Prevalence of Inappropriate Antibiotic Prescribing with or without a Plausible Antibiotic Indication among Safety-Net and Non-Safety Net Populations.

BACKGROUND: Clinicians can prescribe antibiotics inappropriately without coding the indication for antibiotics. Whether the prevalence of inappropriate antibiotic prescribing with or without a plausible indication differs between safety-net and non-safety-net populations is unknown. OBJECTIVE: To assess differences in inappropriate antibiotic prescribing with or without a plausible indication between safety-net and non-safety net populations. DESIGN: Cross-sectional. PARTICIPANTS: Office visits in the 2016, 2018, 2019 National Ambulatory Medical Care Survey with ≥ 1 antibiotic prescription among children (0-17 years) and adults (18-64 years). MAIN MEASURES: Inappropriate antibiotic prescribing with a plausible indication (visits with infection-related diagnosis codes that do not warrant antibiotics, e.g., acute bronchitis); inappropriate prescribing without a plausible indication (visits with codes that are not antibiotic indications, e.g., hypertension). By age group, we used linear regression to assess differences between safety-net (public/no insurance) and non-safety net populations (privately insured), controlling for patient and visit characteristics. KEY RESULTS: Analyses included 67,065,108 and 122,731,809 weighted visits for children and adults, respectively. Among visits for children in the safety-net and non-safety populations, the prevalence of inappropriate antibiotic prescribing with a plausible indication was 11.7% and 22.0% (adjusted difference: -8.0%, 95% CI: -17.1%, 1.0%); the prevalence of inappropriate prescribing without a plausible indication was 11.8% and 8.6% (adjusted difference: -2.0%, 95% CI: -4.6%, 0.6%). Among visits for adults in the safety-net and non-safety populations, the prevalence of inappropriate antibiotic prescribing with a plausible indication was 12.1% and 14.3% (adjusted difference: -0.1%, 95% CI -9.4%, 9.1%); the prevalence of inappropriate prescribing without a plausible indication was 48.2% and 32.3% (adjusted difference: 12.5%, 95% CI: 3.6%, 21.4%). CONCLUSIONS: Inappropriate antibiotic prescribing with or without a plausible antibiotic indication is common in all populations, highlighting the importance of broad-based antibiotic stewardship initiatives. However, targeted initiatives focused on improving coding quality in adult safety-net settings may be warranted.

An opportunity for global antimicrobial stewardship research: Refugee populations.

Antimicrobial resistance is a well-known global health threat that has higher prevalence in the refugee population. Although guidance has been provided by the World Health Organization and Centers for Disease Control and Prevention on implementing antimicrobial stewardship in lower- and middle-income countries, as well as by the United Nations Refugee Agency on other infection prevention and control efforts, no specific guidance exists for implementation of stewardship in this population. We highlight challenges specific to this population, review recent studies of interest within this space, and propose a research agenda to help move stewardship forward in the refugee population. We advocate for the importance of this issue, particularly given recent current events of geopolitical volatility that render this population more vulnerable, in the setting of its already well-known numerous health challenges.

Unequal burden of Zika-associated microcephaly among populations with public and private healthcare in Salvador, Brazil.

OBJECTIVES: To describe the differences in clinical presentation and relative disease burden of congenital Zika syndrome (CZS)-associated microcephaly at 2 large hospitals in Salvador, Brazil that serve patients of different socioeconomic status (SES). METHODS: Clinical and serologic data were collected prospectively from pregnant women and their infants, who delivered at 2 study centers during the 2015-2016 Zika virus (ZIKV) epidemic in Salvador, Brazil. RESULTS: Pregnant women from Salvador, Brazil delivering in a low SES hospital had 3 times higher ZIKV exposure rate than women at a high SES hospital. However, different SES hospitals had similar prevalence of infants with CZS-associated microcephaly (10% vs 6%, p = 0.16) after controlling for ZIKV exposure in their mothers. CONCLUSIONS: Our study supports the positive association between low SES, high maternal ZIKV exposure, and high rates of CZS-associated microcephaly.

Factors associated with low tuberculosis preventive therapy prescription rates among health care workers in rural South Africa.

