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Background: Ordering Clostridioides difficile diagnostics without appropriate clinical indications can result in inappropriate antibiotic prescribing and misdiagnosis of hospital onset C. difficile infection. Manual processes such as provider review of order appropriateness may detract from other infection control or antibiotic stewardship activities. Methods: We developed an evidence-based clinical algorithm that defined appropriateness criteria for testing for C. difficile infection. We then implemented an electronic medical record–based order-entry tool that utilized discrete branches within the clinical algorithm including history of prior C. difficile test results, laxative or stool-softener administration, and documentation of unformed bowel movements. Testing guidance was then dynamically displayed with supporting patient data. We compared the rate of completed C. difficile tests after implementation of this intervention at 5 hospitals to a historic baseline in which a best-practice advisory was used. Results: Using mixed-effects Poisson regression, we found that the intervention was associated with a reduction in the incidence rate of both C. difficile ordering (incidence rate ratio [IRR], 0.74; 95% confidence interval [CI], 0.63–0.88; P = .001) and C. difficile–positive tests (IRR, 0.83; 95% CI, 0.76–0.91; P < .001). On segmented regression analysis, we identified a sustained reduction in orders over time among academic hospitals and a new reduction in orders over time among community hospitals. Conclusions: An evidence-based dynamic order panel, integrated within the electronic medical record, was associated with a reduction in both C. difficile ordering and positive tests in comparison to a best practice advisory, although the impact varied between academic and community facilities.

PURPOSE Parenteral nutrition (PN) has been shown to be a safe method of feeding in the intensive care unit with modern infection prevention practices, but similar analysis in the hematology-oncology setting is lacking. METHODS A retrospective analysis of 1,617 patients with hematologic malignancies admitted and discharged from the Hospital of the University of Pennsylvania during 3,629 encounters from 2017 to 2019 was undertaken to evaluate the association of PN administration with risk of central line–associated bloodstream infection (CLABSI). Proportions of mucosal barrier injury (MBI)-CLABSI and non–MBI-CLABSI were also compared between groups. RESULTS Risk of CLABSI was associated with cancer type and duration of neutropenia but not with PN administration (odds ratio, 1.015; 95% CI, 0.986 to 1.045; P = .305) in a multivariable analysis. MBI-CLABSI comprised 73% of CLABSI in patients exposed to and 70% in patients not exposed to PN, and there was no significant difference between groups (χ2 = 0.06, P = .800). CONCLUSION PN was not associated with increased risk of CLABSI in a sample of patients with hematologic malignancy with central venous catheters when adjusting for cancer type, duration of neutropenia, and catheter days. The high proportion of MBI-CLABSI highlights the effect of gut permeability within this population.

Background Multidrug-resistant organisms (MDROs) frequently contaminate hospital environments. We performed a multicenter, cluster-randomized, crossover trial of 2 methods for monitoring of terminal cleaning effectiveness. Methods Six intensive care units (ICUs) at 3 medical centers received both interventions sequentially, in randomized order. Ten surfaces were surveyed each in 5 rooms weekly, after terminal cleaning, with adenosine triphosphate (ATP) monitoring or an ultraviolet fluorescent marker (UV/F). Results were delivered to environmental services staff in real time with failing surfaces recleaned. We measured monthly rates of MDRO infection or colonization, including methicillin-resistant Staphylococcus aureus, Clostridioides difficile, vancomycin-resistant Enterococcus, and MDR gram-negative bacilli (MDR-GNB) during a 12-month baseline period and sequential 6-month intervention periods, separated by a 2-month washout. Primary analysis compared only the randomized intervention periods, whereas secondary analysis included the baseline. Results The ATP method was associated with a reduction in incidence rate of MDRO infection or colonization compared with the UV/F period (incidence rate ratio [IRR] 0.876; 95% confidence interval [CI], 0.807–0.951; P = .002). Including the baseline period, the ATP method was associated with reduced infection with MDROs (IRR 0.924; 95% CI, 0.855–0.998; P = .04), and MDR-GNB infection or colonization (IRR 0.856; 95% CI, 0.825–0.887; P Conclusions Intensive monitoring of ICU terminal room cleaning with an ATP modality is associated with a reduction of MDRO infection and colonization.

