Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda.

Metagenomic next generation metagenomic sequencing (mNGS) has proven to be a useful tool in the diagnosis and identification of novel human pathogens and pathogens not identified on routine clinical microbiologic tests. In this study, we applied mNGS to characterize plasma RNA isolated from 42 study participants with unexplained acute febrile illness (AFI) admitted to tertiary referral hospitals in Mubende and Arua, Uganda. Study participants were selected based on clinical criteria suggestive of viral infection (i.e., thrombocytopenia, leukopenia). The study population had a median age of 28 years (IQR:24 to 38.5) and median platelet count of 114 x103 cells/mm3 (IQR:66,500 to 189,800). An average of 25 million 100 bp reads were generated per sample. We identified strong signals from diverse virus, bacteria, fungi, or parasites in 10 (23.8%) of the study participants. These included well recognized pathogens like Helicobacter pylori, human herpes virus-8, Plasmodium falciparum, Neisseria gonorrhoeae, and Rickettsia conorii. We further confirmed Rickettsia conorii infection, the cause of Mediterranean Spotted Fever (MSF), using PCR assays and Sanger sequencing. mNGS was a useful addition for detection of otherwise undetected pathogens and well-recognized non-pathogens. This is the first report to describe the molecular confirmation of a hospitalized case of MSF in sub-Saharan Africa (SSA). Further studies are needed to determine the utility of mNGS for disease surveillance in similar settings.

Pathogen genomic surveillance status among lower resource settings in Asia.

Asia remains vulnerable to new and emerging infectious diseases. Understanding how to improve next generation sequencing (NGS) use in pathogen surveillance is an urgent priority for regional health security. Here we developed a pathogen genomic surveillance assessment framework to assess capacity in low-resource settings in South and Southeast Asia. Data collected between June 2022 and March 2023 from 42 institutions in 13 countries showed pathogen genomics capacity exists, but use is limited and under-resourced. All countries had NGS capacity and seven countries had strategic plans integrating pathogen genomics into wider surveillance efforts. Several pathogens were prioritized for human surveillance, but NGS application to environmental and human-animal interface surveillance was limited. Barriers to NGS implementation include reliance on external funding, supply chain challenges, trained personnel shortages and limited quality assurance mechanisms. Coordinated efforts are required to support national planning, address capacity gaps, enhance quality assurance and facilitate data sharing for decision making.

Periprosthetic radius fracture after total wrist arthroplasty: A case report.

Total wrist arthroplasty (TWA) is indicated in select low demand patients with pan-carpal arthritis to decrease pain and preserve motion. Complications of TWA are well described including aseptic loosening, superficial and deep infection, wound issues, component dislocation, stiffness, and both intraoperative and post-operative fracture. With 4th generation implant designs, the incidence of many of these complications have decreased, but these complications remain challenging to address. In particular, sparse literature is available for the treatment of periprosthetic radius fractures after TWA. This report describes the management of an open, periprosthetic distal third radius fracture 18 months after index TWA in a medically complex 53-year-old female with lag screw fixation and a dorsal wrist spanning plate (DSP).

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

A program for real-time surveillance of SARS-CoV-2 genetics.

The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.S.) from March 13th 2020 to July 3rd 2023. Our sequencing protocol ('Virseq') utilizes wet and dry lab procedures to generate mutation-resistant sequencing of the entire SARS-CoV-2 genome, capturing all major lineages. We also characterize 379 clinically relevant SARS-CoV-2 multi-strain co-infections and ensure robust detection of emerging lineages via simulation. The modular infrastructure, sequencing, and analysis capabilities we describe support the U.S. Centers for Disease Control and Prevention national surveillance program and serve as a model for rapid response to emerging pandemics at a national scale.

Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model.

Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PLpro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

Dignity in the Pediatric Population: A Systematic Review.

