyte of interest. In vitro calibration allows for calculations where the current generated with applied potential is proportional to the analyte concentration. Electrochemical biosensor design continues to evolve with the recent implementation of thin needle-type probes that improve spatial resolution within subregions and small nuclei. The enzyme-modified electrodes and fast-scanning cyclic voltammetry behave been notably employed for spatial monitoring of rapid neurotransmitter dynamics. Fast-scanning cyclic voltammetry applies a triangular voltage wave to a carbon fiber electrode at high scan rates  in rats. The somatosensory WBC in rodents processes sensory information from the mystacial whiskers for assessing spatial and textural information through thalamocortical and corticothalamic circuitry . WhiskerAs observed by Thomas et al., TBI-induced pathology in the VPM and S1BF results in maladaptive circuit regeneration resulting in the development of late-onset, persistent sensory sensitivity to whisker stimulation during the whisker nuisance task (WNT) ,154,155.The use of whisker barrel circuit serves as an ideal model to study TBI-induced neurotransmission in a behaviorally relevant circuit, showing hypersensitive glutamate signaling to be associated with the severity of response to the WNT . ElectroPreviously, studies on the contribution of circuit dysfunction to post-traumatic morbidity, in the form of neurotransmission, have relied on measurements obtained by microdialysis techniques. Microdialysis involves implantation of a probe with a 1–2 mm (for rats and mice) semi-permeable membrane for diffusion of extracellular neurotransmitters in artificial cerebral spinal fluid that is collected and assessed using HPLC. The probe and cannula implantation cause tissue damage 300–500 µm away for the probe . NeurotrDuring the decades that neurochemical measurements largely relied on microdialysis, parallel developments were made on methods to measure at second and sub-second timescale with improved resolution. The developments were necessary since the cascade of events that follow signal transduction in neurotransmitter signaling involves a vast range of spatial and temporal scales. Initially, the microelectrodes developed were carbon-fiber based with low reproducibility and not suitable to measure molecules that could not be oxidized or reduced, like glutamate, GABA and ACh. Progress in technical advances over the past several years in microfabrication methods to create electrochemical biosensors containing more than a single microelectrode  have increasingly shown to be effective tools for neurotransmission detection. MEAs have served as an interface to develop amperometric enzyme-based biosensors for measurements of neurotransmitters, including electroactive and non-electroactive neurotransmitters in the extracellular space of the brain . There aIn vivo electrochemical recordings using amperometry in awake, freely moving animals allow for assessment of real-time neurotransmitter changes during behavioral tasks. This remained challenging due to the depth of the brain regions and the necessity to record from freely behaving animals, but advances in microelectrode fabrication allow for probing chemical events for longer-term experiments. Several studies have begun to use this approach to study spontaneous neurochemical events in relation to well-defined, quantifiable behaviors of feeding, reward-seeking, sleep-wake, addictive and attention-deficit hyperactivity behaviors ,194,195.pO2) thereby disrupting neuronal circuits [pO2 in anesthetized rat brain with excellent electrocatalytic activity towards oxygen reduction and low detection limits [Advances in in vivo assessments have enabled us to gain insight into the nature of the subthreshold events that underlie evoked action potential discharge in anesthetized and awake animals. Specifically, direct measurement of brain chemical signaling provides valuable information when combined with other physiological methods to manipulate neuronal activity with sophisticated observational work and casual manipulation studies. Targeting neuronal systems infers causality between neurotransmitter events and behavior in conjunction with optogenetic-based  or chemocircuits ,203. Whicircuits . The cern limits . In a prn limits , implicaExceptional progress had been made towards electrochemical analysis of behaviorally relevant neurotransmitter signaling in the extracellular space as demonstrated by recordings in non-human primates ,207 and Throughout this review, we have focused on circuit reorganization and dysfunction as primary mechanisms towards changes in neurotransmitter signaling after TBI. The following section is intended to acknowledge peripheral influences that have recently been indicated in mediating neurotransmission that may be relevant to treatment and application for in vivo neurotransmitter monitoring.Emerging studies have reported that a substantial population of TBI patients, as high as 25%, report chronic endocrine dysregulation involving growth hormone, gonadal, thyroid, adrenal and antidiuretic hormones ,212,213.Sex-related differences in human brain structure and susceptibility to neuropsychiatric diseases have been documented . Yet, agBifidobacterium and Lactobacillus spp. produce GABA, Bacillus spp. produce DA, Lactobacillus spp. produce ACh and Escherichia spp. produce 5-HT [There has been a growing support of the influence of multiorgan responses on the acute and chronic effects of TBI. Such interactions between the brain and the systemic physiology involving the function of body organs after TBI can indirectly influence neurotransmitter systems or modulators. The sequela of TBI induces significant alterations in metabolic and enteric function, affecting peripheral systems. TBI survivors often report of gastrointestinal dysfunction with reports increased gut permeability , reduceduce 5-HT ,238,239 uce 5-HT . The intuce 5-HT .Mild TBI enhances liver inflammatory markers by altering the redox homeostasis and given their wide implications for their interaction between brain and body, leakage of pro-inflammatory factors through the disrupted BBB can exacerbate brain injury pathology ,243,244.In this review, we have discussed the clinical and experimental evidence on the acute and chronic pathophysiological responses after mild TBI, animal models of diffuse brain injury, alterations of neurotransmitter systems that underlie the circuit deficits which can compromise and/or compensate the processes that enables neuronal response to injury. Alterations in the TBI-induced neurotransmitter systems that form the core machinery expressed by most of the neurons have been considered crucial in the development of PCSs. Multiple in vivo electrochemical measurements with increasing resolution offers a portal to directly quantify and characterize the dynamic real-time neurotransmitter signaling mechanisms that operate during behavior to provide important details of the pathophysiology of circuit function. Advances in the application of interference tools permit more direct modulation of in vivo control of neurotransmitter signaling in animals engaged in freely moving and defined behaviors (see


Advances in electroanatomic mapping (EAM) technology have facilitated improved success and safety profiles in the field of catheter ablation. However, these advances in their current iteration may be of limited value in ablation performed in very small children. The present case report highlights the application of current EAM technologies in an infant with incessant arrhythmias and includes a discussion regarding the application and limitations of newer mapping and ablation technologies in this unique and fragile patient group. Hospital course was notable for circulatory compromise and respiratory distress requiring intubation and invasive vascular line insertion. Additional complicating events included recurrent bloody stools , aspiration and ventilator-associated pneumonias, and urinary tract infection. This also made enteral medication administration difficult and inconsistent. Several antiarrhythmic medications were trialed, including enteral digoxin, propranolol, flecainide, and sotalol, while enteral; intravenous amiodarone; intravenous esmolol; and, additionally, heavy sedation (including dexmedetomidine) were also implemented. However, rhythm control was never achieved despite the use of combination therapies. Furthermore, the observation of an extremely wide QRS tachycardia while on flecainide and digoxin required their cessation. Sotalol uptitration was prevented due to QT prolongation. Although rate control could be achieved with combination intravenous amiodarone and esmolol, multiple attempts at conversion to enteral dosing while preserving stable hemodynamic and respiratory statuses were unsuccessful.A three-month-old, 4.2-kg infant  was brought to the electrophysiology (EP) laboratory for EP testing and catheter ablation of prenatally diagnosed and postnatally recalcitrant atrial tachycardia. A large secundum atrial septal defect (ASD) was also prenatally diagnosed and postnatally confirmed. Focal ectopic atrial tachycardia was documented on 12-lead electrocardiogram Electrophysiology testing and catheter ablation were performed with bilateral femoral venous access  and transesophageal catheter insertion. Vascular access was difficult given the concomitant presence of a right lower-extremity peripherally inserted central catheter (PICC) line, left femoral arterial line, and right internal jugular central venous catheter. The Ensite™ Precision™ system  was incorporated for electroanatomic mapping (EAM) support. A 4-Fr quadripolar catheter was used for initial EAM geometry creation and a 4-Fr deflectable decapolar catheter was then cannulated in the coronary sinus. For activation mapping and ablation, the quadripolar catheter was exchanged with a 7-Fr small-curl, 4-mm nonirrigated-tip radiofrequency (RF) ablation catheter. Fluoroscopy was required during vascular access and catheter advancement/manipulation to avoid entanglement with existing vascular lines, to confirm right versus left atrial positioning during mapping, and to monitor catheter course and stability during ablation. Anticoagulation with serial doses of intravenous heparin was maintained. and 2B. Ablation with a 20-second, 20-W maximum power delivery resulted in arrhythmia suppression during RF application, with subsequent resumption. The catheter tip was adjusted slightly superiorly and a 20-second RF application, this time with a 30-W maximum power delivery, resulted in tachycardia termination in six seconds without further recurrence . A 20-second insurance lesion was also delivered. Postablation testing demonstrated normal atrioventricular conduction, whereas ventricular pacing using the ablation catheter