Background:The spatial and temporal extent of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) environmental contamination has not been precisely defined. We sought to elucidate contamination of different surface types and how contamination changes over time.Methods:We sampled surfaces longitudinally within COVID-19 patient rooms, performed quantitative RT-PCR for the detection of SARS-CoV-2 RNA, and modeled distance, time, and severity of illness on the probability of detecting SARS-CoV-2 using a mixed-effects binomial model.Results:The probability of detecting SARS-CoV-2 RNA in a patient room did not vary with distance. However, we found that surface type predicted probability of detection, with floors and high-touch surfaces having the highest probability of detection: floors (odds ratio [OR], 67.8; 95% credible interval [CrI], 36.3–131) and high-touch elevated surfaces (OR, 7.39; 95% CrI, 4.31–13.1). Increased surface contamination was observed in room where patients required high-flow oxygen, positive airway pressure, or mechanical ventilation (OR, 1.6; 95% CrI, 1.03–2.53). The probability of elevated surface contamination decayed with prolonged hospitalization, but the probability of floor detection increased with the duration of the local pandemic wave.Conclusions:Distance from a patient’s bed did not predict SARS-CoV-2 RNA deposition in patient rooms, but surface type, severity of illness, and time from local pandemic wave predicted surface deposition.

This SHEA white paper identifies knowledge gaps and challenges in healthcare epidemiology research related to coronavirus disease 2019 (COVID-19) with a focus on core principles of healthcare epidemiology. These gaps, revealed during the worst phases of the COVID-19 pandemic, are described in 10 sections: epidemiology, outbreak investigation, surveillance, isolation precaution practices, personal protective equipment (PPE), environmental contamination and disinfection, drug and supply shortages, antimicrobial stewardship, healthcare personnel (HCP) occupational safety, and return to work policies. Each section highlights three critical healthcare epidemiology research questions with detailed description provided in supplementary materials. This research agenda calls for translational studies from laboratory-based basic science research to well-designed, large-scale studies and health outcomes research. Research gaps and challenges related to nursing homes and social disparities are included. Collaborations across various disciplines, expertise and across diverse geographic locations will be critical.

Background:Multidrug-resistant organisms (MDROs) colonizing the healthcare environment have been shown to contribute to risk for healthcare-associated infections (HAIs), with adverse effects on patient morbidity and mortality. We sought to determine how bacterial contamination and persistent MDRO colonization of the healthcare environment are related to the position of patients and wastewater sites.Methods:We performed a prospective cohort study, enrolling 51 hospital rooms at the time of admitting a patient with an eligible MDRO in the prior 30 days. We performed systematic sampling and MDRO culture of rooms, as well as 16S rRNA sequencing to define the environmental microbiome in a subset of samples.Results:The probability of detecting resistant gram-negative organisms, including Enterobacterales, Acinetobacter spp, and Pseudomonas spp, increased with distance from the patient. In contrast, Clostridioides difficile and methicillin-resistant Staphylococcus aureus were more likely to be detected close to the patient. Resistant Pseudomonas spp and S. aureus were enriched in these hot spots despite broad deposition of 16S rRNA gene sequences assigned to the same genera, suggesting modifiable factors that permit the persistence of these MDROs.Conclusions:MDRO hot spots can be defined by distance from the patient and from wastewater reservoirs. Evaluating how MDROs are enriched relative to bacterial DNA deposition helps to identify healthcare micro-environments and suggests how targeted environmental cleaning or design approaches could prevent MDRO persistence and reduce infection risk.

