Phenotypic and genomic analysis of bacteria from war wounds in Dnipro, Ukraine.

Objectives: To better understand the source and potential transmission routes of antibiotic-resistant bacteria infecting injured service members in Ukraine. Methods: Phenotypic and genomic characterizations were performed on 11 Gram-negative pathogens cultured from war wounds at an intermediate evacuation hospital in Dnipro. Results: The analysis revealed both susceptible and extensively drug-resistant strains present in cultures, including high-risk global clones carrying carbapenemases. Conclusions: Globally distributed carbapenemase-producing lineages are being acquired early in the medical evacuation process.

Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Next-generation IEDB tools: a platform for epitope prediction and analysis.

The Next-Generation (NG) IEDB Tools website (https://nextgen-tools.iedb.org) provides users with a redesigned interface to many of the algorithms for epitope prediction and analysis that were originally released on the legacy IEDB Tools website. The initial release focuses on consolidation of all tools related to HLA class I epitopes (MHC binding, elution, immunogenicity, and processing), making all of these predictions accessible from a single application and allowing for their simultaneous execution with minimal user inputs. Additionally, the PEPMatch tool for identifying highly similar epitopes in a set of curated proteomes, as well as a tool for epitope clustering, are available on the site. The NG Tools site allows users to build data pipelines by sending the output of one tool as input for the next. Over the next several years, all pre-existing IEDB Tools, and any newly developed tools, will be integrated into this new site. Here we describe the philosophy behind the redesign and demonstrate the utility and productivity enhancements that are enabled by the new interface.

Dynamics of the Oral Microbiome During Initial Military Training at Fort Benning, Georgia.

INTRODUCTION: Military trainees are at increased risk for infectious disease outbreaks because of the unique circumstances of the training environment (e.g., close proximity areas and physiologic/psychologic stress). Standard medical countermeasures in military training settings include routine immunization (e.g., influenza and adenovirus) as well as chemoprophylaxis [e.g., benzathine penicillin G (Bicillin) for the prevention of group A streptococcal disease] for pathogens associated with outbreaks in these settings. In a population of U.S. Army Infantry trainees, we evaluated changes in the oral microbiome during a 14-week military training cycle. MATERIALS AND METHODS: Trainees were enrolled in an observational cohort study in 2015-2016. In 2015, Bicillin was administered to trainees to ameliorate the risk of group A Streptococcus outbreaks, whereas in 2016, trainees did not receive a Bicillin inoculation. Oropharyngeal swabs were collected from participants at days 0, 7, 14, 28, 56, and 90 of training. Swabs were collected, flash frozen, and stored. DNA was extracted from swabs, and amplicon sequencing of the 16s rRNA gene was performed. Microbiome dynamics were evaluated using the QIIME 2 workflow along with DADA2, SINA with SILVA, and an additional processing in R. RESULTS: We observed that microbiome samples from the baseline (day 0) visit were distinct from one another, whereas samples collected on day 14 exhibited significant microbiome convergence. Day 14 convergence was coincident with an increase in DNA sequences associated with Streptococcus, though there was not a significant difference between Streptococcus abundance over time between 2015 and 2016 (P = .07), suggesting that Bicillin prophylaxis did not significantly impact overall Streptococcus abundance. CONCLUSIONS: The temporary convergence of microbiomes is coincident with a rise in communicable infections in this population. The dynamic response of microbiomes during initial military training supports similar observations in the literature of transient convergence of the human microbiome under cohabitation in the time frame including in this experiment. This population and the associated longitudinal studies allow for controlled studies of human microbiome under diverse conditions.

Profiling of serum factors associated with Staphylococcus aureus skin and soft tissue infections as a foundation for biomarker identification.