BACKGROUND: Despite extensive rollout of tuberculosis preventive therapy (TPT) in South Africa to reduce the incidence of tuberculosis among people living with HIV (PWH), rates of initiation and completion have remained suboptimal. OBJECTIVE: This study aimed to identify factors associated with low TPT prescription rates among health care workers (HCWs) in rural South Africa. METHODS: A cross-sectional study was conducted using an anonymous 39-item questionnaire guided by the Consolidated Framework for Implementation Research (CFIR). HCWs from a government district hospital and 14 primary healthcare clinics (PHCs) in the rural Msinga sub-district of KwaZulu-Natal were surveyed from November 2019 to January 2020. Self-reported data on prescription rates as well as knowledge, attitudes, beliefs, and practices regarding isoniazid preventative therapy, the current TPT regimen, were obtained. Factor analysis and logistic regression were used to determine associations with low prescription rates (< 50% of PWH) for TPT prescribers, and results were placed within CFIR-driven context. RESULTS: Among 160 HCWs, the median (IQR) age was 39 (33-46) years, 76% were women, 78% worked at a PHC, and 44% had experience prescribing TPT. On multivariable analysis, prescribers (n = 71) who believed their patients would not disclose TPT use to others were significantly less likely to prescribe TPT (aOR 4.19 95% CI 1.35-13.00; p = 0.01). Inadequate isoniazid supplies trended towards significance (aOR 10.10 95% CI 0.95-106.92; p = 0.06) in association with low prescription rates. CONCLUSIONS: Strengthening HCW training to emphasize TPT prescription to all eligible PWH regardless of beliefs about patient disclosure and ensuring a consistent isoniazid supply at the health systems-level are both critical steps to enhancing TPT implementation in rural South Africa.

Developmental outcomes in children exposed to Zika virus in utero from a Brazilian urban slum cohort study.

BACKGROUND: The prevalence of developmental alterations associated with in-utero Zika virus (ZIKV) exposure in children is not well understood. Furthermore, estimation of the Population Attributable Fraction (PAF) of developmental alterations attributed to ZIKV has not been performed due to lack of population-based cohorts with data on symptomatic and asymptomatic ZIKV exposures and an appropriate control group. The aim of this study was to characterize neurodevelopmental outcomes of children at 11 to 32 months of age with intrauterine ZIKV exposure and estimate the PAF of alterations secondary to ZIKV exposure. METHODOLOGY/PRINCIPAL FINDINGS: We performed a cohort of biannual community-based prospective serosurveys in a slum community in Salvador, Brazil. We recruited women participating in our cohort, with a documented pregnancy from January 2015 to December 2016 and children born to those mothers. Children were classified as ZIKV exposed in utero (born from women with ZIKV seroconversion during pregnancy) or unexposed (born from women without ZIKV seroconversion or that seroconverted before/after pregnancy) by using an IgG monoclonal antibody blockade-of-binding (BoB). We interviewed mothers and performed anthropometric, audiometric, ophthalmological, neurologic, and neurodevelopmental evaluations of their children at 11 to 32 months of age. Among the 655 women participating in the cohort, 66 (10%) were pregnant during the study period. 46 (70%) of them completed follow-up, of whom ZIKV seroconversion occurred before, during, and after pregnancy in 25 (54%), 13 (28%), and 1 (2%), respectively. The rest of women, 7 (21.2%), did not present ZIKV seroconversion. At 11 to 32 months of life, the 13 ZIKV-exposed children had increased risk of mild cognitive delay (RR 5.1; 95%CI 1.1-24.4) compared with the 33 children unexposed, with a PAF of 53.5%. Exposed children also had increased risk of altered auditory behavior (RR 6.0; 95%CI 1.3-26.9), with a PAF of 59.5%. CONCLUSIONS: A significant proportion of children exposed in utero to ZIKV developed mild cognitive delay and auditory behavioral abnormalities even in the absence of gross birth defects such as microcephaly and other neurodevelopmental domains. Furthermore, our findings suggest that over half of these abnormalities could be attributed to intrauterine ZIKV exposure.

Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe.