OBJECTIVES: Patients undergoing stem cell transplant (SCT) for the treatment of hematologic malignancy are at increased risk for central line-associated bloodstream infections (CLABSIs). The use of prophylactic antibiotics to prevent CLABSIs in the setting of autologous SCT is of unclear benefit. We aimed to evaluate the impact of levofloxacin prophylaxis on reducing CLABSIs in this high-risk population. METHODS: Patients undergoing autologous SCT at a tertiary-care hospital received levofloxacin prophylaxis from January 13, 2016 to January 12, 2017. Levofloxacin was administered from autologous SCT (day 0) until day 13, absolute neutrophil count > 500/mm(3), or neutropenic fever, whichever occurred first. Clinical outcomes were compared to a baseline group who underwent autologous SCT but did not receive antibacterial prophylaxis during the previous year. The primary endpoint was incidence of CLABSIs assessed using Cox proportional hazards regression. RESULTS: A total of 324 patients underwent autologous SCT during the entire study period, with 150 receiving levofloxacin prophylaxis during the intervention period. The rate of CLABSIs was reduced from 18.4% during the baseline period to 6.0% during the intervention period. On multivariable analysis, levofloxacin prophylaxis significantly reduced CLABSI incidence (hazard ratio (HR) 0.33; 95% confidence interval (CI) 0.16–0.69; P=0.003). There was also a reduction in the risk of neutropenic fever (odds ratio (OR) 0.23; 95% CI 0.14–0.39; P

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with coronavirus disease 2019 (COVID-19) and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. Methods We performed amplification of a human gene target (β-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1282 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by late Ct values for the human gene target β-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and β-actin Ct. Results Low-quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio, 0.607 [95% credible interval {CrI}, .487–.753]). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope, 0.181 [95% CrI, .097–.264]), consistent with a reduction in detection of 0.181 cycles for each additional cycle of the β-actin target. COVID-19 disease severity was not associated with β-actin Ct values. Conclusions Variability in NP specimen quality significantly impacts the performance of clinical SARS-CoV-2 assays, and caution should be taken when interpreting quantitative SARS-CoV-2 Ct results. If unrecognized, low-quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the successful application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

BackgroundThe SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR) cycle of threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with COVID-19 and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity.MethodsWe performed amplification of a human gene target (β-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1311 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by high Ct values for the human gene target β-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and β-actin Ct.ResultsLow quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio 0.654, 95%CI 0.523 to 0.802). We observed a positive linear relationship between SARS-CoV-2 and β-actin Ct values (slope 0.169, 95%CI 0.092 to 0.247). COVID-19 disease severity was not associated with β-actin Ct values.ConclusionsVariability in NP specimen quality accounts for significant differences in the sensitivity of clinical SARS-CoV-2 assays. If unrecognized, low quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

In a cohort of inpatients with hematologic malignancy and positive enzyme immunoassay (EIA) or polymerase chain reaction (PCR) Clostridium difficile tests, we found that clinical characteristics and outcomes were similar between these groups. The method of testing is unlikely to predict infection in this population, and PCR-positive results should be treated with concern.Infect Control Hosp Epidemiol 2018;863–866

Background Inappropriate ordering and acquisition of urine cultures leads to unnecessary treatment of asymptomatic bacteriuria (ASB). Treatment of ASB contributes to antimicrobial resistance particularly among hospital-acquired organisms. Our objective was to investigate urine culture ordering and collection practices among nurses to identify key system-level and human factor barriers and facilitators that affect optimal ordering and collection practices. Methods We conducted two focus groups, one with ED nurses and the other with ICU nurses. Questions were developed using the Systems Engineering Initiative for Patient Safety (SEIPS) framework. We used iterative categorization (directed content analysis followed by summative content analysis) to code and analyze the data both deductively (using SEIPS domains) and inductively (emerging themes). Results Factors affecting optimal urine ordering and collection included barriers at the person, process, and task levels. For ED nurses, barriers included patient factors, physician communication, reflex culture protocols, the electronic health record, urinary symptoms, and ED throughput. For ICU nurses, barriers included physician notification of urinalysis results, personal protective equipment, collection technique, patient body habitus, and Foley catheter issues. Conclusions We identified multiple potential process barriers to nurse adherence with evidence-based recommendations for ordering and collecting urine cultures in the ICU and ED. A systems approach to identifying barriers and facilitators can be useful to design interventions for improving urine ordering and collection practices.