confirmed midline, decremental conduction. The catheters and sheaths were removed at the conclusion of the procedure.While simultaneous multipoint acquisition could be used for initial geometry creation, point-by-point contact activation mapping was performed instead herein with the ablation catheter. Use of a multielectrode mapping catheter that would have permitted simultaneous, multipoint mapping was deferred due to vascular access constraints and considerations. Mapping involved a cumulative time of 30 minutes for acquisition and manual timing evaluation, with a total of 49 points included. Focal atrial tachycardia was present, with the site of earliest activation localized to the left atrial aspect of the inferior, anterior rim of the ASD This case represents one of the smallest pediatric patients who has undergone mapping and ablation using the most current iteration of one of the most widely used EAM systems for pediatric and adult electrophysiology procedures. While a reasonable outcome without complications was achieved, the case serves to highlight the progress made as well as the continued limitations inherent in the application of advanced mapping systems for ablative therapy in very small patients.5 Fundamentally, the most widely available and employed EAM systems rely on impedance- and/or magnetic field–based tracking to determine and follow catheter electrode locations. Signal acquisition is typically performed with contact between the catheter electrode(s) and myocardial tissue to record either unipolar or bipolar electrograms. The acquired signals can then be interpreted with regard to their timing (in relation to a prespecified reference site) and amplitude. Timing and amplitude provide the framework for both activation and substrate mapping within the geometric structure created during the movement of catheters within the heart.Electroanatomic mapping systems were developed to facilitate arrhythmia characterization with regard to both mechanism and location and to ensure a better accuracy of ablation delivery. Technology has since progressed to these systems providing real-time visualization of multiple catheters within the cardiac and intravascular spaces; sophisticated cardiac geometry creation with the integration of intracardiac echocardiogram, computed tomography, or magnetic resonance imaging data; dense and rapid mapping of complex arrhythmias within heterogeneous myocardial substrates; real-time monitoring of catheter-tip pressure during ablation delivery; and visualization tools permitting the performance of procedures with very little or no fluoroscopy.8 In addition, advances in ablation technology now permit more accurate local electrogram signal acquisition during ablation delivery, focused open irrigation directed to the site of tip-to-tissue contact during RF energy delivery, and extrapolation of lesion efficacy based on duration and force application data.New and advanced features of EAM systems now permit rapid, automated, multipoint acquisition for faster and simultaneous geometric shell creation, substrate definition , and arrhythmia characterization.Despite many of these advances and features being theoretically at our disposal in this current case, we found that most were of limited utility in such a small patient. Furthermore, the use of these advanced features and tools, particularly in conjunction with the recommended proprietary catheters, would have introduced additional and potential procedural risks as well as substantial increases in procedural costs.9 The authors additionally found that more infant ablation procedures were performed by centers that submitted more procedures overall to the PCAR, which suggests that procedural volume and experience may contribute to these comparable outcomes between age groups. Akdeniz et al. reported on their single-center experience with catheter ablation in very small infants aged younger than one year, with a total of five patients having undergone successful ablation.10 In this small cohort, two had notable congenital heart disease (CHD) and all had accessory pathway–mediated supraventricular tachycardia that was ablated with cryotherapy or RF without major complications. EAM was employed in all elective cases (one case was performed emergently without EAM). Bigelow et al. reported two cases of successful accessory pathway ablation with EAM in infants with CHD and Wolff–Parkinson–White syndrome.11 Ozaki et al. compared outcomes of RF ablation in patients weighing less than 10 kg to those of larger patients.12 Consistent with PCAR data findings, more patients weighing less than 10 kg had CHD as compared with larger patients (45%). Between two and four vascular access sites were employed and catheters of sizes of up to 8.5 Fr were used. RF ablation and EAM were used in all cases with acute success achieved in approximately 91% of patients, with a 15% recurrence rate. Finally, Takeshita et al. reported successful RF ablation of monomorphic right ventricular outflow ventricular tachycardia in a 2.9-kg infant with Ebstein’s anomaly via single catheter access through the right internal jugular vein.13Data on catheter ablation procedures and outcomes in very small patients have generally been favorable. Blaufox et al. on behalf of the Pediatric Catheter Ablation Registry (PCAR) noted comparable success and complication rates between patients aged 18 months or younger and their older pediatric counterparts.. Patch overlap and poor skin contact can lead to suboptimal or misleading data acquisition or problems with EAM functionality during the procedure. Furthermore, many infants undergoing ablation may be critically ill and frequently have associated invasive vascular access and monitoring tools, as was true in the presented case, which further reduces the available space for positioning and applying EAM hardware.Despite the technological advancements that have occurred, however, multiple limitations remain, particularly when applying advanced EAM systems in infant ablation procedures. First, the procedural setup remains rather cumbersome, with a substantial amount of hardware and patches that need to be applied on an extremely limited body surface area Second, the choice and availability of vascular access remains a dominant variable in the planning and performance of infant ablation procedures. Catheter-based procedures in small patients and particularly in those weighing less than 10 kg are heavily impacted by the availability and size of vascular structures. In general, a “less is better” strategy is favored, wherein the smallest/minimum amount of access is used in order to reduce the risk of vascular complications and ensure the highest likelihood of preserving access sites for future procedures. Preexisting or previously inserted vascular lines can also limit or eliminate sites for access and must be accounted for during preprocedural planning. In our patient’s case, internal jugular and lower-extremity venous access were already in active use. Femoral arterial sheath access also distorted anatomical landmarks, making ipsilateral femoral venous access more challenging.Third, newer EAM technology generally requires the application of specific catheters in order to make use of advanced features for mapping and ablation. In our presented case, while a flexible, small-Fr-sized catheter was used for geometry creation with simultaneous, multipoint acquisition, activation mapping was performed in a traditional point-by-point scheme. The open platform of the Ensite™ Precision™ system  permits the use of any catheter for EAM; however, more sophisticated mapping features require the positioning of a reference catheter and a second proprietary magnetic-field-based, sensor-enabled, multielectrode catheter for roving, automated, and multipoint acquisition. This unfortunately also generally necessitates that larger vascular access be available. Proprietary catheters are also often substantially stiffer, thereby increasing the risk of mechanical trauma or perforation in the infant heart.Fourth, although fluoroscopy-free procedures can be performed in many pediatric procedures, the use of fluoroscopy was considered necessary in our case; specifically, the confirmation of appropriate vascular access, avoidance of the entanglement of catheters and wires with the patient’s PICC line, and verification of catheter location in the right or left heart across the ASD all required fluoroscopy. We also encountered some difficulties with EAM distortion, requiring further fluoroscopy to confirm catheter position and for map correction. This distortion may have been related to the limitations seen with field scaling in such a small patient with potentially variable and changing impedance fields during the course of the procedure, as may occur during the course of ventilator changes and shifts in intravascular fluid status.Current RF catheters that employ force-sensing technology are presently only available in irrigated-tip catheter form. Given the size and thickness of the infant heart, irrigation-assisted RF ablation is unnecessary. Furthermore, although it can be argued that force-sensing would be helpful in infant ablation to ensure appropriate applied contact along thin-walled structures or in close proximity to vulnerable structures, this feature alone may not justify the substantially higher cost of these catheters in comparison with standard, nonirrigated models. Although force-sensing catheters could be used without irrigation, this may increase the risk of luminal clot formation or inadvertent air embolus introduction. Further systematic evaluation of the use of force-sensing ablation catheters in infant ablation procedures is needed to better understand their role, safety, efficacy, and cost impact.Significant advances have been and continue to be made with EAM systems and their integration in both common and complex mapping and ablation procedures. Many features have limited applicability in infant ablation, owing to vascular access and size limitations, the risk of myocardial trauma or perforation, and higher procedural costs without associated clear advantages over conventional mapping and ablation tools and techniques. Successful ablation in the presented case was achieved with a careful selection of catheters, the selective integration of available tools during EAM, and the application of standard principles of activation mapping and RF ablation. The development of complimentary technology that permits the integration of newer EAM features specifically in smaller patients could potentially permit earlier ablative therapy in this unique subgroup of patients, thereby avoiding significant morbidity, both cardiac and noncardiovascular organ dysfunction, medication toxicity, and even mortality in the setting of recalcitrant or incessant tachyarrhythmias.