Redox cancer therapeutics target the increased reliance on intracellular antioxidant systems and enhanced susceptibility to oxidant-induced stress of some cancer cells compared to normal cells. Many of these therapeutics are thought to perturb intracellular levels of the oxidant hydrogen peroxide (H2O2), a signaling molecule that modulates a number of different processes in human cells. However, fluorescent probes for this species remain limited in their ability to detect the small perturbations induced during successful treatments. We report a fluorescent sensor based upon human peroxiredoxin-2, which acts as the natural indicator of small H2O2 fluctuations in human cells. The new probe reveals peroxide-induced oxidation in human cells below the detection limit of current probes, as well as peroxiredoxin-2 oxidation caused by two different redox cancer therapeutics in living cells. This capability will be useful in elucidating the mechanism of current redox-based therapeutics and in developing new ones.

A reaction-diffusion model of cytosolic hydrogen peroxide.

As a signaling molecule in mammalian cells, hydrogen peroxide (H2O2) determines the thiol/disulfide oxidation state of several key proteins in the cytosol. Localization is a key concept in redox signaling; the concentrations of signaling molecules within the cell are expected to vary in time and in space in manner that is essential for function. However, as a simplification, all theoretical studies of intracellular hydrogen peroxide and many experimental studies to date have treated the cytosol as a well-mixed compartment. In this work, we incorporate our previously reported reduced kinetic model of the network of reactions that metabolize hydrogen peroxide in the cytosol into a model that explicitly treats diffusion along with reaction. We modeled a bolus addition experiment, solved the model analytically, and used the resulting equations to quantify the spatiotemporal variations in intracellular H2O2 that result from this kind of perturbation to the extracellular H2O2 concentration. We predict that micromolar bolus additions of H2O2 to suspensions of HeLa cells (0.8 × 10(9)cells/l) result in increases in the intracellular concentration that are localized near the membrane. These findings challenge the assumption that intracellular concentrations of H2O2 are increased uniformly throughout the cell during bolus addition experiments and provide a theoretical basis for differing phenotypic responses of cells to intracellular versus extracellular perturbations to H2O2 levels.

Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis.

As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity.

Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model.

Hydrogen peroxide (H2O2) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H2O2 versus disulfide exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H2O2 and its most likely oxidation targets during a sudden increase in H2O2 above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H2O2 diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H2O2 to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H2O2 concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H2O2, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H2O2 within cells.

Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity.

We report the use of HyPer, a genetically encoded, fluorescent sensor that reacts with hydrogen peroxide (H2O2), in a novel screen to engineer enzymes for enhanced production of H2O2. We co-expressed HyPer with cytochrome P450 BM3 variants and, using HyPer's ratiometric signal, found variants that produce greater amounts of H2O2 than the wild-type enzyme through the leakage reaction. The screen avoids lysis procedures and the addition of reagents to assay intracellular contents. Less laborious screening procedures will be useful in engineering more powerful H2O2 generators as tools in quantitative redox biology, and increasing the utility of enzymes that produce H2O2 as a by-product alongside a valuable compound.

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress.

Genetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H2O2), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria. Here, we performed this characterization, specifically in Escherichia coli exposed to external H2O2. We found that the temporal behavior of the signal does not correspond exactly to peroxide concentration in the system as a function of time and expression of the sensor decreases the peroxide scavenging activity of the cell. We also determined the effects of cell number, both before and after normalization of externally added H2O2 to the number of cells. Finally, we report quantitative characteristics of HyPer's signal in this context, including the dynamic range of the signal, the signal-to-noise ratio, and the half saturation constant. These parameters show statistically meaningful differences in signal between two commonly used strains of E. coli, demonstrating how signal can vary with strain. Taken together, our results establish a systematic, quantitative framework for researchers seeking to better understand the role of H2O2 in the immunological response against bacteria, and for understanding potential differences in the details of HyPer's quantitative performance across studies.

CHARMM36 united atom chain model for lipids and surfactants.