We implemented a guideline for appropriate acid suppressant use in hematology-oncology patients. This intervention resulted in a sustained reduction in proton pump inhibitor (PPI) use without an increase in rates of gastrointestinal bleeding. Practice guidelines are effective in reducing PPI use, which is associated with risk of Clostridioides difficile infection.

Background There are limited data regarding SARS-CoV-2 (SC2) environmental contamination in staff areas of healthcare settings. We performed environmental sampling of staff areas in wards where coronavirus disease 19 (COVID-19) patients received care and compared findings to surfaces within COVID-19 patient rooms. Methods The study was conducted at the Hospital of the University of Pennsylvania (Philadelphia, PA) from 9/15/20-1/26/21. Sampling of 20cm2 surfaces in staff common areas (breakroom high-touch surfaces comprising tables and microwave/refrigerator handles; bathroom surfaces comprising toilet, sink, and doorknob; and floors), nurse workstations (computer mice and floors), and COVID-19 patient rooms (high-touch surfaces comprising bedrail, computer mice/keyboards, and doorknobs; bathroom surfaces; and floors) was performed using flocked swabs one or more times per week. Specimens underwent RNA extraction and quantitative real-time polymerase chain reaction to detect the SC2 N1 region. Median comparisons were performed using Wilcoxon rank sum test. Trends in odds were evaluated using Score test. Results Proportions of surface specimens with detectable SC2 RNA are summarized in Table 1. Median copy numbers were lower among staff toilets compared to COVID-19 patient toilets (135.6 vs. 503.8 copies/specimen, p=0.02), lower among staff breakroom compared to patient room high-touch surfaces (104.3 vs. 220.3 copies/specimen, p=0.007), and similar between staff and patient room samples from sinks and floors. At nurse workstations, SC2 RNA was detected among 22/177 (12.4%) computer mouse and 147/178 (82.6%) floor samples. Odds of SC2 detection increased by study week among common area (p< 0.001) and nurse workstation samples (p< 0.001) (Figures 1 and 2). Table 1. SARS-CoV-2 (SC2) RNA detection on staff common area and coronavirus disease 19 (COVID-19) patient room surfaces at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21. Figure 1. Proportion of environmental surface specimens with detectable SARS-CoV-2 RNA from a) staff common areas and b) nurse workstations of inpatient wards where coronavirus disease-19 patients received care at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21. Figure 2. Proportion of environmental surface specimens with detectable SARS-CoV-2 RNA in staff common areas of inpatient wards where coronavirus disease-19 patients received care at the Hospital of the University of Pennsylvania, 9/15/20-1/26/21, by surface type: a) staff breakroom surfaces, b) staff bathroom surfaces, c) staff common area floors. Conclusion A low prevalence of detectable SC2 RNA was observed among staff area high-touch surfaces; however, the likelihood of detection increased over time. Environmental SC2 RNA detection may reflect primary contamination from infected healthcare workers or secondary contamination from contact with infected patients, though a direct relationship between surface SC2 RNA viral detection and transmission risk has not been established. Disclosures Michael Z. David, MD PhD, GSK (Board Member) Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB))