Background: People living in close quarters, such as military trainees, are at increased risk for skin and soft tissue infections (SSTI), especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). The serum immune factors associated with the onset of SSTI are not well understood. Methods: We conducted a longitudinal study of SSTIs, enrolling US Army trainees before starting military training and following up for 14 weeks. Samples were collected on Day 0, 56, and 90. Serum chemokines and cytokines among 16 SSTI cases and 51 healthy controls were evaluated using an electro-chemiluminescence based multiplex assay platform. Results: Of 54 tested cytokines, 12 were significantly higher among SSTI cases as compared to controls. Among the cases, there were correlations between factors associated with vascular injury (i.e., VCAM-1, ICAM-1, and Flt1), the angiogenetic factor VEGF, and IL-10. Unsupervised machine learning (Principal Component Analysis) revealed that IL10, IL17A, C-reactive protein, ICAM1, VCAM1, SAA, Flt1, and VGEF were indicative of SSTI. Conclusion: The study demonstrates the power of immunoprofiling for identifying factors predictive of pre-illness state of SSTI thereby identifying early stages of an infection and individuals susceptible to SSTI.

Unlocking Dendritic Cell-Based Vaccine Efficacy through Genetic Modulation-How Soon Is Now?

The dendritic cell (DC) vaccine anti-cancer strategy involves tumour-associated antigen loading and maturation of autologous ex vivo cultured DCs, followed by infusion into the cancer patient. This strategy stemmed from the idea that to induce a robust anti-tumour immune response, it was necessary to bypass the fundamental immunosuppressive mechanisms of the tumour microenvironment that dampen down endogenous innate immune cell activation and enable tumours to evade immune attack. Even though the feasibility and safety of DC vaccines have long been confirmed, clinical response rates remain disappointing. Hence, the full potential of DC vaccines has yet to be reached. Whether this cellular-based vaccination approach will fully realise its position in the immunotherapy arsenal is yet to be determined. Attempts to increase DC vaccine immunogenicity will depend on increasing our understanding of DC biology and the signalling pathways involved in antigen uptake, maturation, migration, and T lymphocyte priming to identify amenable molecular targets to improve DC vaccine performance. This review evaluates various genetic engineering strategies that have been employed to optimise and boost the efficacy of DC vaccines.

Six Extensively Drug-Resistant Bacteria in an Injured Soldier, Ukraine.

Blood and surveillance cultures from an injured service member from Ukraine grew Acinetobacter baumannii, Klebsiella pneumoniae, Enterococcus faecium, and 3 distinct Pseudomonas aeruginosa strains. Isolates were nonsusceptible to most antibiotics and carried an array of antibiotic resistant genes, including carbapenemases (blaIMP-1, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-72) and 16S methyltransferases (armA and rmtB4).

Identification of an Outbreak Cluster of Extensively Antibiotic-Resistant GC1 Acinetobacter baumannii Isolates in U.S. Military Treatment Facilities.

An outbreak involving an extensively antibiotic-resistant Acinetobacter baumannii strain in three military treatment facilities was identified. Fifty-nine isolates recovered from 30 patients over a 4-year period were found among a large collection of isolates using core genome multilocus sequence typing (MLST). They differed by only 0 to 18 single nucleotide polymorphisms (SNPs) and carried the same resistance determinants except that the aphA6 gene was missing in 25 isolates. They represent a novel sublineage of GC1 lineage 1 that likely originated in Afghanistan. IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals.

A panel of diverse Klebsiella pneumoniae clinical isolates for research and development.