Coronavirus disease 2019 (COVID-19) represents an unprecedented challenge in modern public health practice. Having spread to over 180 countries and having affected millions of individuals, the COVID-19 pandemic requires a coordinated, effective response without sacrifice to quality or availability of other essential medical services . In counMinimizing nosocomial  infections is integral to an effective COVID-19 response. The World Health Organization (WHO) has published interim COVID-19 infection prevention and control (IPC) guidance, which focuses on administrative controls , environmental controls (including ventilation and waste management) and personal protective equipment  . As moreAside from responding to this global emergency, countries must also prepare for future outbreaks and continue to support and strengthen existing infectious disease programs . In thisWHO recently published interim IPC guidance for COVID-19 describes standard airborne IPC practices for hospital settings, which mirror the general WHO minimum requirements for IPC programs ,13. The Establish a well-equipped triage station at the entrance of health facilities which is supported by trained staffUse screening questionnaires according to the most updated case definitionPost signs in public areas asking symptomatic patients to alert health care workers (HCWs)Ensure social distancing  or screening people as they enterEnsure that patients cover their nose and mouth with a tissue or elbow, scarf or home-made maskOffer a medical mask to patients with suspected COVID-19 in waiting rooms or in cohorting areasEncourage good hand hygiene with handwashing with soap and water or alcohol gelProvide adequate training to HCWs with weekly in-service trainingEstablish a surveillance process for possible COVID-19 infection among HCWsMonitor HCW compliance with standard IPC precautionsRestrict visiting hours to facilities as well as the number of people that can visit health facilitiesEnsure posters, patient education and handwashing/sanitizing.Ensure adequate ventilation in rooms where aerosol-generating procedures are performedLimit room capacity to the absolute minimum for patient care and supportImplement standard PPE precautionsImplement additional contact and droplet precautions before entering the room of a patient with suspected or confirmed COVID-19Use particulate respirators (at least as protective as N95 or FFP2)In the context of these interim guidelines, WHO published a dashboard summarizing the readiness of countries in the WHO African Region for COVID-19 response . These qIn examining readiness for COVID-19 spread, we now review the outbreak in South Africa, currently the largest on the continent . South ASouth Africa is uniquely positioned to respond swiftly and broadly to emerging community transmission of COVID-19. With the largest HIV burden in the world and a high TB and MDR-TB burden, IPC practices are well understood and buy-in from providers, health officials and other stakeholders is high. To most effectively prepare for COVID-19 spread in hospital facilities, however, South Africa must look at current innovations within the national TB control program and those of TB technical assistance partners.Mycobacterium tuberculosis, which spreads when someone with TB expels the bacteria into the air, most commonly through coughing. The national TB control program in South Africa, along with key technical assistance partners like the USAID Tuberculosis South Africa Project, continue to develop and implement innovative TB IPC policies and interventions. One of these interventions is an administrative control measure meant to improve triage practices for patients with suspected TB while reducing time from hospital presentation to TB testing, from testing to diagnosis and from diagnosis to treatment initiation. This strategy, known as Finding cases Actively, Separating safely and Treating effectively (FAST), is currently implemented in 84% (n = 86) of hospitals in eleven priority districts in South Africa, including OR Tambo, Sarah Baartman, Nelson Mandela Health Metro, Waterberg, Sekhukhune, Mangaung, Fezile Dabi, Johannesburg Health Metro, Tshwane Health Metro, eThekwini and uMkhanyakude [According to the most recent WHO Global TB report, South Africa is one of eight countries accounting for two thirds of the global TB burden  and is listed as one of the 30 high TB burden countries . TB is tanyakude .Finding cases actively: The first pillar of the FAST strategy is to identify patients with suspected TB upon entry to a supported hospital. Upon entering a facility, a health care worker greets each patient and identifies those with TB symptoms, including those with an active cough, into an area separate from the general waiting area for patients. This process is conducted regardless of hospital ward and is not restricted to TB clinics in order to find as many patients with TB symptoms as possible, even those who may be at the hospital for an unrelated reason.Separating safely: After identifying symptomatic patients, these patients are separated into a waiting area which is separate from the general waiting area, are given a surgical mask and are educated on cough hygiene. Patients are then asked to produce a sputum sample which is used for molecular testing  for identification of TB and resistance to rifampicin, a common first-line TB treatment. After the sample is given to laboratory technicians, a nurse will further separate patients into a separate waiting area. Laboratory technicians then test the sample immediately, and results are available on the same day, sometimes in as little as four hours  [porting) .Treating effectively: If a sputum sample indicates presence of active TB, a nurse will then explain the test result to the patient and begin anti-TB treatment. If rifampicin resistance is indicated, further drug susceptibility testing (DST) including line probe assay (LPA) and other testing may be indicated. Patients are generally started on treatment on the same day before leaving the facility and are referred to a lower-level health center for local, decentralized follow up care.The FAST approach has identified 12 636 additional TB cases which would have otherwise been missed. Additionally, the Tuberculosis South Africa Project has achieved high screening, testing and treatment initiation rates among patients entering supported facilities as shown in Aside from the FAST initiative, the Tuberculosis South Africa Project works extensively in governance and policy guidance, contact management, environmental controls and awareness building activities to reduce stigma surrounding TB and to encourage people to get tested and start TB treatment. Taken together, these TB-specific IPC interventions serve as useful entry points for COVID-19 IPC responses. Importantly, these TB interventions are functional, have political buy-in and funding, have trained staff and facility-level champions, and have proven results. Health officials must look to functioning programs like these to mount a wide-ranging, agile response to the public health emergency that is COVID-19.While South Africa serves as one example, other countries will necessarily be affected by the COVID-19 pandemic. Other studies including ecological analyses should be implemented to understand possible country-level associations between TB and COVID-19 burden, especially among high TB burden countries. As the body of evidence grows, more specific recommendations can be made to more effectively prepare for future airborne infection threats.COVID-19 represents an unprecedented challenge for infection prevention and control. As shown in settings like South Africa, however, there are successful and functional TB IPC programs in place which can be mobilized quickly and universally to prevent widespread hospital-based infections. In using these strategies, TB program managers and other stakeholders must harness this opportunity for a sustained focus on acute respiratory infections and airborne infection control programs. TB is the biggest infectious disease killer globally and, while COVID-19 is a global public health emergency, we must maintain this urgency