Transcriptome-wide quantitative profiling of PUS7-dependent pseudouridylation by nanopore direct long read RNA sequencing.

Pseudouridylation is a prevalent post-transcriptional RNA modification that impacts many aspects of RNA biology and function. The conversion of uridine to pseudouridine (Ψ) is catalyzed by the family of pseudouridine synthases (PUSs). Development of robust methods to determine PUS-dependent regulation of Ψ location and stoichiometry in low abundant mRNA is essential for biological and functional understanding of pseudouridylation. Here, we present a framework, NanoPsiPy, for identifying Ψ sites and quantify their levels in poly-A RNA at single-nucleotide resolution using direct RNA long-read Nanopore sequencing, based on the observation that Ψ can cause characteristic U-to-C basecalling errors in Nanopore direct RNA sequencing data. Our method was able to detect low and high stoichiometric Ψ sites in human mRNA. We validated our method by transcriptome-wide quantitative profiling of PUS7-dependent Ψ sites in poly-A RNA from a MYCN -amplified neuroblastoma cell line. We identified 8,625 PUS7-dependent Ψ sites in 1,246 mRNAs that encode proteins involved primarily in ribosome biogenesis, translation, and mitochondrial energy metabolism. Our work provides the first example of using direct RNA long-read Nanopore sequencing for transcriptome-wide quantitative profiling of mRNA pseudouridylation regulated by a PUS. We envision that our method will facilitate functional interrogation of PUSs in biological and pathological processes.

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subset-defining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCRαß clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

Production and transport of plasma-generated hydrogen peroxide from gas to liquid.

In this work, the transport of hydroxyl radicals and hydrogen peroxide from a humid atmospheric pressure plasma jet into plasma-treated liquids is analysed. The concentration of H2O2 was measured by a spectrophotometric approach using the reagent ammonium metavanadate. OH was measured by the terephthalic acid dosimeter and the chemiluminescence of luminol. The plasma jet used is based on the design of the well-investigated COST reference jet and is extended by a capillary between the two electrodes. In addition to the experiments, the 0-dimensional plasma-chemical kinetics code GlobalKin was used to analyse the plasma chemistry in the gas phase in more detail. After 5 min plasma treatment, a maximum H2O2 concentration of 1 mM was found in the liquid, while the OH concentration was a factor 50 lower. The concentrations of both species in the liquid increased with plasma power, and the H2O2 concentration also increased with the humidity concentration of the feed gas, while the OH concentration first increased with humidity admixture and then decreased. The transport of both species could be controlled by the treatment distance, the gas flow rate and low frequency pulsing of the RF jet in such a way that the selectivity towards the long-lived species H2O2 was increased. Qualitative trends in the simulated number densities of gas phase H2O2 and OH at the location of the gas-liquid interface fit relatively well to the experimental measurements in the liquid.

Glycolysis: An early marker for vancomycin-specific T-cell activation.

BACKGROUND: Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients. METHODS: CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with in vitro assays. RESULTS: Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction. CONCLUSION: These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Combined central serous chorioretinopathy, hypermetropia, short axial length, chorioretinal folds, enlarged/thickened ocular coats, with varying association of scleral changes (CHAFES).

PURPOSE: To describe a condition with the following features: chronic central serous chorioretinopathy (CCSC), chorioretinal folds, scleral changes (including any of the following flattened or 'squared off' posterior pole, 'T sign', or thickened ocular coats), accompanied by a short axial length and hypermetropia in a series of 7 patients. METHODS: The case notes of 7 patients presenting with a combination of CSC, choroidal folds scleral changes and hypermetropia were reviewed as part of a retrospective case series. Corrected visual acuities, serial refraction, colour imaging, fluorescein and indocyanine green angiography findings, together with B-ultrasound scan features were recorded, with axial length measurements as available (< 23.3 mm was defined as short). RESULTS: The study included 14 eyes of 7 subjects (2 females and 5 males) with a primary presentation of central vision disturbance. All patients showed signs of previous or current episodes of the following features in at least one eye: CSC (5/7 bilateral); choroidal folds (6/7 bilateral), thickening of ocular coats in the 5 in whom this was measured, at least one scleral abnormality on ultrasound in at least one eye. A short axial length at final appointment was recorded in 13/14 eyes. CONCLUSIONS AND RELEVANCE: The combination of CCSC with choroidal folds, hypermetropia with apparent shortening of the eyeball associated with one or more scleral abnormalities such as a flattened or 'squared off 'appearance of the B ultrasound may be a specific ocular condition. The aetiology of this particular combination of posterior segment manifestations is unknown; the choroid could be the primary focus of disease with secondary involvement of the sclera. Alternatively, the features observed may result from a chronic inflammatory process affecting the sclera with secondary effects on the choroid, retinal pigment epithelium and retina. In our case series, the final vision was not significantly different from vision at presentation.