Klebsiella pneumoniae are a leading cause of healthcare-associated infections worldwide. In particular, strains expressing extended-spectrum ß-lactamases (ESBLs) and carbapenemases pose serious treatment challenges, leading the World Health Organization (WHO) to designate ESBL and carbapenem-resistant Enterobacteriaceae as 'critical' threats to human health. Research efforts to combat these pathogens can be supported by accessibility to diverse and clinically relevant isolates for testing novel therapeutics. Here, we describe a panel of 100 diverse K. pneumoniae isolates that are publicly available to assist the research community in this endeavour. Whole-genome sequencing (WGS) was performed on 3878 K. pneumoniae clinical isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. The isolates were cultured from 63 facilities in 19 countries between 2001 and 2020. Core-genome multilocus sequence typing and high-resolution single-nucleotide polymorphism-based phylogenetic analyses captured the genetic diversity of the collection and were used to select the final panel of 100 isolates. In addition to known multidrug-resistant (MDR) pandemic lineages, the final panel includes hypervirulent lineages and isolates with specific and diverse resistance genes and virulence biomarkers. A broad range of antibiotic susceptibilities, ranging from pan-sensitive to extensively drug-resistant isolates, are described. The panel collection, and all associated metadata and genome sequences, are available at no additional cost and will be an important resource for the research community and for the design and development of novel antimicrobial agents and diagnostics against this important pathogen.

The Emergence and Persistence of Candida auris in Western New York With No Epidemiologic Links: A Failure of Stewardship?

Reports of Candida auris infection in patients without epidemiologic links to prior outbreaks are scarce. We describe the genomic epidemiology of such a case in Western New York. Before emergence, the patient received >60 days of excess antibiotics. Candida auris was recovered on near-patient surfaces after enhanced terminal cleanings.

A one-year genomic investigation of Escherichia coli epidemiology and nosocomial spread at a large US healthcare network.

BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are a leading cause of bloodstream and urinary tract infections worldwide. Over the last two decades, increased rates of antibiotic resistance in E. coli have been reported, further complicating treatment. Worryingly, specific lineages expressing extended-spectrum ß-lactamases (ESBLs) and fluoroquinolone resistance have proliferated and are now considered a serious threat. Obtaining contemporary information on the epidemiology and prevalence of these circulating lineages is critical for containing their spread globally and within the clinic. METHODS: Whole-genome sequencing (WGS), phylogenetic analysis, and antibiotic susceptibility testing were performed for a complete set of 2075 E. coli clinical isolates collected from 1776 patients at a large tertiary healthcare network in the USA between October 2019 and September 2020. RESULTS: The isolates represented two main phylogenetic groups, B2 and D, with six lineages accounting for 53% of strains: ST-69, ST-73, ST-95, ST-131, ST-127, and ST-1193. Twenty-seven percent of the primary isolates were multidrug resistant (MDR) and 5% carried an ESBL gene. Importantly, 74% of the ESBL-E.coli were co-resistant to fluoroquinolones and mostly belonged to pandemic ST-131 and emerging ST-1193. SNP-based detection of possible outbreaks identified 95 potential transmission clusters totaling 258 isolates (12% of the whole population) from ≥ 2 patients. While the proportion of MDR isolates was enriched in the set of putative transmission isolates compared to sporadic infections (35 vs 27%, p = 0.007), a large fraction (61%) of the predicted outbreaks (including the largest cluster grouping isolates from 12 patients) were caused by the transmission of non-MDR clones. CONCLUSION: By coupling in-depth genomic characterization with a complete sampling of clinical isolates for a full year, this study provides a rare and contemporary survey on the epidemiology and spread of E. coli in a large US healthcare network. While surveillance and infection control efforts often focus on ESBL and MDR lineages, our findings reveal that non-MDR isolates represent a large burden of infections, including those of predicted nosocomial origins. This increased awareness is key for implementing effective WGS-based surveillance as a routine technology for infection control.

Quality and Integrated Service Delivery: A Cross-Sectional Study of the Effects of Malaria and Antenatal Service Quality on Malaria Intervention Use in Sub-Saharan Africa.