and momentum for TB IPC after the threat has passed.As the world comes together to tackle the COVID-19 pandemic, it is important to ensure that essential services and operations for dealing with long-standing health problems continue to protect the lives of people with TB and other diseases or health conditions. Health services, including national programs to combat TB, need to be actively engaged in ensuring an effective and rapid response to COVID-19 while ensuring that TB services are maintained .In funding and bolstering these existing TB IPC strategies for COVID-19 responses, country governments can improve airborne IPC interventions in general, to improve TB case detection, to reduce nosocomial infections and to reduce TB deaths generally. While the initial benefit in expanding these programs lies in preventing wide community transmission of COVID-19, we must recognize the opportunity in maintaining this level of response against TB to continue to reduce new TB infections and deaths from TB.As COVID-19 continues to spread globally, we must look to existing health system capacity for a rapid and robust response. As shown by WHO, IPC readiness on the African continent varies and shows only moderate preparedness for impending outbreaks, in which South Africa represents the ideal scenario. In high TB burden countries like South Africa, IPC interventions, especially like those implemented by the USAID Tuberculosis South Africa Project, offer a ready-made entry point to broad airborne IPC responses with trained providers, guidance documents and facility-level champions. These programs must be expanded and resourced to minimize nosocomial infection and death. In expanding these programs, however, we must keep the future in mind and realize the 2-fold benefit of IPC programs: the immediate benefit of preventing COVID-19 outbreaks, and the long-term benefit of reducing ARI death in general, including deaths form TB. In the wake of World TB Day and in the context of a rapidly moving pandemic, there is no better resource for high burden TB countries than national TB control programs. We must rapidly expand and resource these programs to evade unnecessary death and suffering due to COVID-19 and other ARIs.


Background: To determine the magnitude of difference between manufacturer-submitted and pan-Canadian Oncology Drug Review (pCODR) calculated incremental cost-effectiveness ratios (ICERs), incremental cost (ΔC), and incremental effectiveness (ΔE); to examine whether there is a significant difference in the proportion of ICERs deemed cost-effective; to evaluate trends in the ICERs over time; and to identify methodological issues in manufacturer-submitted economic models. Methods: Economic guidance reports for all drug indications submitted from July 2011–November 2018 were extracted from the pCODR database. Cumulative distribution plots were constructed to compare the manufacturer-submitted economic values with both the pCODR lower- and upper-reanalyzed estimates. The proportion of drug reviews considered cost-effective at varying willingness-to-pay (WTP) thresholds by the manufacturer and pCODR were calculated. Manufacturer changes in ICERs over time from 2012 to 2018 were determined. Recurring methodological issues with manufacturer submissions were tallied. Results: There were 73 unique indications that were included. Manufacturer-submitted ICERs were consistently lower than pCODR estimates for most indications. Manufacturer-submitted ICERs were generally more cost-effective over a range of WTP thresholds. From 2012 to 2018, manufacturer and economic guidance panel (EGP) lower limit reanalyzed ICERs did not change significantly over time. However, EGP upper limit re-analyses did show decreasing cost-effectiveness (increasing ICERs). The two most common issues identified in the manufacturer-submitted models were related to survival time horizon and utility estimates. Conclusions: Manufacturers tend to overestimate the cost-effectiveness of their therapies when submitting economic models to pCODR. Although certain methodological issues are still common in manufacturer-submitted models, revision rates are high for most issues raised by pCODR. Anti-cancer therapies are a significant financial burden on both patients and healthcare systems. As the incidence of cancer continues to rise and new therapies are rapidly introduced, public drug plans with limited budgets continue to face difficult resource allocation decisions . AssessiBecause of limited healthcare resources, evidence of cost-effectiveness is a critical component of drug funding decisions . Novel tIn the pCODR review process, the cost-effectiveness data used in pERC’s deliberations are delineated through a two-step submission process, as follows: manufacturers first submit their economic models to pCODR, specifying their best estimate of the drug’s ICER, ΔC, and ΔE. pCODR subsequently convenes an economic guidance panel (EGP), consisting of independent health economists or HTA-experienced panel members ,8. The EPrevious studies have examined the methodological issues in manufacturer submitted economic models to pCODR, the components commonly revised by the EGP, and their potential correlation with final pERC recommendations ,10,11. MIdentifying the recurring problems faced by economic models submitted to HTA review committees is an important step in the development of more suitable models in the future. While previous studies have examined some of these methodological issues, to our knowledge, a direct comparison of manufacturer-calculated and independently reanalyzed ICERs has not been formally conducted in the Canadian context. Therefore, in this study, we investigate if manufacturer-submitted economic models are generating ICERs that are more optimistic in comparison with pCODR-generated ICERs. The objectives of this study are to determine the magnitude of difference between manufacturer and pCODR ICERs, ΔC, and ΔE; to examine whether there is a significant difference in the proportion of ICERs deemed to be cost-effective; and to evaluate the trends in the ICERs over time. To further explore why manufacturer-submitted and EGP-reanalyzed ICERs may differ, we also present more recent data on the common methodological issues revised by the EGP.All drug indications submitted from 13 July 2011, when pCODR first started accepting submissions, to 1 November 2018, were identified from the publicly available pCODR database . IndicatFor each drug review, two reviewers (T.J. and R.S.) independently extracted the generic drug name, pCODR number, indication, route of administration, comparator drug, pERC final recommendation , date of final recommendation, manufacturer-submitted economic values reported as point estimates , and EGP’s lower and upper estimates . Methodological issues were identified by examining final economic guidance reports, and recurring issues were grouped into categories based on theme and whether the issue was revised by EGP during reanalysis. To ensure accuracy, all data extractions were compared between the two reviewers, with disagreements being resolved through discussion with a third reviewer (K.C.).Key data from included drug reviews, including the ICER, ΔC, and ΔE values, were summarized using descriptive statistics. Cumulative distribution plots were constructed to compare the manufacturer-submitted economic values with both the lower and upper EGP reanalyzed estimates. The absolute differences in ICERs were calculated by subtracting the manufacturer-submitted value from the EGP reanalyzed estimates; therefore, a positive difference indicated that the manufacturer’s ICERs were lower and more optimistic. The proportion of drug reviews considered to be cost-effective at varying willingness-to-pay (WTP) thresholds  by the manufacturer and EGP reanalyzed models were also calculated. As Canada does not endorse an explicit societal WTP threshold, a range was used in this analysis based on the World Health Organization’s suggestion of one to three times gross domestic product per capita . ICERs tOverall, 73 unique indications were included in this analysis, from 149 submissions initially identified from