Assessing the effect of a canine surgical-neutering educational programme on the knowledge and confidence of Indian veterinary participants.

India has a large, free-roaming dog population, encompassing both owned and stray dogs. Canine surgical neutering is often a central component of dog population management and rabies control initiatives. The provision of practical, surgical training opportunities remains a major challenge for veterinary educational establishments worldwide to ensure competency in this routine procedure. A 12-day educational programme, focusing on surgical neutering skills, was developed to address this need. A questionnaire comprising 26 questions covering surgical and clinical topics, and a self-assessment of confidence in undertaking five common surgical procedures, was completed immediately before and after finishing the programme. A total of 296 participants attended, with 228 achieving the inclusion criteria for the study. Total knowledge scores increased significantly after the training programme (mean score pre-18.94, 95% CI 18.13-19.74; post-28.11, 95% CI 27.44-28.77, p < 0.05) with improvements seen in all categories (surgical principles, anaesthesia, antibiotic use and wound management). After accounting for other participants' characteristics, scores increased, on average, by 9 points after training. Being female was associated with significantly higher overall scores, while compared to younger and older age groups, those aged 25-34 were associated with lower overall scores. Amongst those with post-graduate qualifications, overall scores increased with age. Furthermore, there was an increase in self-rated confidence by participants in undertaking all five procedures. This study demonstrates that a targeted training programme can improve veterinary participants' knowledge and confidence in canine surgical neutering and may provide an effective way to develop surgical expertise amongst veterinarians engaged in dog population management initiatives.

Corrigendum: Electronic application for rabies management improves surveillance, data quality, and investigator experience in Haiti.

[This corrects the article DOI: 10.3389/fvets.2023.1052349.].

Electronic application for rabies management improves surveillance, data quality, and investigator experience in Haiti.

Background: Integrated bite case management (IBCM) is a multi-sectoral response to animal-bites which reduces human and canine rabies mortality through animal quarantine, bite-victim counseling, and vaccination tracking. Haiti's national rabies surveillance program was established in 2013 using paper-based IBCM (pIBCM) with adoption of an electronic smartphone application (eIBCM) in 2018. Methods: We evaluated the feasibility of implementing the electronic app in Haiti and compared pIBCM and eIBCM data quality collected January 2013-August 2019. Deaths prevented, cost-per-death averted, and cost-per-investigation during use of pIBCM and eIBCM were estimated using a previously validated rabies cost-effectiveness tool that accounted for bite-victim demographics; probability of acquiring rabies; post-exposure prophylaxis; and costs including training, supplies, and salaries. We compared pIBCM and eIBCM based on data comprehensiveness, completeness, and reporting efficiency. Surveys were administered to IBCM staff to evaluate the usefulness, simplicity, flexibility, and acceptability of eIBCM. Results: Of 15,526 investigations, 79% were paper-based and 21% electronic. IBCM prevented 241 (estimated) human rabies deaths. Using pIBCM, cost-per-death averted was $2,692 and the cost-per-investigation was $21.02; up to 55 data variables were collected per investigation; data transmission took 26 days to reach national staff, and 180 days until analysis. Using eIBCM, the cost-per-death averted was $1,247 and the cost-per-investigation was $22.70; up to 174 data variables were collected per investigation; data transmission took 3 days to reach national staff, and 30 days until analysis. Among 12,194 pIBCM investigations, 55% were mappable by commune, compared to 100% of eIBCM investigations mappable by GPS. Animal case definitions were incorrectly ascribed by investigators in 5.5% of pIBCM investigations and zero for eIBCM; typically, errors were in determining probable vs. suspect case assignments. Overall, eIBCM was well-accepted by staff, who reported the app is easy-to-use, facilitates investigations, and compared to pIBCM hastens data reporting. Discussion: In Haiti, eIBCM showed improved data completeness, data quality, and shorter notification times with minimal increase in operational cost. The electronic app is simple-to-use and facilitates IBCM investigations. Rabies endemic countries could refer to eIBCM in Haiti as a cost-effective means to reduce human rabies mortality and improve surveillance capacity.