Using regionally linked facility and household surveys, we measured the quality of integrated antenatal care and malaria in pregnancy services in Kenya, Namibia, Senegal, and Tanzania. We examined country heterogeneities for the association of integrated antenatal and malaria service quality scores with insecticide-treated bed net (ITN) use in pregnant women and children under-five and intermittent preventive treatment in pregnancy (IPTp-2) uptake. Malaria in pregnancy service quality was low overall. Our findings suggest modest, positive associations between malaria in pregnancy quality and ITN use and IPTp-2 uptake across pooled models and for most studied countries, with evidence of heterogeneity in the strength of associations and relevant confounding factors. Antenatal care quality generally was not associated with the study outcomes, although a positive interaction with malaria in pregnancy quality was present for pooled ITN use models. The improved quality of malaria services delivered during formal antenatal care can help address low coverage and usage rates of preventive malaria interventions in pregnancy and childhood. Study findings may be used to target quality improvement efforts at the sub-national level. Study methods may be adapted to identify low-performing facilities for intervention and adaption to other areas of care, such as HIV/AIDS, child immunizations, and postnatal care.

IS26-mediated plasmid reshuffling results in convergence of toxin-antitoxin systems but loss of resistance genes in XDR Klebsiella pneumoniae from a chronic infection.

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB-IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin-antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.

Nasal microbiota evolution within the congregate setting imposed by military training.

The human microbiome is comprised of a complex and diverse community of organisms that is subject to dynamic changes over time. As such, cross-sectional studies of the microbiome provide a multitude of information for a specific body site at a particular time, but they fail to account for temporal changes in microbial constituents resulting from various factors. To address this shortcoming, longitudinal research studies of the human microbiome investigate the influence of various factors on the microbiome of individuals within a group or community setting. These studies are vital to address the effects of host and/or environmental factors on microbiome composition as well as the potential contribution of microbiome members during the course of an infection. The relationship between microbial constituents and disease development has been previously explored for skin and soft tissue infections (SSTIs) within congregate military trainees. Accordingly, approximately 25% of the population carries Staphylococcus aureus within their nasal cavity, and these colonized individuals are known to be at increased risk for SSTIs. To examine the evolution of the nasal microbiota of U.S. Army Infantry trainees, individuals were sampled longitudinally from their arrival at Fort Benning, Georgia, until completion of their training 90 days later. These samples were then processed to determine S. aureus colonization status and to profile the nasal microbiota using 16S rRNA gene-based methods. Microbiota stability differed dramatically among the individual trainees; some subjects exhibited great stability, some subjects showed gradual temporal changes and some subjects displayed a dramatic shift in nasal microbiota composition. Further analysis utilizing the available trainee metadata suggests that the major drivers of nasal microbiota stability may be S. aureus colonization status and geographic origin of the trainees. Nasal microbiota evolution within the congregate setting imposed by military training is a complex process that appears to be affected by numerous factors. This finding may indicate that future campaigns to prevent S. aureus colonization and future SSTIs among high-risk military trainees may require a 'personalized' approach.

Multidrug-Resistant and Virulent Organisms Trauma Infections: Trauma Infectious Disease Outcomes Study Initiative.