pCODR . No indiIn examining the ICERs, manufacturer-submitted ICERs were consistently lower than both the EGP low and high estimates for most indications A. The meSeparating the ICERs into their two components (ΔC and ΔE), it was observed that the values for ΔC were generally similar between all three economic analyses see B. In terUsing manufacturer-submitted ICERs, more interventions were considered cost-effective over a range of willingness-to-pay thresholds . Based op = 0.78; p = 0.94), while ICERs in the EGP high re-analyses increased significantly by 19% annually  review committee, the precursor to pCODR. A previous study that examined issues encountered by JODR when evaluating manufacturer-submitted economic models reported an uncertainty of comparative clinical benefits, costing assumptions that favored manufacturers and a lack of robustness for the submitted analyses to be the most common concerns . ManufacOver the six-year period analyzed, the trend in cost-effectiveness was similar between the manufacturer and EGP lower limit re-analyses, with ICERs not significantly changing over time. However, the EGP upper limit re-analyses did show decreasing cost-effectiveness (increasing ICERs) over this six-year period. Previous research has shown that the cost-effectiveness of novel anticancer drugs is decreasing over time, as the cost of these therapies has been increasing dramatically without a proportional increase in clinical effectiveness ,19. ThesIn terms of the methodological issues with manufacturer-submitted economic models, time horizon issues were the most commonly identified and revised by the EGP. The EGP commonly re-computed the ICERs using a different (lifetime) time horizon, especially in cancer settings, where the models inappropriately projected clinically implausible survival tails of 30 to 40 years. These “long and thick” extrapolated survival tails are not properly validated by data, but are unfortunately created by the submitting modeler under the misguided defense of a “lifetime horizon”. In cases where submitted models project a substantial proportion of patients surviving many years beyond the expected survival based on data and clinical experience in these cancer settings, EGPs will adjust the model to a different lifetime time horizon in order to minimize the inappropriate effects of the overestimation of long-term survivors on the cost-effectiveness results. Overall, submitted models that incorporate inappropriately long time horizons may overestimate long-term survival benefits. This is similar to Masucci et al., suggesting that manufacturers continue to overestimate survival when submitting their drugs to pCODR . Issues Our study is not without limitations: firstly, our analysis included 109 economic guidance reports with 73 indications due to the exclusion of submissions that did not publish all of their ICER data. While incorporating these additional indications might impact our findings, a potential limitation is that it is unclear whether the excluded reviews are methodologically similar to those included. Nevertheless, there is insufficient publicly available data for these excluded submissions to allow for further in-depth analysis. This highlights the importance of transparency in the reporting of economic models that inform public-funding decisions in order to enhance accountability and public scrutiny. Secondly, the categories used to classify commonly identified issues in our study were chosen subjectively based on how frequently they were reported in the economic guidance reports. Therefore, any issues discussed by the EGP and manufacturers  that were not published in these reports were not captured. Another limitation is that EGP provides upper and lower limit estimates of ICERs, without providing a best point estimate in many cases. In certain indications, the difference between EGP upper and lower estimates can vary widely, making the comparison between the EGP and manufacturer ICERs less precise. Furthermore, there is also no evidence to show that EGP re-analyses are necessarily the gold standard for comparison, but the consistent finding of higher EGP ICERs that were derived from multiple health economists and academic researchers independent of manufacturers and payers across most submissions suggested that these ICERs were less likely to be subjected to perceived or actual influence from any stakeholders. For instance, there is still controversy regarding the appropriateness of adjusting the time horizon in the submitted economic models. Ideally, the time horizon should represent a life-time horizon, but two main issues emerge, namely: (1) there is no consensus on the correct life-time horizon for different cancer settings, and (2) most randomized controlled trials have a short-term follow-up, limiting the extent to which clinical benefits can be extrapolated. In revising the economic models, EGP may reduce the time horizons to remove future projected benefits that were unproven and subject to considerable uncertainty. However, there is no clear consensus if this approach taken by the EGP is the ideal approach to explore uncertainty in the over-extrapolation of potential long-term benefits when only short-term data exist. Finally, economic studies for oncology drugs conducted in the province of Quebec were not included in this analysis, as pCODR does not provide drug funding recommendations to Quebec. Future studies can be performed to expand this analysis to include economic reports from Quebec, and to compare our current results. As the number of submissions to pCODR increases and the sample size becomes larger, other areas of future research include evaluating methodological issues in submitted economic models based on specific disease areas or drug types .With the increasing cost of oncology drugs, manufacturer-submitted economic models are scrutinized by the EGP before pERC makes its final funding recommendations. When compared with EGP-calculated estimates, manufacturers tend to overestimate the cost-effectiveness of their therapies when submitting economic models to pCODR. Although certain methodological issues are still common in manufacturer-submitted models, revision rates are high for most issues raised by the EGP. Given the ongoing methodological issues identified in this analysis and in previous studies, one natural option to consider is whether payers, such as pCODR, should directly construct economic models instead of modifying manufacturer submissions. However, this would require manufacturers to provide proprietary data access to pCODR, which is unlikely. Furthermore, pCODR would require greater resources to construct economic models for every drug submission. Because of resource limitations, it is currently feasible and is more convenient for pCODR to revise existing models from manufacturers rather than to build their own. Therefore, manufacturers should continue submitting economic models in accordance with the published guidelines. To improve the quality of these submitted models, and to better align EGP and manufacturer values, pCODR should continue revising their submission guidelines, highlighting and proposing solutions to commonly found methodological issues in submitted economic models. While manufactures tend to follow these pCODR guidelines, there is still room for interpretation, which can result in the model being revised by the EGP. For instance, the lack of consensus on what is considered a life-time horizon for specific cancers may currently result in model projections that do not reflect the clinical reality. This extrapolation of time horizon is a recurring issue identified in this analysis and in other literature. As manufacturers increasingly use surrogate endpoints and shorter follow-up times in clinical trials, potentially to gain early regulatory approval, correcting the time horizon issue remains an ongoing challenge. A potential solution to tackle this issue is implementing a process for lifecycle revaluation. For instance, when clinical trial data becomes mature, manufacturers should be encouraged to re-evaluate their initial model projections to see if they fit with the current data.