CONTEXT: Children are a uniquely vulnerable patient population with restricted abilities for self-advocacy and autonomy, risking infringement upon their dignity. Yet the concept of dignity in pediatrics remains underexplored relative to the adult literature and other outcome measures. OBJECTIVES: To characterize how dignity is defined, evaluated, and/or measured in pediatrics. METHODS: We conducted a systematic review following PRISMA guidelines across the following databases: MEDLINE, Embase, Cumulative Index of Nursing and Allied Health, PsycINFO, Global Health, Social Science Premium Collection, and Dissertation and Theses. We included publications from database inception through April 2023, in English, involving children aged 0-18 years, and prioritizing dignity as a central theme with a focus on defining, evaluating, or measuring dignity. Study descriptions and pertinent characteristics were extracted and synthesized using a predefined form. RESULTS: Forty-four articles met inclusion criteria; fewer than half comprised original research (20/44, 45%). Most studies (38/44, 86%) included description of the meaning of dignity, with emergence of salient themes around respect, communication, agency/autonomy, and privacy. Less than half (19/44, 43%) included a measurement or evaluation of dignity; approximately one-third described dignity therapy. More than one-third of publications focused on dignity at end of life (17/44, 39%) and included discussions of palliative medicine and hospice (15/44, 34%). CONCLUSION: Relatively few published studies describe dignity in pediatrics. Opportunities exist to broaden scholarship on this topic in partnership with patients, families, and clinicians, with the goal of assessing and strengthening dignity-centered care across the illness course and at the end of life.

Methylome-wide studies of six metabolic traits.

Exploring the molecular correlates of metabolic health measures may identify the shared and unique biological processes and pathways that they track. Here, we performed epigenome-wide association studies (EWASs) of six metabolic traits: body mass index (BMI), body fat percentage, waist-hip ratio (WHR), and blood-based measures of glucose, high-density lipoprotein (HDL) cholesterol, and total cholesterol. We considered blood-based DNA methylation (DNAm) from >750,000 CpG sites in over 17,000 volunteers from the Generation Scotland (GS) cohort. Linear regression analyses identified between 304 and 11,815 significant CpGs per trait at P<3.6×10-8, with 37 significant CpG sites across all six traits. Further, we performed a Bayesian EWAS that jointly models all CpGs simultaneously and conditionally on each other, as opposed to the marginal linear regression analyses. This identified between 3 and 27 CpGs with a posterior inclusion probability ≥ 0.95 across the six traits. Next, we used elastic net penalised regression to train epigenetic scores (EpiScores) of each trait in GS, which were then tested in the Lothian Birth Cohort 1936 (LBC1936; European ancestry) and Health for Life in Singapore (HELIOS; Indian-, Malay- and Chinese-ancestries). A maximum of 27.1% of the variance in BMI was explained by the BMI EpiScore in the subset of Malay-ancestry Singaporeans. Four metabolic EpiScores were associated with general cognitive function in LBC1936 in models adjusted for vascular risk factors (Standardised ßrange: 0.08 - 0.12, PFDR < 0.05). EpiScores of metabolic health are applicable across ancestries and can reflect differences in brain health.

Understanding how young people transitioning from out-of-home care acquire and develop independent living skills and knowledge: A systematic review of longitudinal studies.

Young people leaving state care often experience hardship in many areas of their life. At a population level, their outcomes in early adulthood are poorer compared to general populations. Effective preparation for leaving care and post-care support systems is vital to improving outcomes. Individual and systemic support for young people to acquire Independent Living Skills (ILS) in the following eight ILS domains have been identified: Financial Management, Knowledge of Accessing Available Supports, Managing Housing, Education Planning, Job Seeking, Health Risk Management, Domestic and Self-help Task, and Managing Relationships. This systematic review aims to identify, summarise, and appraise longitudinal studies that address ILS across these ILS domains to understand better how outcomes could be improved. Seven databases (CINAHL, Embase, ProQuest, PsychINFO, PubMed, Scopus, and Web of Science) were searched on 20th July 2023. In total, twenty-seven studies published between 1994 and 2022 from various countries met the eligibility criteria. The included studies reported on 2-4 waves and adopted different methodological approaches. Study quality was scored using Qualsyst. Study characteristics and details of the interventions are presented in tables. Studies cover overlapping ILS domains, which are mapped in a matrix. Results revealed that nearly three-quarters (74% or 20 out of 27) of studies explored four or fewer of the eight ILS domains. The most frequent ILS domain covered was 'Knowledge of Accessing Available Supports' (19/27 studies). The main conclusion considers the concept of independence as a misnomer, with ILS covering multiple, intersecting, and interdependent domains, which ultimately help and hinder one another. Further research is required to adopt a more comprehensive approach encompassing all the domains to better inform policy, programs, and practice. A limitation is that a meta-analysis was not conducted for this review. This study registered a 'Protocol' with OSF Registries (DOI: 10.17605/OSF.IO/MJ3ZX) on June 5th, 2022.

Recent advances in understanding the mechanisms in skeletal muscle of interaction between exercise and frontline antihyperglycemic drugs.

