Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

BACKGROUND: The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. METHODS: Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. RESULTS: Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917-2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. CONCLUSION: These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Therapeutic response to artemisinin combination therapies among individuals with Plasmodium falciparum single infection vs mixed Plasmodium species infections: a retrospective posthoc analysis in Kisumu County, western Kenya.

OBJECTIVES: This study examined the treatment response of mixed vs single-species Plasmodium falciparum infections to artemisinin-based combination therapies (ACTs). METHODS: A total of 1211 blood samples collected on days 0, 7, 14, 21, 28, 35, and 42 from 173 individuals enrolled in two randomized ACT efficacy studies were tested for malaria using 18s ribosomal RNA-based real-time polymerase chain reaction. All recurrent parasitemia were characterized for Plasmodium species composition and time to reinfection during 42-day follow-up compared across ACTs. RESULTS: Day 0 samples had 71.1% (116/163) single P. falciparum infections and 28.2% (46/163) coinfections. A total of 54.0% (88/163) of individuals tested positive for Plasmodium at least once between days 7-42. A total of 19.3% (17/88) of individuals with recurrent infections were infected with a different Plasmodium species than observed at day 0, with 76.5% (13/17) of these "hidden" infections appearing after clearing P. falciparum present at day 0. Artesunate-mefloquine (16.4 hours) and dihydroartemisinin-piperaquine (17.6 hours) had increased clearance rates over artemether-lumefantrine (21.0 hours). Dihydroartemisinin-piperaquine exhibited the longest duration of reinfection prophylaxis. Cure rates were comparable across each species composition. CONCLUSION: No differences in clearance rates were found depending on whether the infection contained species other than P. falciparum. Significantly longer durations of protection were observed for individuals treated with dihydroartemisinin-piperaquine.

Impact of parasite genomic dynamics on the sensitivity of Plasmodium falciparum isolates to piperaquine and other antimalarial drugs.

BACKGROUND: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials. METHODS: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined. RESULTS: The PPQ median IC50 interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC50s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC50s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC50 and Pfcrt K76T allele (P=0.0026). CONCLUSIONS: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.

Burden of malaria infection among individuals of varied blood groups in Kenya.

BACKGROUND: The ABO blood groups consist of A, B, and H carbohydrate antigens, which regulate protein activities during malaria infection in humans. Understanding the interplay between the malaria parasite and blood group antigens is essential in understanding new interventions to reduce the global burden of malaria. This study assessed the burden of malaria infection among individuals with varying blood groups seeking treatment at selected hospitals in Kenya. METHODS: A total of 366 samples from an ongoing malaria surveillance study were diagnosed for malaria by microscopy and further typed for blood group using ABO blood grouping. Age and sex were recorded in a data sheet, and analysed using R software version 4. Groups' proportions (blood group, malaria infection, age and sex) were compared using Pearson's Chi-square and Fischer exact tests. Wilcoxon and Kruskal-Wallis tests were performed and P-value < 0.05 was considered significant after Bonferroni correction for multiple comparisons. To understand the effect of each blood group on parasitaemia, multivariate logistic regression was used to model ABO blood group in relation to parasitaemia. RESULTS: Of the 366 samples analysed, 312 were malaria positive, mean age was 9.83 years (< 5 years n = 152 (48.41%), 6 to 17 years n = 101 (32.16%) and > 18 years n = 61 (19.43%)). Malaria prevalence was higher among females than males, 54.46% and 45.54%, respectively. Kisumu enrolled the highest number 109 (35%)) of malaria cases, Kombewa 108 (35%), Malindi 32 (10%), Kisii 28 (9%), Marigat 23 (7%), and Kericho 12 (4%). Blood group O+ was the most prevalent among the enrolled individuals (46.50%), A+ (27.71%), B+ (21.02%) and AB+ (4.78%) respectively. Compared to blood group O+, blood group B+ individuals were (14%) were more likely to habour Plasmodium falciparum infection as opposed to A+ and AB+ individuals, that were 7% and 20%, respectively,. Those living in malaria-endemic zones presented with higher parasite densities compared to those living in malaria-epidemic (p = 0.0061). Individuals bearing B + blood group are more likely to habour high parasitaemia compared to O + blood group bearers (OR = 4.47, CI = 1.53-13.05, p = 0.006). CONCLUSION: Individuals of blood group B harbour high parasitaemia compared with the blood group O, Additionally, blood group A and B present with symptoms at lower parasitaemia than blood group O. Regardles of malaria transmission zones, individuals from endemic zones showed up with high parasitaemia and among them were more individuals of blood groups A and B than individuals of blood group O. Implying that these individuals were more at risk and require additional attention and effective case management.