Schizophrenia is a disorder with a heterogeneous etiology involving complex interplay between genetic and environmental risk factors. The immune system is now known to play vital roles in nervous system function and pathology through regulating neuronal and glial development, synaptic plasticity, and behavior. In this regard, the immune system is positioned as a common link between the seemingly diverse genetic and environmental risk factors for schizophrenia. Synthesizing information about how the immune-brain axis is affected by multiple factors and how these factors might interact in schizophrenia is necessary to better understand the pathogenesis of this disease. Such knowledge will aid in the development of more translatable animal models that may lead to effective therapeutic interventions. Here, we provide an overview of the genetic risk factors for schizophrenia that modulate immune function. We also explore environmental factors for schizophrenia including exposure to pollution, gut dysbiosis, maternal immune activation and early-life stress, and how the consequences of these risk factors are linked to microglial function and dysfunction. We also propose that morphological and signaling deficits of the blood-brain barrier, as observed in some individuals with schizophrenia, can act as a gateway between peripheral and central nervous system inflammation, thus affecting microglia in their essential functions. Finally, we describe the diverse roles that microglia play in response to neuroinflammation and their impact on brain development and homeostasis, as well as schizophrenia pathophysiology. Schizophrenia (SCZ) is a prevalent mental illness without satisfactory treatment options. Approximately 20 million people worldwide are afflicted by this chronic and debilitating mental disorder . SCZ is Genome-wide association studies (GWAS) have identified novel susceptibility loci that confer greater risk for SCZ . These bMicroglia are central nervous system (CNS) phagocytes that, among their other roles, orchestrate innate immunity in the brain. Microglia have well-described roles in rapidly responding to inflammatory insults through dynamic surveillance of the CNS parenchyma  and cleaIn this review, we discuss the genetic and environmental risk factors for SCZ and how they converge to alter microglial function in response to systemic and central inflammation. Additionally, we highlight how these risk factors alter the indispensable functions of microglia during development, adolescence and adulthood. Limitations of the current knowledge are also addressed, and key future experiments are proposed. Understanding how the heterogeneous genetic and environmental risk factors for SCZ interact to reach a disease threshold and determine its progression is necessary for the development of more effective therapeutics.Schizophrenia is driven by genetic factors, as the risk for developing this disorder increases from 1% in the general populationto 50% in individuals with a diagnosed twin . Recent C4A), located in the MHC locus, is highly associated with SCZ: specific structural variants and regulatory regions that increase the expression of C4A confer a greater risk for SCZ (C4A could directly contribute to SCZ pathology (The major histocompatibility (MHC) locus is located on chromosome 6 and has the highest association to SCZ compared to any other loci across the genome . This re for SCZ . The com for SCZ . Besides for SCZ . Ground-athology .C4A, C4b, in medial prefrontal cortex (mPFC) layer (L) 2/3 pyramidal neurons led to a marked reduction in connectivity and decreased sociability in juvenile and adult mice, both of which mirrored the deficits seen in SCZ  is an important regulator of C4 that is expressed during early postnatal development  (IFN-γ), an important regulator of viral propagation (IL)-1α  , which ipagation . In supp (IL)-1α , IL-1β  . TLRs plCD14, CLU, DPP4, EGR1, HSPD1, MHC and C4 genes  was first found in a Scottish family with SCZ , which can be generated from nitrogen oxide, can also be changed photochemically after their release from motor vehicles (The environment is becoming increasingly polluted from multiple sources. Traffic-related air pollution (TRAP), such as diesel exhaust , is the vehicles . Altogetvehicles ; Table 1vehicles . Indeed,vehicles . Interesvehicles .3  system to maintain homeostasis, for instance by regulating hunger and digestion processes at steady state . There hThe microbiota not only plays a key role in regulating host metabolism but also modulates inflammatory responses and neural function. Germ-free mice have multiple deficits in nervous system function including heightened hypothalamic-pituitary-adrenal (HPA) axis responses , alteredLactobacillus compared to healthy controls, and levels of this bacterium correlate with psychosis severity , which facilitates increased neuroinflammation. The presence of gut microbes is necessary for the proper formation of the BBB during early development. Mice from germ-free dams have disrupted BBB maturation, which is evident by decreased tight junction expression both prenatally and postnatally. The hyperpermeability of the BBB in germ-free mice persists into adulthood, but can be rescued by microbiota transplantation from controls or through the administration of bacteria that produce short chain fatty acids (SCFAs) , which aGut microbes produce metabolites that can cross the BBB and inhibit the function of mitochondria in the CNS . A decreGut microbiota dysbiosis can alter the maturation and function of microglia in the CNS, thus contributing to neuroinflammation . Germ-frMicroglia also show sex-dependent differences in response to gut microbe sterility. Microglia from germ-free male mice displayed altered expression of immune genes and a more immature phenotype at juvenile stages whereas microglia from female mice were more affected in adulthood . These fWithout a doubt, the gut microbiome influences the development and maintenance of the immune and nervous systems, with significant crosstalk. In the context of SCZ, the metabolites and diversity of gut microbes may impact multiple disease symptoms. The microbiome links multiple risk factors for SCZ, including stress responses, by promoting immune activation and BBB disruption. Innate immunity of the brain, including microglial function, is sensitive to gut dysbiosis, making the gut microbiota an interesting target in SCZ. Probiotics and microbiome transplants should be further explored to improve symptom severity in people with SCZ. Additionally, precautionary steps could be taken in pregnant mothers to improve diversity of gut microflora, considering its profound impact on brain development. Future work should further explore the role of SCFA-producing microbes, considering that they exert anti-inflammatory effects and improve brain function and behavior. Taken together, gut microbes are positioned to alter immune responses to environmental challenges by regulating neuronal function, behavior, and microglial responses, all of which are altered in SCZ.It has become increasingly clear that immune challenges occurring during pregnancy increases offspring risk for varied neurodevelopmental and neuropsychiatric disorders, including SCZ. Specifically, maternal exposure during pregnancy to bacterial  or viralMIA impacts brain function in a circuit-specific manner and interacts with other risk factors for SCZ. These early immune insults can elicit a vast array of phenotypes in mice that are relevant to SCZ and ASD, including abnormalities in ultrasonic vocalization and sociability, increased repetitive behaviors, motor dysfunction, and deficits in sensorimotor gating and cognitive abilities such as working memory . Some ofGiven that microglia are the primary innate immune cells of the brain, they provide rapid responses to immune insults and are greatly affected by systemic inflammation. MIA exerts its effects on neurodevelopment largely by disrupting microglial function and by priming them for altered responses later in life. Changes in the density of microglia are found in early postnatal MIA offspring in multiple cortical and subcortical regions including the anterior cingulate cortex, striatum and hippocampus . MicroglP2ry6, Sirpa, Siglece, Cx3cr1, Fcgr1, Itgav)  . These r, Itgav) . Inflamm, Itgav) . Importa, Itgav) . These fMultiple studies targeting microglial signaling pathways were able to reverse MIA-associated neuropathology, suggesting that microglia are the main culprit in inducing neurological dysfunction in response to immune challenges. For example, a study that targeted colony stimulating factor 1 receptor (CSF-1R), which plays a role in microglial proliferation, was successful in reversing some MIA-induced phenotypes . DepletiMIA is a risk factor for SCZ that depends on maternal immune signaling relayed to the fetal brain through the placenta. Maternal gut microorganisms have been found to play an important role in MIA-mediated deficits. A ground-breaking study showed that MIA phenotypes in exposed offspring are dependent on the presence of segmented filamentous bacteria in the maternal gut which promote Th17 cell differentiation, leading to increased IL-17a production . MIA pheMIA is an important model that has increased our understanding of how immune insults occurring during embryonic development can alter brain development. Although there is variability in data obtained using mouse models of MIA, notably due to differences in immunogen manufacture , timing of immunogen administration, dosage, route of administration, housing conditions, timing of cage cages and mouse strain used , understC4 