Evaluating the benefit of early patient and public involvement for product development and testing with small companies.

INTRODUCTION: There is a growing understanding of the benefits of patient and public involvement (PPI), and its evaluation, in research. An online version of the CUBE PPI evaluation framework has been developed. We sought to use the CUBE to evaluate the value of early PPI with two small healthcare companies during product development. METHODS: Contributors were recruited online and had lived experience of either type 1 diabetes or obesity. Two 1-h sessions were run with a company developing a smartphone application to manage diabetes (DEE-EM): one with young people (YP; n = 5) and one with parents (n = 7). Two 1-h sessions were run with a company developing a weight-loss product, both with adults (n = 7 in each session). Sessions were facilitated by an independent University researcher and attended by company representatives, who presented their product. One facilitator led the evaluation of the session by giving a demonstration of the CUBE and asking simple questions in the YP session. RESULTS: A high proportion of contributors completed the CUBE (80.5% DEE-EM; 93% Oxford Medical Products). Responses were positive to all four CUBE dimensions (in italics). Contributors felt there were diverse ways to contribute to the sessions, and that they had a strong voice to add to the discussion. Balance was achieved regarding whose concerns (public or company) led the agenda, and contributors felt that both companies would make changes based on the discussion. The supportive attitude of both companies resulted in most contributors feeling comfortable participating in PPI sessions with the industry, while recognising the profit-making aspect of their work. CONCLUSIONS: PPI with small healthcare companies is both feasible and worthwhile. The CUBE framework facilitated the evaluation of the interaction between experts in different knowledge spaces. We provide recommendations for future projects, including considerations of who should participate and the level of implicit endorsement of the product that participation implies. PATIENT OR PUBLIC CONTRIBUTION: People with lived experience of type 1 diabetes or obesity were invited to contribute to one of four PPI sessions, which they then evaluated. One contributor agreed to contribute to the analysis of the evaluation data and interpretation and preparation of the manuscript.

Vibrational kinetics in repetitively pulsed atmospheric pressure nitrogen discharges: average-power-dependent switching behaviour.

Characterisation of the vibrational kinetics in nitrogen-based plasmas at atmospheric pressure is crucial for understanding the wider plasma chemistry, which is important for a variety of biomedical, agricultural and chemical processing applications. In this study, a 0-dimensional plasma chemical-kinetics model has been used to investigate vibrational kinetics in repetitively pulsed, atmospheric pressure plasmas operating in pure nitrogen, under application-relevant conditions (average plasma powers of 0.23-4.50 W, frequencies of 1-10 kHz, and peak pulse powers of 23-450 W). Simulations predict that vibrationally excited state production is dominated by electron-impact processes at lower average plasma powers. When the average plasma power increases beyond a certain limit, due to increased pulse frequency or peak pulse power, there is a switch in behaviour, and production of vibrationally excited states becomes dominated by vibrational energy transfer processes (vibration-vibration (V-V) and vibration-translation (V-T) reactions). At this point, the population of vibrational levels up to v ⩽ 40 increases significantly, as a result of V-V reactions causing vibrational up-pumping. At average plasma powers close to where the switching behaviour occurs, there is potential to control the energy efficiency of vibrational state production, as small increases in energy deposition result in large increases in vibrational state densities. Subsequent pathways analysis reveals that energy in the vibrational states can also influence the wider reaction chemistry through vibrational-electronic (V-E) linking reactions (N + N 2 ( 40 ⩽ v ⩽ 45 ) → N ( 2 D ) + N 2 ( A ) and N + N 2 ( 39 ⩽ v ⩽ 45 ) → N + N 2 ( a ' ) ), which result in increased Penning ionisation and an increased average electron density. Overall, this study investigates the potential for delineating the processes by which electronically and vibrationally excited species are produced in nitrogen plasmas. Therefore, potential routes by which nitrogen-containing plasma sources could be tailored, both in terms of chemical composition and energy efficiency, are highlighted.