A new benzophenone, and the antiplasmodial activities of the constituents of Securidaca longipedunculata fresen (Polygalaceae).

Extracts from Securidaca longipedunculata showed antiplasmodial activities against reference clones and clinical isolates using SYBR Green I method. A new benzophenone, 2,3,4,5-tetramethoxybenzophenone (1) was isolated and characterized along with seven known compounds: 4-hydroxy-2,3-dimethoxybenzophenone (2); 3-hydroxy-5-methoxybiphenyl (3), methyl-2-hydroxy-6-methoxybenzoate (4), benzyl-2-hydroxy-6-methoxybenzoate (5), 2-hydroxy-6-methoxybenzoic acid (6), 2,4,5-trimethoxybenzophenone (7) and 2-methoxy-3,4-methylenedioxybenzophenone (8). Compounds 1 and 2 showed ex vivo antiplasmodial activities (IC50 28.8 µM and 18.6 µM, respectively); while 5 and 8 showed in vivo activities (IC50 19.7 µM and 14.5 µM, respectively) against D6 strain. In a cytotoxicity assay, all the extracts (with an exception of the MeOH extract of the leaves) and pure compounds were not toxic to the normal LO2 and BEAS cell-lines, while the methanol roots extract (IC50 66.4 µg/mL against A549, and 77.4 µg/mL against HepG2), compounds 6 (IC50 22.2 µM against A549) and 7 (IC50 45.2 µM against HepG2) were weakly active against cancerous cell-lines.

Molecular characterization of enterotoxigenic Escherichia coli toxins and colonization factors in children under five years with acute diarrhea attending Kisii Teaching and Referral Hospital, Kenya.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infectious diarrhea in children. There are no licensed vaccines against ETEC. This study aimed at characterizing Escherichia coli for ETEC enterotoxins and colonization factors from children < 5 years with acute diarrhea and had not taken antibiotics prior to seeking medical attention at the hospital. METHODS: A total of 225 randomly selected archived E. coli strains originally isolated from 225 children with acute diarrhea were cultured. DNA was extracted and screened by multiplex polymerase chain reaction (PCR) for three ETEC toxins. All positives were then screened for 11 colonization factors by PCR. RESULTS: Out of 225 E. coli strains tested, 23 (10.2%) were ETEC. Heat-stable toxin (ST) gene was detected in 16 (69.6%). ETEC isolates with heat-stable toxin of human origin (STh) and heat-stable toxin of porcine origin (STp) distributed as 11 (68.8%) and 5 (31.2%) respectively. Heat-labile toxin gene (LT) was detected in 5 (21.7%) of the ETEC isolates. Both ST and LT toxin genes were detected in 2 (8.7%) of the ETEC isolates. CF genes were detected in 14 (60.9%) ETEC strains with a majority having CS6 6 (42.9%) gene followed by a combination of CFA/I + CS21 gene detected in 3 (21.4%). CS14, CS3, CS7 and a combination of CS5 + CS6, CS2 + CS3 genes were detected equally in 1 (7.1%) ETEC isolate each. CFA/I, CS4, CS5, CS2, CS17/19, CS1/PCFO71 and CS21 genes tested were not detected. We did not detect CF genes in 9 (39.1%) ETEC isolates. More CFs were associated with ETEC strains with ST genes. CONCLUSION: ETEC strains with ST genes were the most common and had the most associated CFs. A majority of ETEC strains had CS6 gene. In 9 (39.1%) of the evaluated ETEC isolates, we did not detect an identifiable CF.