gene that led to enhanced C4 expression  and LPS , and glucose transporter GLUT4, all of which have been implicated in immune system function  in healthy subjects leads to psychotic symptoms and cognitive deficits that resemble those observed in SCZ (Abnormal activity in multiple brain networks and regions are observedin SCZ . There id in SCZ . Additiod in SCZ . Recent d in SCZ . Circulad in SCZ . An incrd in SCZ . StudiesBeyond the BBB, peripheral inflammatory responses can gain access tothe CNS via the meninges, the multi-layered protective tissue thatsurrounds the brain and spinal cord [reviewed in There is growing evidence from both human and animal studies that many of the risk factors for SCZ converge on their ability to promote neuroinflammation, and that these effects are mediated in part by microglia. However, is there a pro-inflammatory phenotype in SCZ? Post-mortem and clinical studies show an increase in pro-inflammatory markers in people with SCZ compared to controls . Moreovein vivo markers for neuroinflammation would be useful. Ideally, additional work should be done to specifically interrogate the extent of neuroinflammation in SCZ, in addition to peripheral inflammation, to determine if increased inflammation correlates with all or only a percentage of SCZ cases.Schizophrenia cases can be sub-divided using either serum or post-mortem brain tissue levels of pro-inflammatory cytokines, which reveal that about 40% of SCZ cases have a high inflammatory expression signature . Althougin vitro model from patient-derived neuronal cultures while decreasing the risk for SCZ when administered to young adults (Given that SCZ is a highly heterogeneous disease, it is not surprising that there are different disease subtypes. Studies that have divided individuals with SCZ based on inflammatory markers have found more severe symptomology in those with higher levels of pro-inflammatory markers. Specifically, there is evidence for greater gray matter loss and poorer performance in language tasks  and incrg adults , suggestin vivo imaging study in awake mice has shown that microglial contacts with synapses increase synaptic activity thus enhancing neuronal network synchronization (In line with this, microglia are significantly altered in SCZ and contribute to neural dysfunction by responding and contributing to neuroinflammatory signaling . In SCZ nization . In thisnization , suggestC1q and C3 (Although there is evidence that microglia contribute to excessive synaptic pruning in SCZ, it is not clear if microglia-dependent synapse formation is also altered. Since much of the data collected from individuals with SCZ is from post-mortem samples, it is difficult to discern what is occurring on the synaptic level earlier in development. Recent studies suggest that more immature spine types can be differentially targeted in SCZ , therefoq and C3 . This inq and C3 . These sin vivo studies can be performed to gain greater understanding into how microglia functionally impact synaptic development and circuit function in pathological states.Indeed, there are multiple disease-associated microglial subtypes such as those seen in neurodegenerative disorders  and darkAlthough there is no doubt that the immune system plays a critical role in shaping brain development and contributes to disease states when dysregulated, there is a need to understand which specific circuits and neuromodulatory systems in particular are most impacted by abnormal immune signaling. It is clear that complement proteins facilitate the removal of synapses  and thatThere is evidence that inflammatory responses target specific neuromodulatory systems and brain circuits. For example, changes to the gut microbiome driven by inflammation can alter the production of serotonin , which iC4 led to a decrease in excitatory connectivity with no loss of inhibitory transmission, suggesting that excitatory synapses might be more vulnerable to elimination (The glutamate hypothesis of SCZ evolved from observations that NMDA receptor antagonists, such as ketamine, produce behavioral states similar to SCZ negative and positive symptoms in healthy human subjects . As discmination . In suppmination , providimination . Togethemination .Although these studies suggest that risk factors for SCZ can exert specific effects on different CNS circuits, more work is needed to fully understand the mechanisms by which inflammation alters specific neuromodulatory systems and circuits. Studies that combine mouse models of SCZ and inflammation with whole-brain or mesoscopic imaging could shed light into how specific neuronal networks are impaired in SCZ .It is well-established that spine dysfunction is present in SCZ and is at least partially mediated by reductions in excitatory synaptic connectivity and plasticity . HoweverC4. In this in vivo model, synaptic loss observed in the PFC was specifically due to a loss of smaller spine-types while mushrooms spines were unaffected (in vivo (How are certain synapses selectively removed while others are protected? In the developing brain, there is a period of enhanced synaptogenesis followed by critical developmental periods characterized by experience-dependent refinement of synapses . Synaptiaffected . This evaffected . Small s(in vivo . Additio(in vivo . In thisin vivo human imaging studies notably through specific markers could help answer this question. It is encouraging that previous studies show a similar phenotype in mice that is seen in humans in terms of weaker synapses preferentially being eliminated (It is also possible that in SCZ, spine loss is driven by the inability of circuits to produce “appropriate” connections. Therefore, the subsequent excessive pruning that occurs in SCZ could be due to the fact that neurons fail to produce adequate connections in the first place. This is relevant in the context of microglia since they regulate synaptogenesis . Since miminated . This coiminated .Here, we have highlighted the diverse risk factors for SCZ and how they impact the CNS by altering immune signaling. Likely, these risk factors act additively on certain signaling pathways to push vulnerable individuals past a certain threshold into a disease state. This field would benefit from future studies that aim to elucidate how the immune system regulates specific circuits and neuromodulatory systems to drive the diverse phenotypes observed in SCZ. Additionally, an in-depth understanding of the specific signaling networks compromised in SCZ may enable the restoration of typical immune-driven neurodevelopment after exposure to the various genetic and environmental risk factors described in this review.AC, MC, M-ÈT, and AC-M wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurological disorders caused by neuroviral infections are an obvious pathogenic manifestation. However, non-neurotropic viruses or peripheral viral infections pose a considerable challenge as their neuropathological manifestations do not emerge because of primary infection. Their secondary or bystander pathologies develop much later, like a syndrome, during and after the recovery of patients from the primary disease. Massive inflammation caused by peripheral viral infections can trigger multiple neurological anomalies. These neurological damages may range from a general cognitive and motor dysfunction up to a wide spectrum of CNS anomalies, such as Acute Necrotizing Hemorrhagic Encephalopathy, Guillain-Barré syndrome, Encephalitis, Meningitis, anxiety, and other audio-visual disabilities. Peripheral viruses like Measles virus, Enteroviruses, Influenza viruses (HIN1 series), SARS-CoV-1, MERS-CoV, and, recently, SARS-CoV-2 are reported to cause various neurological manifestations in patients and are proven to be neuropathogenic even in cellular and animal model systems. This review presents a comprehensive picture of CNS susceptibilities toward these peripheral viral infections and explains some common underlying themes of their neuropathology in the human brain. The common thread among herpes simplex virus (HSV), West Nile virus (WNV), enteroviruses like Poliovirus (PV), Coxsackievirus (CV), Influenza virus (IAV), Measles virus (MV), and especially human respiratory viruses like Coronaviruses (CoV) is that their primary site of infection is not the human Central Nervous System (CNS), yet they are all “Neuropathogenic” and exhibit fatal neurological abnormalities in humans. These viruses infect millions worldwide and cause a spectrum of neurological and psychiatric illnesses –3. If weThe Central Nervous System (CNS) is strictly guarded by the blood-brain barrier (BBB) and effective immune surveillance. An intriguing question emerges over how these non-neurotropic viruses (most of them respiratory viruses) cause their respective primary pathology in the distal part of the host body, ultimately disrupting the sanctity of CNS. Is there a common pathway for all these viruses to enter CNS and cause neurological disorders or do they all use a unique route for causing their neuropathology?In the usual course of infection, viruses usually cross the infected tissue and travel back to the bloodstream and lymph nodes and, through a hematogenous route, they can gain access to the CNS. Clinical observations show that any virus that is exposed to epithelial and endothelial linings usually breach the CNS because almost all human mucosal epithelial linings are heavily embedded with peripheral sensory nerves. However, in the case of non-neurotropic viral infections, it all seems opportunistic, because these viruses first exhibit their primary disease symptoms, followed by a secondary set of pathologies which include