Molecular simulations of lipids and surfactants require accurate parameters to reproduce and predict experimental properties. Previously, a united atom (UA) chain model was developed for the CHARMM27/27r lipids (Hénin, J., et al. J. Phys. Chem. B. 2008, 112, 7008-7015) but suffers from the flaw that bilayer simulations using the model require an imposed surface area ensemble, which limits its use to pure bilayer systems. A UA-chain model has been developed based on the CHARMM36 (C36) all-atom lipid parameters, termed C36-UA, and agreed well with bulk, lipid membrane, and micelle formation of a surfactant. Molecular dynamics (MD) simulations of alkanes (heptane and pentadecane) were used to test the validity of C36-UA on density, heat of vaporization, and liquid self-diffusion constants. Then, simulations using C36-UA resulted in accurate properties (surface area per lipid, X-ray and neutron form factors, and chain order parameters) of various saturated- and unsaturated-chain bilayers. When mixed with the all-atom cholesterol model and tested with a series of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol mixtures, the C36-UA model performed well. Simulations of self-assembly of a surfactant (dodecylphosphocholine, DPC) using C36-UA suggest an aggregation number of 53 ± 11 DPC molecules at 0.45 M of DPC, which agrees well with experimental estimates. Therefore, the C36-UA force field offers a useful alternative to the all-atom C36 lipid force field by requiring less computational cost while still maintaining the same level of accuracy, which may prove useful for large systems with proteins.

Influence of ester-modified lipids on bilayer structure.

Lipid membranes function as barriers for cells to prevent unwanted chemicals from entering the cell and wanted chemicals from leaving. Because of their hydrophobic interior, membranes do not allow water to penetrate beyond the headgroup region. We performed molecular simulations to examine the effects of ester-modified lipids, which contain ester groups along their hydrocarbon chains, on bilayer structure. We chose two lipids from those presented in Menger et al. [J. Am. Chem. Soc. 2006, 128, 14034] with ester groups in (1) the upper half of the lipid chain (MEPC) and (2) the middle and end of the lipid chain (MGPC). MGPC (30%)/POPC bilayers formed stable water pores of diameter 5-7 Å, but MGPC (22%)/POPC and MEPC (30%)/POPC bilayers did not form these defects. These pores were similar to those formed during electroporation; i.e., the head groups lined the pore and allowed water and ions to transport across the bilayer. However, we found that lateral organization of the MGPC lipids into clusters, instead of an electric field or charge disparity as in electroporation, was essential for pore formation. On the basis of this, we propose an overall mechanism for pore formation. The similarities between the ester-modified lipids and byproducts of lipid peroxidation with multiple hydrophilic groups in the middle of the chain suggest that free radical reactions with unsaturated lipids and sterols result in fundamental changes that may be similar to what is seen in bilayers with ester-modified lipids.

Update of the cholesterol force field parameters in CHARMM.

A modification of the CHARMM36 lipid force field (C36) for cholesterol, henceforth, called C36c, is reported. A fused ring compound, decalin, was used to model the steroid section of cholesterol. For decalin, C36 inaccurately predicts the heat of vaporization (~10 kJ/mol) and molar volume (~10 cc/mol), but C36c resulted in near perfect comparison with experiment. MD simulations of decalin and heptane at various compositions were run to estimate the enthalpy and volumes of mixing to compare to experiment for this simple model of cholesterol in a chain environment. Superior estimates for these thermodynamic properties were obtained with C36c versus C36. These new parameters were applied to cholesterol, and quantum mechanical calculations were performed to modify the torsional potential of an acyl chain torsion for cholesterol. This model was tested through simulations of DMPC/10% cholesterol, DMPC/30% cholesterol, and DOPC/10% cholesterol. The C36 and C36c results were similar for surface areas per lipid, deuterium order parameters, electron density profiles, and atomic form factors and generally agree well with experiment. However, C36 and C36c produced slightly different cholesterol angle distributions with C36c adopting a more perpendicular orientation with respect to the bilayer plane. The new parameters in the C36c modification should enable more accurate simulations of lipid bilayers with cholesterol, especially for those interested in the free energy of lipid flip/flop or transfer of phospholipids and/or cholesterol.

Lipid chain branching at the iso- and anteiso-positions in complex Chlamydia membranes: a molecular dynamics study.

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso- and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC). These three membranes were simulated at 323K and simulations of DPhPC at 298K were also performed for better comparison to existing experimental data. Two simulations of representative EB and RB membranes of C. trachomatis composed of nine different lipid components were performed at 310.15K, to accurately reflect compositions determined by experiment and physiological conditions. Based on nearly 0.5µs of simulation data, we report that branching increases average lipid surface area, area elastic moduli, and lipid axial relaxation times, while decreasing lipid chain order. Branching also has a distinct effect on electron density profiles. Due to their high cholesterol concentrations, the EB and RB membranes were found to have relatively high area elastic moduli, which may have important biological implications.