Regular exercise and antihyperglycemic drugs are front-line treatments for type-2 diabetes and related metabolic disorders. Leading drugs are metformin, sodium-glucose cotransporter-2 inhibitors, and glucagon-like peptide 1 receptor agonists. Each class has strong individual efficacy to treat hyperglycemia, yet the combination with exercise can yield varied results, some of which include blunting of expected metabolic benefits. Skeletal muscle insulin resistance contributes to the development of type-2 diabetes while improvements in skeletal muscle insulin signaling are among key adaptations to exercise training. The current review identifies recent advances into the mechanisms, with an emphasis on skeletal muscle, of the interaction between exercise and these common antihyperglycemic drugs. The review is written toward researchers and thus highlights specific gaps in knowledge and considerations for future study directions.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Intramuscular diacylglycerol accumulates with acute hyperinsulinemia in insulin-resistant phenotypes.

Elevated skeletal muscle diacylglycerols (DAGs) and ceramides can impair insulin signaling, and acylcarnitines (acylCNs) reflect impaired mitochondrial fatty acid oxidation, thus, the intramuscular lipid profile is indicative of insulin resistance. Acute (i.e., postprandial) hyperinsulinemia has been shown to elevate lipid concentrations in healthy muscle and is an independent risk factor for type 2 diabetes (T2D). However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to or exacerbating insulin resistance. We therefore investigated the impact of acute hyperinsulinemia on the skeletal muscle lipid profile to help characterize the physiological basis in which hyperinsulinemia elevates T2D risk. In a cross-sectional comparison, endurance athletes (n = 12), sedentary lean adults (n = 12), and individuals with obesity (n = 13) and T2D (n = 7) underwent a hyperinsulinemic-euglycemic clamp with muscle biopsies. Although there were no significant differences in total 1,2-DAG fluctuations, there was a 2% decrease in athletes versus a 53% increase in T2D during acute hyperinsulinemia (P = 0.087). Moreover, C18 1,2-DAG species increased during the clamp with T2D only, which negatively correlated with insulin sensitivity (P < 0.050). Basal muscle C18:0 total ceramides were elevated with T2D (P = 0.029), but not altered by clamp. Acylcarnitines were universally lowered during hyperinsulinemia, with more robust reductions of 80% in athletes compared with only 46% with T2D (albeit not statistically significant, main effect of group, P = 0.624). Similar fluctuations with acute hyperinsulinemia increasing 1,2 DAGs in insulin-resistant phenotypes and universally lowering acylcarnitines were observed in male mice. In conclusion, acute hyperinsulinemia elevates muscle 1,2-DAG levels with insulin-resistant phenotypes. This suggests a possible dysregulation of intramuscular lipid metabolism in the fed state in individuals with low insulin sensitivity, which may exacerbate insulin resistance.NEW & NOTEWORTHY Postprandial hyperinsulinemia is a risk factor for type 2 diabetes and may increase muscle lipids. However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to insulin resistance. We observed that acute hyperinsulinemia elevates muscle 1,2-DAGs in insulin-resistant phenotypes, whereas ceramides were unaltered. Insulin-mediated acylcarnitine reductions are also hindered with high-fat feeding. The postprandial period may exacerbate insulin resistance in metabolically unhealthy phenotypes.

Advancing Research on and Treatment of Dissociative Identity Disorder With People With Lived Experience.

Dissociative identity disorder is a posttraumatic, psychobiological syndrome that develops over time during childhood. Despite empirical evidence supporting the validity of this diagnosis and its relation to trauma, the disorder remains a misunderstood and stigmatized condition. This article highlights expert consensus guidelines and current empirical research on the treatment of dissociative identity disorder. In addition, the authors describe the Lived Experience Advisory Panel (LEAP), which was designed to leverage the expertise of individuals with dissociative identity disorder to combat stigma and improve research, clinical programming, professional education, and public outreach related to the disorder. This article also describes how LEAP members have partnered with other researchers to create new knowledge through participatory action research in order to advance equitable service provision and effect positive change.

Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study.

Background: In Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. Methods: Between September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 µg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. Results: A total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 µg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 µg/ml, 12/15 were mcr-1. Conclusions: We found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Diagnostic accuracy of DPP Fever Panel II Asia tests for tropical fever diagnosis.