Background Identifying risk factors for environmental contamination with multidrug-resistant organisms (MDROs) is essential to prioritize methods for prevention of hospital transmission. Methods Patients admitted to an ICU with an MDRO detected on clinical culture in the prior 30 days were enrolled. Patients (4 body sites) and high-touch objects (HTO) (3 composite sites) in ICU rooms were sampled. Environmental transmission was defined by shared MDRO species cultured on patient and HTO cultures obtained on multiple time points during the patient’s stay. Risk factors for environmental transmission were identified with logistic regression. Results Forty-five patients were included (median 2 days of longitudinal sampling [IQR 1–4 days]). Enrollment anatomic cultures included extended-spectrum beta-lactamase-producing Enterobacterales (ESBLE) (n=12, 27%), carbapenem-resistant organisms (CRO) (n=4, 9%), methicillin-resistant S.aureus (MRSA) (n=11, 24%), vancomycin-resistant Enterococci (VRE) (n=4, 9%), and C.difficile (CDIFF) (n=14, 31%). Patient colonization during serial sampling was common with CRO (n=21, 47%), ESBLE (n=16, 36%), and VRE (n=16, 36%) and less so with MRSA (n=7, 16%) and CDIFF (n=5, 11%). Detection of MDROs on environmental surfaces was also common with identification of CRO in 47% of patient rooms (n=21) and ESBLE in 29% (n=13); MRSA (n=2, 4%), VRE (n=9, 20%), and CDIFF (n=3, 7%) were rarer. Patient to environment transmission was observed in 40% of rooms (n=18). Thirteen (29%) rooms had foreign MDRO contamination (i.e., one not detected on a body culture), most (n=10) with CRO. Environmental MDROs were most common in bathroom/sinks (n=22), followed by surfaces near the patient (n=10), and least common surfaces often touched by staff within the room (n=6). On multivariable logistic regression, naïve to clustering by patient, recent receipt of a proton pump inhibitor (OR 2.35, 95% CI 1.00 – 5.52, P=0.049) and presence of one or more wounds (OR 2.56, 95% CI 1.05 – 6.26, P=0.038) were significantly associated with environmental transmission (OR 1.56, 95% CI 1.01 – 2.43, P=0.046) (Table 1). Conclusion MDRO contamination of patient rooms is common with detection of organisms attributed to, and foreign to, the occupant. Disclosures Michael Z. David, MD PhD, GSK (Consultant)

We implemented a guideline for appropriate acid suppressant use in hematology-oncology patients. This intervention resulted in a sustained reduction in proton pump inhibitor (PPI) use without an increase in rates of gastrointestinal bleeding. Practice guidelines are effective in reducing PPI use, which is associated with risk of Clostridioides difficile infection.

Background We evaluated the differential impact of levofloxacin administered for the prophylaxis of bloodstream infections compared with broad-spectrum beta-lactam (BSBL) antibiotics used for the treatment of neutropenic fever on the gut microbiome in patients with hematologic malignancy. Methods Stool specimens were collected from patients admitted for chemotherapy or stem cell transplant in the setting of the evaluation of diarrhea from February 2017 until November 2017. Microbiome characteristics were compared among those exposed to levofloxacin prophylaxis vs those who received BSBL antibiotics. Results Sixty patients were included, most with acute myeloid leukemia (42%) or multiple myeloma (37%). The gut microbiome of patients with BSBL exposure had significantly reduced Shannon’s alpha diversity compared with those without (median [interquartile range {IQR}], 3.28 [1.73 to 3.71] vs 3.73 [3.14 to 4.31]; P = .01). However, those with levofloxacin exposure had increased alpha diversity compared with those without (median [IQR], 3.83 [3.32 to 4.36] vs 3.32 [2.35 to 4.02]; P = .03). Levofloxacin exposure was also associated with a trend toward lower risk of dominance of non-Bacteroidetes genera compared with those without levofloxacin exposure (3 [14%] vs 15 [38%]; P = .051). Conclusions The impact of antibiotics on the gut microbiome varies by class, and levofloxacin may disrupt the gut microbiome less than BSBLs in this patient population.