Synergism in Antiplasmodial Activities of Artemether and Lumefantrine in Combination with Securidaca longipedunculata Fresen (Polygalaceae).

Malaria is the most lethal parasitic disease in the world. The frequent emergence of resistance by malaria parasites to any drug is the hallmark of sustained malaria burden. Since the deployment of artemisinin-based combination therapies (ACTs) it is clear that for a sustained fight against malaria, drug combination is one of the strategies toward malaria elimination. In Sub-Saharan Africa where malaria prevalence is the highest, the identification of plants with a novel mechanism of action that is devoid of cross-resistance is a feasible strategy in drug combination therapy. Thus, artemether and lumefantrine were separately combined and tested with extracts of Securidaca longipedunculata, a plant widely used to treat malaria, at fixed extract-drug ratios of 4:1, 3:1, 1:1, 1:2, 1:3, and 1:4. These combinations were tested for antiplasmodial activity against three strains of Plasmodium falciparum (W2, D6, and DD2), and seven field isolates that were characterized for molecular and ex vivo drug resistance profiles. The mean sum of fifty-percent fractional inhibition concentration (FIC50) of each combination and singly was determined. Synergism was observed across all fixed doses when roots extracts were combined with artemether against D6 strain (FIC50 0.403 ± 0.068) and stems extract combined with lumefantrine against DD2 strain (FIC50 0.376 ± 0.096) as well as field isolates (FIC50 0.656 ± 0.067). Similarly, synergism was observed in all ratios when leaves extract were combined with lumefantrine against W2 strain (FIC50 0.456 ± 0.165). Synergism was observed in most combinations indicating the potential use of S. longipedunculata in combination with artemether and lumefantrine in combating resistance.

Status Update on Infection Prevention and Control at Deployed Medical Treatment Facilities.

INTRODUCTION: Infections with multidrug resistant organisms that spread through nosocomial transmission complicate the care of combat casualties. Missions conducted to review infection prevention and control (IPC) practices at deployed medical treatment facilities (MTFs) previously showed gaps in best practices and saw success with targeted interventions. An IPC review has not been conducted since 2012. Recently, an IPC review was requested in response to an outbreak of multidrug resistant organisms at a deployed facility. MATERIALS AND METHODS: A Joint Service team conducted onsite IPC reviews of MTFs in the U.S. Central Command area of operations. Self-assessments were completed by MTF personnel in anticipation of the onsite assessment, and feedback was given individually and at monthly IPC working group teleconferences. Goals of the onsite review were to assist MTF teams in conducting assessments, review practices for challenges and successes, provide on the spot education or risk mitigation, and identify common trends requiring system-wide action. RESULTS: Nine deployed MTFs participated in the onsite assessments, including four Role 3, three Role 2 capable of surgical support, and two Role 1 facilities. Seventy-eight percent of sites had assigned IPC officers although only 43% underwent required predeployment training. Hand hygiene and healthcare associated infection prevention bundles were monitored at 67% and 29% of MTFs, respectively. Several challenges including variability in practices with turnover of deployed teams were noted. Successes highlighted included individual team improvements in healthcare associated infections and mentorship of untrained personnel. CONCLUSIONS: Despite successes, ongoing challenges with optimal deployed IPC were noted. Recommendations for improvement include strengthening IPC culture, accountability, predeployment training, and stateside support for deployed IPC assets. Variability in IPC practices may occur from rotation to rotation, and regular reassessment is required to ensure that successes are sustained through times of turnover.

Mindfulness-Based Cognitive Therapy Intervention for Young Adults with Cancer: A Pilot Mixed-Method Study.