BACKGROUND: Fever is the most frequent symptom in patients seeking care in South and Southeast Asia. The introduction of rapid diagnostic tests (RDTs) for malaria continues to drive patient management and care. Malaria-negative cases are commonly treated with antibiotics without confirmation of bacteraemia. Conventional laboratory tests for differential diagnosis require skilled staff and appropriate access to healthcare facilities. In addition, introducing single-disease RDTs instead of conventional laboratory tests remains costly. To overcome some of the delivery challenges of multiple separate tests, a multiplexed RDT with the capacity to diagnose a diverse range of tropical fevers would be a cost-effective solution. In this study, a multiplex lateral flow immunoassay (DPP Fever Panel II Assay) that can detect serum immunoglobulin M (IgM) and specific microbial antigens of common fever agents in Asia (Orientia tsutsugamushi, Rickettsia typhi, Leptospira spp., Burkholderia pseudomallei, Dengue virus, Chikungunya virus, and Zika virus), was evaluated. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood (WB) and serum samples from 300 patients with undefined febrile illness (UFI) recruited in Vientiane, Laos PDR were tested using the DPP Fever Panel II, which consists of an Antibody panel and Antigen panel. To compare reader performance, results were recorded using two DPP readers, DPP Micro Reader (Micro Reader 1) and DPP Micro Reader Next Generation (Micro Reader 2). WB and serum samples were run on the same fever panel and read on both micro readers in order to compare results. ROC analysis and equal variance analysis were performed to inform the diagnostic validity of the test compared against the respective reference standards of each fever agent (S1 Table). Overall better AUC values were observed in whole blood results. No significant difference in AUC performance was observed when comparing whole blood and serum sample testing, except for when testing for R. typhi IgM (p = 0.04), Leptospira IgM (p = 0.02), and Dengue IgG (p = 0.03). Linear regression depicted R2 values had ~70% agreement across WB and serum samples, except when testing for leptospirosis and Zika, where the R2 values were 0.37 and 0.47, respectively. No significant difference was observed between the performance of Micro Reader 1 and Micro Reader 2, except when testing for the following pathogens: Zika IgM, Zika IgG, and B pseudomallei CPS Ag. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the diagnostic accuracy of the DPP Fever Panel II is comparable to that of commonly used RDTs. The optimal cut-off would depend on the use of the test and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This multiplex RDT offers diagnostic benefits in areas with limited access to healthcare and has the potential to improve field testing capacities. This could improve tropical fever management and reduce the public health burden in endemic low-resource areas.

Border Control: The Role of the Microbiome in Regulating Epithelial Barrier Function.

The gut mucosal epithelium is one of the largest organs in the body and plays a critical role in regulating the crosstalk between the resident microbiome and the host. To this effect, the tight control of what is permitted through this barrier is of high importance. There should be restricted passage of harmful microorganisms and antigens while at the same time allowing the absorption of nutrients and water. An increased gut permeability, or "leaky gut", has been associated with a variety of diseases ranging from infections, metabolic diseases, and inflammatory and autoimmune diseases to neurological conditions. Several factors can affect gut permeability, including cytokines, dietary components, and the gut microbiome. Here, we discuss how the gut microbiome impacts the permeability of the gut epithelial barrier and how this can be harnessed for therapeutic purposes.

Treatment of dissociative identity disorder: leveraging neurobiology to optimize success.

INTRODUCTION: Dissociative identity disorder (DID) is a treatable mental health condition that is associated with a range of psychobiological manifestations. However, historical controversy, modern day misunderstanding, and lack of professional education have prevented accurate treatment information from reaching most clinicians and patients. These obstacles also have slowed empirical efforts to improve treatment outcomes for people with DID. Emerging neurobiological findings in DID provide essential information that can be used to improve treatment outcomes. AREAS COVERED: In this narrative review, the authors discuss symptom characteristics of DID, including dissociative self-states. Current treatment approaches are described, focusing on empirically supported psychotherapeutic interventions for DID and pharmacological agents targeting dissociative symptoms in other conditions. Neurobiological correlates of DID are reviewed, including recent research aimed at identifying a neural signature of DID. EXPERT OPINION: Now is the time to move beyond historical controversy and focus on improving DID treatment availability and efficacy. Neurobiological findings could optimize treatment by reducing shame, aiding assessment, providing novel interventional brain targets and guiding novel pharmacologic and psychotherapeutic interventions. The inclusion of those with lived experience in the design, planning and interpretation of research investigations is another powerful way to improve health outcomes for those with DID.