Background:Clostridioides difficile infection (CDI) is a major contributor to morbidity and mortality in patients with hematologic malignancy. Due to both immunosuppression and frequent antibiotic exposures, up to one-third of inpatients receiving chemotherapy or stem-cell transplant develop CDI. Transmission of C. difficile in healthcare facilities occurs due to environmental surface contamination and hand carriage by healthcare workers from colonized and infected patients. We investigated the effectiveness of enhanced room cleaning in collaboration with environmental services (EVS) staff to prevent CDI transmission and infection.Methods: From April 1, 2018, to September 30, 2018, a multimodal enhanced cleaning intervention was implemented on 2 oncology units at the Hospital of the University of Pennsylvania. This intervention included real-time feedback to EVS staff following ATP bioluminescence monitoring. Additionally, all rooms on the intervention units underwent UV disinfection after terminal cleaning. We performed a system-level cohort study, comparing rates of CDI on the 2 study units to historic and 2 concurrent control units. Historic and concurrent control units received UV disinfection only for rooms with prior occupants with MRSA or CDI. All units during the intervention period received education on the importance of environmental cleaning for infection prevention. Mixed-effects Poisson regression was used to adjust for system-level confounders. Results: A median of 1.34 CDI cases per 1,000 patient days (IQR, 1.20–3.62) occurred during the 12-month baseline period. There was a trend toward a reduced rate of CDI across all units during the intervention period (median, 1.19; IQR, 0.00–2.47; P = .13) compared with all units during the historical period. Using mixed-effects Poisson regression, accounting for the random effects of study units, the intervention was associated with an incidence rate ratio for C. difficile of 0.72 compared to control units (95% CI, 0.53–0.97; P = .03). Average room turnaround time (TAT) increased across all units during the study period, from 78 minutes (IQR 74–81) to 92 minutes (IQR, 85–96; P < .001). Within the intervention period, TAT was higher on intervention units (median, 94 minutes; IQR, 92–98) compared to concurrent control units (median, 85; IQR, 80–92; P = .005). Conclusions: Enhanced environmental cleaning, including UV disinfection of all patient rooms and ATP bioluminescent monitoring with real-time feedback, was associated with a reduction in the incidence of CDI.Funding: NoneDisclosures: None

To identify the attributable mortality of central line associated blood stream infections (CLABSI) through meta-analysis. Meta-analysis of case control and cohort studies, matched and unmatched, that reported on mortality of patients with and without CLABSI was performed. MEDLINE, CENTRAL, CINAHL were searched. Non-interventional studies of all languages that reported mortality in patients with CLABSI were included. Data were extracted on patient population, study setting, design, diagnostic criteria for CLABSI, and mortality. Results from studies comparing mortality due to CLABSI were pooled using a random effects model with assessment of heterogeneity. Heterogeneity of studies was assessed with an I 2 statistic and a funnel plot was generated to assess for publication bias. Eighteen studies were included with 1,976 CLABSI cases. Of the included studies, 17 took place in intensive care unit settings, most involved a mixed population of medical and surgical patients, and ten were matched using an illness severity index. Our findings show an odds ratio of in hospital death associated with CLABSI as 2.75 (CI 1.86–4.07) and 1.51 (CI 1.08–2.09) in the subgroup of the ten matched studies. Those studies where greater than 30 % of CLABSI were attributed to coagulase-negative Staphylococcus had an odds ratio of death of 1.64 (95 % CI 1.02–2.65) compared with 4.71 (95 % CI 1.54–14.39). CLABSI is associated with a significantly increased risk of death supporting the use of extensive efforts to reduce these infections.

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.