Purpose: Group mindfulness-based interventions are emerging as a promising, nonstigmatizing, and cost-effective strategy that may improve the well-being of individuals living with cancer. This study is a pilot pre-post mixed-method study to examine the feasibility, acceptability, and effects of an 8-week Mindfulness-based Cognitive Therapy group for Young Adults with Cancer (YA-MBCT). Methods: We approached young adults with cancer, who enrolled in YA-MBCT groups, offered at a large cancer hospital in Toronto, Ontario, Canada. Feasibility and acceptability were assessed through attendance rate and a postintervention satisfaction scale. Psychosocial outcomes were evaluated with a pre-post questionnaire package, with validated self-report measures, assessing depression, anxiety, perceived stress, quality of life, mindfulness, and self-compassion. Qualitative interviews were completed among a subset of participants to gain additional feedback. Results: Participants were 70 young adults with cancer, recruited from five YA-MBCT groups. Sixty participants (85%) attended a minimum of six of eight sessions, and overall satisfaction rates were high. All psychosocial outcomes demonstrated statistically significant changes (p < 0.01), with medium to large effect sizes (Cohen's d > 0.5). Qualitative interviews (n = 14) demonstrated overall positive views about the intervention, and provided insight into unique age-specific benefits, including reducing fear of cancer recurrence, improving body image, and creating a sense of belonging. Conclusion: The YA-MBCT is feasible and acceptable among young adults with cancer, with the potential to improve psychosocial outcomes. Our preliminary results should be replicated with larger studies with an active control group.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds.  Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.

Antimicrobial Stewardship Challenges in the Deployed Setting.

INTRODUCTION: Up to 34% of combat trauma injuries are complicated by infection with multidrug-resistant organisms. Overutilization of antibiotics has been linked to increased multidrug-resistant organisms in combat-injured patients. Antimicrobial stewardship efforts at deployed medical treatment facilities have been intermittently reported; however; a comprehensive assessment of antimicrobial stewardship practices has not been performed. MATERIALS AND METHODS: A survey tool was modified to include detailed questions on antimicrobial stewardship practices at medical treatment facilities. A Joint Service, multidisciplinary team conducted on-site assessments and interviews to assess the status of antimicrobial stewardship best practices, with particular emphasis on antibiotic prophylaxis in combat injured, in the U.S. Central Command operational theaters. Limitations to implementing stewardship to the national standards were explored thematically. RESULTS: Nine Role 1, 2, and 3 medical facilities representing the range of care were assessed on-site. A total of 67% of the sites reported a formal antimicrobial stewardship program and 56% of the sites had an assigned head of antimicrobial stewardship. No military personnel in theater received training on antimicrobial stewardship and laboratory assets were limited. Personnel at these sites largely had access to Joint Trauma System guidelines describing antimicrobial prophylaxis for combat injured (89%), yet infrequently received feedback on their implementation and adherence to these guidelines (11%). CONCLUSIONS: Antimicrobial stewardship programs in theater are in the early stages of development in theater. Areas identified for improvement are access to expertise, development of a focus on high-impact lines of effort, laboratory support, and the culture of antimicrobial prescribing. Risks can be mitigated through theater level formalization of efforts, expert mentoring through telehealth, and a focus on implementation and adherence and feedback to national guidelines.

Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro.

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.

(Queer) Family Values and "Reciprocal IVF": What Difference Does Sexual Identity Make?

In this paper I employ the case of "reciprocal IVF" (R-IVF)-in which a female-female couple uses in vitro fertilization to allow one woman to be the genetic mother and the other the gestational mother of their child(ren)-to illuminate the role sexual identity might productively play in bioethics. Bioethicists who have taken up this issue have largely focused on the moral permissibility and availability of the technology, and so defend R-IVF through analogy to commonly accepted different-sex uses of IVF. In this way, they position sexual identity as largely irrelevant to the primary bioethical questions raised by R-IVF. My approach diverges on these counts as I focus on the ethics of R-IVF through the lens of queer family values specifically. I ask what the practice of R-IVF as an option might mean for the queer community at large, and so view sexual identity as integral to a full bioethical evaluation of the practice.