Updates on the immunopathology and genomics of severe cutaneous adverse drug reactions.

Severe cutaneous adverse reactions (SCARs) such as Stevens-Johnson syndrome, toxic epidermal necrolysis (SJS/TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS)/drug-induced hypersensitivity syndrome (DIHS) cause significant morbidity and mortality and impede new drug development. HLA class I associations with SJS/TEN and drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome have aided preventive efforts and provided insights into immunopathogenesis. In SJS/TEN, HLA class I-restricted oligoclonal CD8+ T-cell responses occur at the tissue level. However, specific HLA risk allele(s) and antigens driving this response have not been identified for most drugs. HLA risk alleles also have incomplete positive and negative predictive values, making truly comprehensive screening currently challenging. Although, there have been key paradigm shifts in knowledge regarding drug hypersensitivity, there are still many open and unanswered questions about SCAR immunopathogenesis, as well as genetic and environmental risk. In addition to understanding the cellular and molecular basis of SCAR at the single-cell level, identification of the MHC-restricted drug-reactive self- or viral peptides driving the hypersensitivity reaction will also be critical to advancing premarketing strategies to predict risk at an individual and drug level. This will also enable identification of biologic markers for earlier diagnosis and accurate prognosis, as well as drug causality and targeted therapeutics.

Chronic anti-coagulation therapy reduced mortality in patients with high cardiovascular risk early in COVID-19 pandemic.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with provoked thrombo-inflammatory responses. Early in the COVID-19 pandemic this was thought to contribute to hypercoagulability and multi-organ system complications in infected patients. Limited studies have evaluated the impact of therapeutic anti-coagulation therapy (AC) in alleviating these risks in COVID-19 positive patients. Our study aimed to investigate whether long-term therapeutic AC can decrease the risk of multi-organ system complications (MOSC) including stroke, limb ischemia, gastrointestinal (GI) bleeding, in-hospital and intensive care unit death in COVID-19 positive patients hospitalized during the early phase of the pandemic in the United States. METHODS: A retrospective analysis was conducted of all COVID-19 positive United States Veterans between March 2020 and October 2020. Patients receiving continuous outpatient therapeutic AC for a least 90 days prior to their initial COVID-19 positive test were assigned to the AC group. Patients who did not receive AC were included in a control group. We analyzed the primary study outcome of MOSC between the AC and control groups using binary logistic regression analysis (Odd-Ratio; OR). RESULTS: We identified 48,066 COVID-19 patients, of them 879 (1.8%) were receiving continuous therapeutic AC. The AC cohort had significantly worse comorbidities than the control group. On the adjusted binary logistic regression model, therapeutic AC significantly decreased in-hospital mortality rate (OR; 0.67, p = 0.04), despite a higher incidence of GI bleeding (OR; 4.00, p = 0.02). However, therapeutic AC did not significantly reduce other adverse events. CONCLUSION: AC therapy reduced in-hospital death early in the COVID-19 pandemic among patients who were hospitalized with the infection. However, it did not decrease the risk of MOSC. Additional trials are needed to determine the effectiveness of AC in preventing complications associated with ongoing emerging strains of the COVID-19 virus.

NEWS2 in out-of-hospital settings, the ambulance and the emergency department.

As more healthcare is provided in non-hospital settings, it is essential to support clinicians in recognising early signs of clinical deterioration to enable prompt intervention and treatment.There are intuitive reasons why the use of the National Early Warning Score 2 (NEWS2) in out-of-hospital settings may enhance the community response to acute illness by using a common language across healthcare. An additional advantage of the use of NEWS2 in community settings is that it is not disease specific and requires no expensive technology or great expertise to take a full set of observations that can be an indicator of clinical acuity.However, concerns have been expressed as NEWS2 was developed in acute hospital settings that it may not be applicable in community settings; this review shares some of the practical ways that NEWS2 can support clinical practice along with the emerging published evidence.