Background C. difficile infection is common in patients with hematologic malignancy. There is increasing recognition that molecular (polymerase chain reaction, PCR) based testing lacks specificity for infection, while detecting patients with colonization. The objective of our study was to evaluate characteristics of patients with toxin enzyme immunoassay (EIA) vs. PCR positive C. difficile test results. Methods A retrospective review of inpatients at a tertiary care academic center with hematologic malignancy and a positive C. difficile test from 1/2015 to 1/2016 was performed. Data on demographics, comorbidities, clinical features, and outcomes were collected using medical record review. Characteristics were compared between patients with EIA vs. PCR positive test results using chi-squared or Fisher’s exact test for categorical variables and Wilcoxon rank-sum test for continuous variables. Results A total of 130 patients were included: 51% and 49% had a PCR positive and EIA positive result, respectively. Diagnoses included AML (42%), multiple myeloma (22%), and Non-Hodgkin’s lymphoma (13%). Antibiotic exposure was similar, with a median of 4 days of anti-pseudomonal antibiotics received in the prior 30 days. There was no difference in history of a positive C. difficile test in the prior year (12% in the EIA group, 10% in the PCR group, P = 0.71). Patients with EIA positive results were more likely to have a WBC ≥15/mm3 (18% vs. 6%, P = 0.02). However, there were no differences in presence of fever, stool frequency, or imaging evidence of colitis at the time of testing. Medications in the prior 72 hours were similar, including the use of proton pump inhibitors of ~40% and of laxatives of 28%. Clinical outcomes were also similar between patients with EIA vs. PCR positive tests: all-cause death (22% vs. 20%), recurrent CDI (9% vs. 13%), colectomy (1% vs. 4%), and megacolon (0% vs. 3%). Most patients received treatment with oral vancomycin for a median duration of 14 days. Conclusion In patients with hematologic malignancy, those with EIA vs. PCR positive C. difficile test results were clinically similar. These findings suggest that algorithms for testing and treatment of C. difficile in hematologic malignancy patients will need to be specifically targeted towards this immunocompromised population. Disclosures All authors: No reported disclosures.

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.

Lipid bilayers, normally a barrier to charged species and large molecules, are permeabilized by electric fields, a phenomenon exploited by cell biologists and geneticists for porating and transfecting cells and tissues. Recent molecular simulation studies have advanced our understanding of electroporation, but the relative contributions of atomically local details (interface water and headgroup dipole and counterion configurations) and medium- and long-range electrostatic gradients and changes in membrane structural shifts to the initiating conditions and mechanisms of pore formation remain unclear. Molecular dynamics simulations of electroporation in several lipid systems presented here reveal the effects of lipid hydrocarbon tail length and composition on the magnitude of the field required for poration and on the location of the initial sites of field-driven water intrusion into the bilayer. Minimum porating external fields of 260 mV nm(-1), 280 mV nm(-1), 320 mV nm(-1), and 380 mV nm(-1) were found for 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), and 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC) bilayers, respectively, and correlated most strongly with the bilayer thickness. These phospholipid systems share several common features including a wide, dynamic distribution of the headgroup dipole angle with the bilayer normal ranging from 0 to 155 degrees that is only slightly shifted in a porating electric field, and similar electric field-induced shifts in water dipole orientation, although the mean water dipole moment profile at the aqueous-membrane interface is more sensitive to the electric field for DOPC than for the other phospholipids. The location of pore initiation, at the anode- or cathode-facing leaflet, varies with the composition of the bilayer and correlates with a change in the polarity of the localized electric field at the interface.

To investigate the mechanism of biological cell membrane electroporation at the nanosecond and nanometer scale, we tracked pore-forming lipids and water in molecular dynamics simulations of a palmitoyloleoylphosphatidylcholine bilayer in a minimum porating electric field. Although the field-generated torque tilts the mean head group dipole a few degrees away from its equilibrium, zero-field position relative to the bilayer plane, this change in conformation does not appear to contribute directly to the development of the pore-initiating aggregation of lipid head groups and water that leads to the formation of a membrane-spanning hydrophilic pore. Field-directed rotation of the head group dipoles in the plane of the incipient pore wall, in combination with water dipole and solvation interactions at the aqueous-lipid interface, is one component in the coordinated ensemble of electroporation events.