INTRODUCTION: During the wars in Iraq and Afghanistan, increased incidence of multidrug-resistant (MDR) organisms, as well as polymicrobial wounds and infections, complicated the management of combat trauma-related infections. Multidrug resistance and wound microbiology are a research focus of the Trauma Infectious Disease Outcomes Study (TIDOS), an Infectious Disease Clinical Research Program, Uniformed Services University, research protocol. To conduct comprehensive microbiological research with the goal of improving the understanding of the complicated etiology of wound infections, the TIDOS MDR and Virulent Organisms Trauma Infections Initiative (MDR/VO Initiative) was established as a collaborative effort with the Brooke Army Medical Center, Naval Medical Research Center, U.S. Army Institute of Surgical Research, and Walter Reed Army Institute of Research. We provide a review of the TIDOS MDR/VO Initiative and summarize published findings. METHODS: Antagonism and biofilm formation of commonly isolated wound bacteria (e.g., ESKAPE pathogens-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), antimicrobial susceptibility patterns, and clinical outcomes are being examined. Isolates collected from admission surveillance swabs, as part of infection control policy, and clinical infection workups were retained in the TIDOS Microbiological Repository and associated clinical data in the TIDOS database. RESULTS: Over the TIDOS study period (June 2009 to December 2014), more than 8,300 colonizing and infecting isolates were collected from military personnel injured with nearly one-third of isolates classified as MDR. At admission to participating U.S. military hospitals, 12% of wounded warriors were colonized with MDR Gram-negative bacilli. Furthermore, 27% of 913 combat casualties with ≥1 infection during their trauma hospitalization had MDR Gram-negative bacterial infections. Among 335 confirmed combat-related extremity wound infections (2009-2012), 61% were polymicrobial and comprised various combinations of Gram-negative and Gram-positive bacteria, yeast, fungi, and anaerobes. Escherichia coli was the most common Gram-negative bacilli isolated from clinical workups, as well as the most common colonizing MDR secondary to extended-spectrum ß-lactamase resistance. Assessment of 479 E. coli isolates collected from wounded warriors found 188 pulsed-field types (PFTs) from colonizing isolates and 54 PFTs from infecting isolates without significant overlap across combat theaters, military hospitals, and study years. A minority of patients with colonizing E. coli isolates developed subsequent infections with the same E. coli strain. Enterococcus spp. were most commonly isolated from polymicrobial wound infections (53% of 204 polymicrobial cultures). Patients with Enterococcus infections were severely injured with a high proportion of lower extremity amputations and genitourinary injuries. Approximately 65% of polymicrobial Enterococcus infections had other ESKAPE organisms isolated. As biofilms have been suggested as a cause of delayed wound healing, wound infections with persistent recovery of bacteria (isolates of same organism collected ≥14 days apart) and nonrecurrent bacterial isolates were assessed. Biofilm production was significantly associated with recurrent bacteria isolation (97% vs. 59% with nonrecurrent isolates; P < 0.001); however, further analysis is needed to confirm biofilm formation as a predictor of persistent wound infections. CONCLUSIONS: The TIDOS MDR/VO Initiative provides comprehensive and detailed data of major microbial threats associated with combat-related wound infections to further the understanding of wound etiology and potentially identify infectious disease countermeasures, which may lead to improvements in combat casualty care.

Analysis of Calcaneal Bone Mineral Density (cBMD) in Healthy College Students.

BACKGROUND: There is limited evidence describing the relationship between calcaneal bone mineral density (cBMD) and activity level, menstrual history, or the development of bone stress injury (BSI). HYPOTHESIS/PURPOSE: The purposes of this study were to: 1) examine the influence of physical activity on cBMD in healthy college students (HCS), 2) determine if there is an association between cBMD, body mass index (BMI), sex, menstrual history, and history of BSI in HCS, and 3) compare the cBMD of HCS to cBMD data collected on intercollegiate athletes (ICA) from a previous study. STUDY DESIGN: Cross-sectional design. METHODS: This cross-sectional study recruited a convenience sample of HCS at one institution. Subjects provided self-reported injury and menstrual history, completed a physical activity questionnaire, and cBMD and BMI measures were obtained. Descriptive statistics, statistical analyses of relationships (Chi-square and relative risk), logistic regression, and differences (t-tests) were used in the statistical analyses. RESULTS: One hundred three HCS (82 female, 21 male; age 21.9 ± 1.13) consented to participate. The composite score for work, leisure, and sport activity ranged from 5.6 to 11.1 (7.9 ± 1.1) for HCS subjects. There was no significant correlation between cBMD and physical activity in HCS, however, a significant correlation was found between reported age of onset of menstruation and left and right cBMD (r = -0.22 and r = -0.23; p < 0.05) and history of secondary amenorrhea and history of BSI (r = 0.32; p < 0.05). There was no difference in cBMD between the male ICA and male HCS, but highly significant differences in cBMD between the female ICA and female HCS groups (p < 0.000). CONCLUSIONS: Age of menarche and secondary amenorrhea are significantly associated with cBMD and history of BSI in HCS subjects, respectively. Differences in cBMD among the HCS subjects were not related to activity level. cBMD was significantly lower in female HCS as compared to female ICA. This difference in cBMD between ICA and HCS may be activity related. LEVEL OF EVIDENCE: Level 3.