Chronic Anti-Coagulation Therapy Reduced Mortality In Patients With High Cardiovascular Risk Early In COVID-19 Pandemic.

Background: Coronavirus disease 2019 (COVID-19) is associated with provoked thrombo-inflammatory responses. Early in the COVID-19 pandemic this was thought to contribute to hypercoagulability and multi-organ system complications in infected patients. Limited studies have evaluated the impact of therapeutic anti-coagulation therapy (AC) in alleviate these risks in COVID-19 positive patients. Our study aimed to investigate whether long-term therapeutic AC can decrease the risk of multi-organ system complications (MOSC) including stroke, limb ischemia, gastrointestinal (GI) bleeding, in-hospital and intensive care unit death in COVID-19 positive patients during the early phase of the pandemic in the United States. Methods: A retrospective analysis was conducted of all COVID-19 positive United States Veterans between March 2020 and October 2020. Patients receiving continuous therapeutic AC for a least 30 days prior to or after their initial COVID-19 positive test were assigned to the AC group. Patients who did not receive AC were included in a control group. We analyzed the primary study outcome of MOSC between the AC and control groups using binary logistic regression analysis (Odd-Ratio; OR). Results: We identified 48,066 COVID-19 patients, of them 879 (1.8%) were receiving continuous therapeutic AC. The AC cohort had significantly worse comorbidities than the control group. On the adjusted binary logistic regression model, therapeutic AC significantly decreased in-hospital mortality rate (OR; 0.67, p = 0.04), despite a higher incidence of GI bleeding (OR; 4.00, p = 0.02). However, therapeutic AC did not significantly reduce other adverse events. Conclusion: AC therapy reduced in-hospital death early in the COVID-19 pandemic among patients who were hospitalized with the infection. However, it did not decrease the risk of MOSC. Additional trials are needed to determine the effectiveness of AC in preventing complications associated with ongoing emerging strains of the COVID-19 virus.

A low-cost, sensitive and specific PCR-based tool for rapid clinical detection of HLA-B*35 alleles associated with delayed drug hypersensitivity reactions.

HLA (HLA) alleles are risk factors for CD8+ T-cell-mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence-based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low-cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi-disease risk; exemplified by associations with diverse alleles in HLA-B*35, including HLA-B*35:01 and green tea- or co-trimoxazole-induced liver injury. Here, we developed a cost-effective (<$10USD) qPCR assay for rapid (<2.5 h) clinical detection of HLA-B*35 alleles. The assay was validated using 430 DNA samples with previous American society for histocompatibility and immunogenetics-accredited sequence-based high-resolution HLA typing, positively detecting all HLA-B*35 allelic variants in our cohort, and as expected by primer design, the six samples that expressed low-frequency B*78:01. The assay did not result in positive detection for any negative control allele. With expected detection of B*35 and B*78, our assay sensitivity (95% CI, 95.07%-100.00%) and specificity (95% CI, 98.97%-100.00%) of 100% using as low as 10 ng of DNA provides a reliable HLA-B*35 screening tool for clinical validation and HLA-risk-based prevention and diagnostics.

First study on molecular detection of three major canine tick-borne pathogens in subclinically infected dogs in Chiang Mai, Thailand.