Discovery of novel inhibitors of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug-resistant Acinetobacter baumannii strains and the non-efficacy of currently available antibiotics against such infections have led to an urgent need for the development of novel antibacterials. In an effort to address this problem, we have identified three novel inhibitors, namely, D5, D12 and D6 using in silico screening with a homology model of the outer membrane protein W2 (OmpW2) from A. baumannii, as the proposed new drug target. OmpW is an eight-stranded ß-barrel protein involved in the transport of hydrophobic molecules across the outer membrane and maintenance of homeostasis under cellular stress. The antimicrobial activities of compounds D5, D12 and D6 were evaluated against a panel of clinical isolates of A. baumannii strains. These compounds inhibited the growth of the strains with minimum inhibitory concentration (MIC) ranges of 1-32µg/mL. Time-kill kinetic studies with the highly virulent and multidrug-resistant strain, A. baumannii 5075, indicated that D6 exhibited the highest bactericidal activity asa≥3log10 CFU/mL (99.9%) reduction in colony count from the initial inoculum was observed after 30min incubation. D5 and D12 reduced at least 1log10 CFU/mL (90%) of the initial inoculum after 24h. In conclusion, these three lead inhibitors have provided two distinct chemical scaffolds for further analog design and optimizations, using chemical synthesis, to develop more potent inhibitors of the pathogen.

Complete Genome Sequence of Staphylococcus epidermidis 1457.

Staphylococcus epidermidis 1457 is a frequently utilized strain that is amenable to genetic manipulation and has been widely used for biofilm-related research. We report here the whole-genome sequence of this strain, which encodes 2,277 protein-coding genes and 81 RNAs within its 2.4-Mb genome and plasmid.

General paediatric inpatient deaths over a 15-year period.

OBJECTIVE: To retrospectively review trends of general paediatric inpatient deaths at a tertiary care children's hospital over a 15-year period. METHODS: Data were collected for all patients who died on the general paediatric wards or paediatric intensive care unit (PICU) during 1998, 2005 or 2012 and had a 'general paediatric condition'-an underlying condition or diagnosis that would normally result in admission to a general paediatric ward. Data were related to: demographics, health services utilization, information about provision and orders related to cardiopulmonary resuscitation (CPR) at time of death and involvement of palliative care services. RESULTS: Eighty-five inpatients met inclusion criteria: 35 in 1998, 27 in 2005 and 23 in 2012. Nearly 95% of general paediatric patients who died in 1998 did so in the PICU, 59.3% in 2005 and 69.6% in 2012. The median age of death decreased from 3 years in 1998 to 2 years in 2012. The proportion of patients with 'no CPR' orders at time of death increased from 31.4% in 1998 to 87.0% in 2012. Similarly, the proportion of patients with palliative care team involvement prior to death increased from less than 10% in 1998 to 73.9% in 2012. CONCLUSIONS: The absolute number of inpatient general paediatric deaths has decreased from 1998 to 2012 at this hospital. A larger proportion of these deaths are occurring on the general paediatric wards rather than in the PICU over time. 'No CPR' orders and palliative care consultations are becoming more prevalent in these patients prior to death.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice.

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense.

OBJECTIVE: We sought to: 1) provide an overview of the genomic epidemiology of an extensive collection of carbapenemase-producing bacteria (CPB) collected in the U.S. Department of Defense health system; 2) increase awareness of the public availability of the sequences, isolates, and customized antimicrobial resistance database of that system; and 3) illustrate challenges and offer mitigations for implementing next generation sequencing (NGS) across large health systems. DESIGN: Prospective surveillance and system-wide implementation of NGS. SETTING: 288-hospital healthcare network. METHODS: All phenotypically carbapenem resistant bacteria underwent CarbaNP® testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and open-source software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. RESULTS: From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly blaKPC, blaNDM, and blaOXA23). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones. CONCLUSION: Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.

Airworthiness testing of medical maggots.

An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certification, including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.