Atomic-resolution molecular dynamics simulations of lipid bilayers containing 7% phosphatidylserine (PS) on one leaflet are consistent with experimental observations of membrane poration and PS externalization in living cells exposed to nanosecond, megavolt-per-meter electric pulses. Nanometer-diameter aqueous pores develop within nanoseconds after application of an electric field of 450 mV/nm, and electrophoretic transport of the anionic PS headgroup along the newly constructed hydrophilic pore surface commences even while pore formation is still in progress.

To aid in understanding the mechanism of electric field-driven pore formation in lipid bilayers, we propose a scheme for characterizing the life cycle of a transient membrane electropore, from the formation of the initial defect to the restoration of the intact bilayer. We apply this analysis to heterogeneous phospholipid bilayers (phosphatidylcholine:phosphatidylserine, PC:PS) in the presence of calcium ions. Previous reports of molecular dynamics (MD) simulations of similar PC:PS systems containing Ca2+ [1,2] are consistent with experimental observations [3]. In this study we assembled systems containing varying amount of PC, PS, and Ca2+ and observed how long it took each bilayer to porate after the application of an electric field. We also measured the effect of Ca2+ on pore lifetime (the time it takes to restore the porated bilayer after removal of the electric field). We find that systems containing Ca2+ are more difficult to electroporate, and that this effect is magnified in systems containing PS. We also observe that Ca2+ has little effect on pore lifetime, but that PC:PS systems have shorter lifetimes than pure PC systems. Finally, we report binding isotherms for Ca2+ and PC:PS bilayers, an additional metric for the validity of phospholipid bilayer simulations containing calcium.[1] Bockmann, R. A., and H. Grubmuller. 2004. Multistep binding of divalent cations to phospholipid bilayers: A molecular dynamics study. Angewandte Chemie-International Edition 43:1021-1024.[2] Vernier, P. T., M. J. Ziegler, and R. Dimova. 2009. Calcium binding and head group dipole angle in phosphatidylserine-phosphatidylcholine bilayers. Langmuir 25:1020-1027.[3] Sinn, C. G., M. Antonietti, and R. Dimova. 2006. Binding of calcium to phosphatidylcholine-phosphatidylserine membranes. Colloids and Surfaces A-Physicochemical and Engineering Aspects 282:410-419.

Molecular dynamics (MD) studies showing that oxidized lipids increase the frequency of water defects in phospholipid bilayers suggest that the presence of oxidized lipids in a bilayer will also increase the sensitivity of the bilayer to electropermeabilization. To investigate this possibility we applied external electric fields during MD simulations of PLPC (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine) bilayers containing varying concentrations of oxidized PLPC species - the peroxidized linoleic acid derivatives 12-oxo-9-dodecenoic acid (12-al), which contains an aldehyde group, and 13-trans, cis-hydroperoxide linoleic acid (13-tc), which contains a hydroperoxide group. Systems with higher concentrations of oxidized lipids form hydrophilic electropores in significantly shorter times than do systems with lower oxidized lipid concentrations, and at lower electric fields. Furthermore, bilayers containing 12-al electroporate more quickly than bilayers containing 13-tc, possibly a result of the decreased thickness of membranes containing 12-al. Sites of water defect formation and subsequent electroporation appear to coincide with local clustering of oxidized lipids in the bilayer. In large-area simulations containing localized high oxidized lipid concentrations, pores formed preferentially in these oxidized regions. The tendency of the oxidized lipids to bend their sn-2 tail toward the aqueous interface, which may result in membrane thinning and a decrease in the lipid areal density, was not noticeably enhanced by the application of an external electric field, but the presence of the aldehyde and hydroperoxy oxygens on the otherwise nonpolar lipid tails appears to facilitate the penetration of water into the bilayer interior. Simulation results were verified by experimental observations of enhanced permeabilization of oxidized membranes in living cells.