Background and Aim: Canine tick-borne pathogens (CTBPs) are an important cause of morbidity in dogs in Thailand. This study aimed to evaluate the occurrence of three CTBPs in clinically normal, owned dogs to understand the risk for the general canine population. We also examined sex, age, tick infestation, and packed cell volume (PCV) of the animals in association with active infection of the CTBPs. Materials and Methods: A total of 139 dogs were included in the study. Blood samples were collected for thin blood smear, PCV and nested polymerase chain reaction (PCR) assay. Statistical analyses were performed to examine the association between individual factors and CTBP infection status determined by PCR. In addition, sensitivity, specificity, and Cohen's kappa were calculated to assess the utility of routine blood smear. Results: The PCR results showed that 31 dogs (22.3%) were infected with at least one of the three pathogens. The occurrence rate for Ehrlichia canis, Anaplasma platys, and Hepatozoon canis was 2.2% (3/139), 18.7% (24/139), and 2.8% (4/139), respectively. There were two cases of coinfection with A. platys and E. canis. The univariate analyses did not yield any associations between recorded variables and the active infection. Microscopic examination showed good sensitivity and agreement only for H. canis (Sn: 75%, 95% confidence interval: 24.9-98.7, k=0.85). Conclusion: Our findings confirmed the endemicity of the CTBPs in owned canine population in the study site. In-depth epidemiological investigation would be warranted to elucidate environmental risk factors for CTBP infection.

Practical Implementation of Genetics: New Concepts in Immunogenomics to Predict, Prevent, and Diagnose Drug Hypersensitivity.

Delayed drug hypersensitivities are CD8+ T cell-mediated reactions associated with up to 50% mortality. Human leukocyte antigen (HLA) alleles are known to predispose disease and are specific to drug, reaction, and patient ethnicity. Pretreatment screening is recommended for a handful of the strongest associations to identify and prevent drug use in high-risk patients. However, an incomplete predictive value implicates other HLA-imposed risk factors, and low carriage of many identified HLA-risk alleles combined with the high cost of sequence-based typing has limited economic viability for similar recommendation of screening across drugs and health care systems. For mitigation, an expanding armory of low-cost polymerase chain reaction-based screens is being developed, and HLA-imposed risk factors are being discovered. These include (1) polymorphic variants of metabolic and endoplasmic reticulum aminopeptidase enzymes toward multiallelic screening with increased predictivity; (2) regulation by immune checkpoint inhibitors, enabling detolerized animal models of human disease; and (3) immunodominant T cell receptors (TCR) on clonally expanded CD8+ T cells. For the latter, HLA risk-restricted TCR provides immunogenomic strategies and samples from a single patient to identify novel HLA-risk associations in underserved minority populations, tissue-relevant effector biomarkers toward earlier diagnosis and treatment, and HLA-TCR-presented immunogenic structures to aid future drug development.

Impact of Socioeconomic Status on Major Amputation in Patients with Peripheral Vascular Disease and Diabetes Mellitus.

BACKGROUND: Both peripheral vascular disease (PVD) and diabetes mellitus (DM) are leading causes of lower extremity amputation. The Area Deprivation Index (ADI) is a tool used to estimate socioeconomic status (SES) based off a person's 9-digit zip code, and this value has been shown to correlate with poor health outcomes. We sought to understand the effect of SES on major amputation in diabetic patients with PVD in a single healthcare system. METHODS: All patients presenting to a single healthcare system with dual diagnosis of PVD and DM from January 2012 to December 2017 were identified using International Classification of Diseases (ICD) 9/10 codes. Patients undergoing major amputation (below-knee and above-knee) were identified by Current Procedural Terminology (CPT) codes and compared to those who did not have amputation. The ADI score and comorbid disease processes were identified. The Mann-Whitney U-test was performed to compare ADI scores between the amputation and nonamputation groups. Categorical variables were analyzed using the Chi-squared or Fisher's exact test, and t-tests were used for continuous variables. A logistic regression was performed to test the association between SES and amputation status. RESULTS: A total of 2,009 patients were identified, of which 85 underwent major amputation. After adjusting for comorbidities, patients in the amputation group had higher ADI scores as compared to those who did not have amputation (median ADI score 8 vs. 6, P < 0.05). Logistic regression modeling demonstrated an Odds Ratio of 1.10 (95% confidence interval: 1.01-1.19), indicating the odds of being in the amputation group are increased by 10% for every 1-point increase in the ADI score. CONCLUSIONS: After controlling for comorbidities, patients with PVD and DM residing in neighborhoods with lower SES have increased odds of undergoing major lower-limb amputation than those from neighborhoods with higher SES despite receiving care at the same healthcare system. Further study is warranted to determine factors contributing to this difference.