Cholesterol flip-flop: insights from free energy simulation studies.

The mechanism of lipid flip-flop motion is important for maintaining the asymmetric distribution of lipids in a biological membrane. To explore the energetics and mechanism of passive cholesterol flip-flop and its dependence on chain saturation, we performed two-dimensional umbrella sampling simulations in DPPC, POPC, and DAPC (dipalmitoyl-, palmitoyloleoyl-, and diarachidonylphosphatidylcholine) and used the string method to identify the most probable flip-flop paths based on the two-dimensional free energy maps. The resulting paths indicate that cholesterol prefers to tilt first and then move to the bilayer center where the free energy barrier exists. The barrier is lower in DAPC than in DPPC or POPC, and the calculated flip-flop rates show that cholesterol flip-flop in a poly-unsaturated bilayer is faster than in more saturated bilayers. The free energy barrier results from the unfavorable enthalpic contribution arising from cholesterol-water/lipid interactions and the favorable entropic contribution due to increased lipid dynamics. While the cholesterol-water interaction has similar contributions to the barrier due to desolvation of the cholesterol hydroxyl group in all lipids, the cholesterol-lipid interaction has a much lower barrier in DAPC than in DPPC or POPC, resulting in the lower free energy barrier in DAPC.

Sterol binding and membrane lipid attachment to the Osh4 protein of yeast.

Osh4 is an oxysterol-binding protein homologue found in yeast that is essential for the intracellular transport of sterols and cell life. In this study, molecular dynamics simulations were used to investigate the binding of ergosterol, 25-hydroxycholesterol, and lipid moieties to Osh4. The binding energies between both sterols and Osh4 were dominated by van der Waals interactions with residues within the sterol binding pocket, and were further stabilized by water-mediated interactions with polar residues at the bottom of the binding pocket (W46, Q96, Y97, N165, Q181). Only Q96 was able to form direct hydrogen bonds with each sterol. Possible lipid binding sites on the surface of Osh4 were identified by docking four lipid moieties modeled from different lipid head groups (phosphatidylcholine, phosphatidylserine, phosphatidylinositol(4,5)biphosphate, and phosphatidylinositol(3,4,5)triphosphate). Though lipids docked to several regions along the protein surface, the most commonly encountered regions were found along the ß-barrel region, a loop formed by residues 236-244, and the mouth of the sterol binding pocket. Several residues identified in these regions, such as K168, A169, K173, and E412, are either included or adjacent to residues experimentally implicated in Osh4 membrane binding. Lipid docking did not result in favorable binding regions on the α(7) helix or the distal binding surface, suggesting that protein backbone conformational changes are needed for membrane attachment in these regions. Ultimately, understanding how Osh4 attaches to cellular membranes and binds sterol will lead to a clear understanding of how this protein transports sterols between organelles in vivo.

CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes.

The CHARMM-GUI Membrane Builder (http://www.charmm-gui.org/input/membrane), an intuitive, straightforward, web-based graphical user interface, was expanded to automate the building process of heterogeneous lipid bilayers, with or without a protein and with support for up to 32 different lipid types. The efficacy of these new features was tested by building and simulating lipid bilayers that resemble yeast membranes, composed of cholesterol, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylamine, and palmitoyloleoylphosphatidylserine. Four membranes with varying concentrations of cholesterol and phospholipids were simulated, for a total of 170 ns at 303.15 K. Unsaturated phospholipid chain concentration had the largest influence on membrane properties, such as average lipid surface area, density profiles, deuterium order parameters, and cholesterol tilt angle. Simulations with a high concentration of unsaturated chains (73%, membrane(unsat)) resulted in a significant increase in lipid surface area and a decrease in deuterium order parameters, compared with membranes with a high concentration of saturated chains (60-63%, membrane(sat)). The average tilt angle of cholesterol with respect to bilayer normal was largest, and the distribution was significantly broader for membrane(unsat). Moreover, short-lived cholesterol orientations parallel to the membrane surface existed only for membrane(unsat). The membrane(sat) simulations were in a liquid-ordered state, and agree with similar experimental cholesterol-containing membranes.