the neurological aberrations.Backed up by over 100 years of extensive research and abundant literature, we can dig deeper into various viral strategies to disrupt CNS and make a wider comparison to find some commonly exploited pathways. In the following discussion, we will try to explore this issue on whether their entry into the CNS is opportunistic and accidental or whether these peripheral viruses are equipped to run such a multifaceted pathogenesis.Major routes for entering the CNS are broadly categorized into two options. First, the hematogenous route, which means entry of peripheral infected blood cells like monocytes/macrophages as a “Trojan Horse” via crossing the BBB  . The AmeDijkman et al. reported that human coronaviruses (HCoV) cause far fewer cytopathic effects on primary human respiratory epithelial cells . They alBy classical definitions, a pathogen and their pathogenesis represent a typical cause-and-effect relationship, described as “germ theory.” It was a gold standard established by Robert Koch in 1890, also known as the Koch’s Postulates. However, viruses seem to easily defy Koch’s Postulates when causing neurological disorders. In the case of viral infections, we have multiple examples where neurological sequelae of viral infections happened decades after the primary infection, when all the signature of viral genome, proteins, and toxins are long gone from the host. This gives us a serious challenge in assigning and linking the viral infection and resulting neuropathogenesis. Viruses are known to be an original root cause of several neurological syndromes either directly or indirectly. The difference between neuropathology caused by direct viral infection and that caused by potential bystander factors is still a highly explorable field.encephalitis lethargica  in flu patients after the recovery. These neurological perturbations could reduce the quality of a patient’s life for decades  have been reported in human brain autopsy samples which clearly established that, being primarily a respiratory virus, it is naturally neuroinvasive in human hosts and can successfully replicate within the brain . FurtherOther strains of coronavirus, such as HCoV-229E, also infects human primary monocytes and activates them . These HIn the neuronal transmission route, the virus typically infects the neurons located peripherally and actively transports itself using the cellular actin-myosin machinery of neurons to enter the CNS . This roSpeculations about viral etiology behind common neurological diseases such as Parkinson’s disease (PD), ADEM , and multiple sclerosis (MS) has gained much strength in recent times. Particularly with various coronaviruses, such as HCoV-229E and HCoV-OC43, being detected within Parkinson’s disease (PD), ADEM , and multiple sclerosis (MS) patient’s brains, it is now discussed as a link between these neurodegenerative diseases and viral infections . Even muDuring coronavirus replication and proliferation in lung epithelial cells, the alveolar gas exchange phenomenon is severely hampered because of damaged lung epithelial cells . AfterwaThis could be true for persistent infections of other coronaviruses within the CNS, however, more experimental evidence is needed to establish such a correlation for all the recent coronaviruses, such as SARS-CoV-1, MERS-CoV, and SARS-CoV-2.Cytokines were once given the analogy of rain. As moderate and timely rain is needed to sustain life on earth, similarly a baseline amount of cytokines are actually needed for multiple cellular and physiological processes during the life of an organism , 69. Cyttrans-cellular crossing of the BBB , Multiple sclerosis (MS), Parkinson’s Disease (PD), Huntington’s Disease (HD), ischemia, and strokes, , 68. ManThe secondary stimulus which activates microglia can begin in the CNS by many means, such as any neuroviral infections or bacterial invasion. However, it has been observed that conditions which contribute to systemic inflammation such as diabetes, ischemic conditions, and arthritis, are a major cause of priming microglia, and this can be particularly seen in elderly populations –82. ThisOther than microglia, astrocytes and neurons can also produce multiple cytokines upon exposure to external stimuli , 78. SucWe will now briefly list the neurological anomalies caused by the following viral invaders.SARS-CoV-1 was the reason for the 2003, 2009, and 2012 SARS epidemics, which started in Asia and spread worldwide. This virus causes the characteristic respiratory illness, which includes high fever, dry cough, and difficult breathing, and severe cases manifested by respiratory failure and death . This SAAs it occurred 17 years ago, research communities have had enough time to investigate the immediate and long-term impacts of SARS-CoV on the human CNS. SARS-CoV was found to trigger many neurological abnormalities such as encephalitis, aortic ischemic stroke, and polyneuropathy . InteresMERS-CoV is a relatively recent member of the coronavirus family, identified in 2015 in the Arabian Peninsula. It originated from bats and used camels as an intermediate host for many years before jumping to human hosts . It has The latest addition of novel coronavirus, first observed in 2019 in Wuhan city of China, has been named SARS-CoV-2 based on its genome sequence analysis , 96. ThiACE2 is an established receptor for many human coronaviruses and influenza virus , 98. InsSince the pandemic is still going on, long-term secondary effects of SARS-CoV-2 infection on patient’s health is yet to be observed by clinicians. Neurological abnormalities are still recorded at patient levels, and their molecular mechanisms and animal model studies and cellular data are yet to be performed and validated. Clinical reports have shown that SARS-CoV-2 infected patients display the symptoms of intracranial infection like headache, loss of consciousness, epileptic seizures, etc. A majority of patients reported loss of smell and taste, a condition known as anosmia and dysgeusia –102. IntHuman coronaviruses share a strong similarity with neuroinvasive animal coronaviruses, such as PHEV  , FCoV f, and JHMAs the COVID-19 pandemic has crippled socio-economic activities and human interactions globally, the world needs urgent measures to contain and suppress the viral transmission as well as get an effective vaccine as a long-term goal. Based on available clinical and epidemiological data so far, the World Health Organization (WHO) has already issued many guidelines which has helped in designing the prevention strategies to suppress the general viral transmission and mortality rate due to SARS-CoV-2. A controlled movement of population, increased and rapid testing followed by isolation, and a wider availability of healthcare services to reduce mortality are a few commonly practiced suggestions which have controlled this disease to a great extent. Since neurological disturbances were more frequent in severely ill and critical patients of COVID-19 as compared to mild ones , this suInfluenza viruses are a member of the Orthomyxoviridae family and contain negative-sense single-strand RNA as their genome . There aIn the case of Influenza infections, there is an extensive history of patients reporting a variety of CNS disorders which collectively suggested that Influenza virus is a potentially neurotropic virus and is capable of giving long-lasting neurological sequelae . Even soHowever, this paradox can be explained from a neuroinflammation perspective. Peripheral “Cytokine storm” caused by viral infections can explain why being neurotropic is not an essential criteria for neurological abnormalities. The peripheral cytokines can travel to the CNS and indirectly activate or prime the brain resident microglia (as explained in the above sections) , 126. InNipah virus (NiV) is one of the most recent emerging members of the paramyxoviridae family and causes acute respiratory illness in human hosts . In critMeasles virus (MV) is a common name for genus Morbillivirus, belonging to the subfamily Paramyxovirinae of the family Paramyxoviridae. MV falls in the category of an enveloped virus with a genome of a single-strand, negative sense RNA . Measlestrans-synaptic fashion. The paradox of their traveling route has been reviewed in detail by Young et al.  or even viral toxic proteins can cross the blood-brain barrier and aggravate the neuroinflammatory response within the CNS. We have also highlighted in our previous work how extracellular vesicles released by peripheral immune cells after Dengue virus infection have the capacity to trigger a cytokine storm phenomenon in the CNS. These recent works provide us with more clues about how we can block the interconnections of periphery cells and the CNS. We can further explore the trafficking biology to control the extracellular vesicle secretion which transfers the signals and transcriptional activators between organs. We can also explore various temporary measure to regulate BBB permeability, so that SARS-CoV-2 crossing over to CNS could be checked. Enhancers for mucosal immunity should be explored, which can contain SARS-CoV-2 at epithelial surfaces itself and not allow them to invade all the vital organs of the host, including the CNS.This awareness would offer us an opportunity to adopt a much more holistic approach of neurological healthcare management while the acute phase of the SARS-Cov-2 is still ongoing, in order to improve the upcoming life-quality of SARS-CoV-2 and other respiratory patients.RM and AB conceptualized the framework of this review article, corrected, read, and finalized the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.