Safety and immunogenicity of a plant-derived recombinant protective antigen (rPA)-based vaccine against Bacillus anthracis: A Phase 1 dose-escalation study in healthy adults.

BACKGROUND: The potential use of Bacillus anthracis as a bioterrorism weapon requires a safe and effective vaccine that can be immediately distributed for mass vaccination. Protective antigen (PA), a principal component of virulence factors edema toxin and lethal toxin of B. anthracis, has been the topic of extensive research. Previously, full-length PA (PA83) was manufactured using a transient plant-based expression system. Immunization with this PA83 antigen formulated with Alhydrogel® adjuvant elicited strong neutralizing immune responses in mice and rabbits and protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. This Phase 1 study evaluates this vaccine's safety and immunogenicity in healthy human volunteers. METHODS: This first-in-human, single-blind, Phase 1 study was performed at a single center to investigate the safety, reactogenicity, and immunogenicity of the plant-derived PA83-FhCMB vaccine at four escalating dose levels (12.5, 25, 50 or 100 µg) with Alhydrogel® in healthy adults 18-49 years of age (inclusive). Recipients received three doses of vaccine intramuscularly at 28-day intervals. Safety was evaluated on days 3, 7, and 14 following vaccination. Immunogenicity was assessed using an enzyme-linked immunosorbent assay (ELISA) and a toxin neutralizing antibody (TNA) assay on days 0, 14, 28, 56, 84, and 180. RESULTS: All four-dose ranges were safe and immunogenic, with no related serious adverse events observed. Peak ELISA Geometric Mean Concentration (GMC) and TNA ED50 Geometric Mean Titer (GMT) were noted at Day 84, 1 month after the final dose, with the most robust response detected in the highest dose group. Antibody responses decreased by Day 180 across all dose groups. Long-term immunogenicity data beyond six months was not collected. CONCLUSIONS: This is the first study demonstrating a plant-derived subunit anthrax vaccine's safety and immunogenicity in healthy adults. The results support further clinical investigation of the PA83-FhCMB vaccine. ClinicalTrials.gov identifier. NCT02239172.

Metabolites of Cannabis Induce Cardiac Toxicity and Morphological Alterations in Cardiac Myocytes.

Cannabis is one of the most commonly used recreational drugs worldwide. Rrecent epidemiology studies have linked increased cardiac complications to cannabis use. However, this literature is predominantly based on case incidents and post-mortem investigations. This study elucidates the molecular mechanism of Δ9-tetrahydrocannabinol (THC), and its primary metabolites 11-Hydroxy-Δ9-THC (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Treatment of cardiac myocytes with THC-OH and THC-COOH increased cell migration and proliferation (p < 0.05), with no effect on cell adhesion, with higher doses (250-100 ng/mL) resulting in increased cell death and significant deterioration in cellular architecture. Conversely, no changes in cell morphology or viability were observed in response to THC. Expression of key ECM proteins α-SMA and collagen were up-regulated in response to THC-OH and THC-COOH treatments with concomitant modulation of PI3K and MAPK signalling. Investigations in the planarian animal model Polycelis nigra demonstrated that treatments with cannabinoid metabolites resulted in increased protein deposition at transection sites while higher doses resulted in significant lethality and decline in regeneration. These results highlight that the key metabolites of cannabis elicit toxic effects independent of the parent and psychoactive compound, with implications for cardiotoxicity relating to hypertrophy and fibrogenesis.

Measurements of 5,6-Orthoquinone, a Surrogate for the Presumed Active Primaquine Metabolite 5-Hydroxyprimaquine, in the Urine of Cambodian Adults.

The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.