The influence of microbial colonization on inflammatory versus pro-healing trajectories in combat extremity wounds.

A combination of improved body armor, medical transportation, and treatment has led to the increased survival of warfighters from combat extremity injuries predominantly caused by blasts in modern conflicts. Despite advances, a high rate of complications such as wound infections, wound failure, amputations, and a decreased quality of life exist. To study the molecular underpinnings of wound failure, wound tissue biopsies from combat extremity injuries had RNA extracted and sequenced. Wounds were classified by colonization (colonized vs. non-colonized) and outcome (healed vs. failed) status. Differences in gene expression were investigated between timepoints at a gene level, and longitudinally by multi-gene networks, inferred proportions of immune cells, and expression of healing-related functions. Differences between wound outcomes in colonized wounds were more apparent than in non-colonized wounds. Colonized/healed wounds appeared able to mount an adaptive immune response to infection and progress beyond the inflammatory stage of healing, while colonized/failed wounds did not. Although, both colonized and non-colonized failed wounds showed increasing inferred immune and inflammatory programs, non-colonized/failed wounds progressed beyond the inflammatory stage, suggesting different mechanisms of failure dependent on colonization status. Overall, these data reveal gene expression profile differences in healing wounds that may be utilized to improve clinical treatment paradigms.

Targeted metagenomic assessment reflects critical colonization in battlefield injuries.

IMPORTANCE: Microbial contamination in combat wounds can lead to opportunistic infections and adverse outcomes. However, current microbiological detection has a limited ability to capture microbial functional genes. This work describes the application of targeted metagenomic sequencing to profile wound bioburden and capture relevant wound-associated signatures for clinical utility. Ultimately, the ability to detect such signatures will help guide clinical decisions regarding wound care and management and aid in the prediction of wound outcomes.

Host CD3+ T-cells can significantly modulate phage treatment effects on bacterial bioburden in mouse models.

Wound healing is a complex system including such key players as host, microbe, and treatments. However, little is known about their dynamic interactions. Here we explored the interplay between: (1) bacterial bioburden and host immune responses, (2) bacterial bioburden and wound size, and (3) treatments and wound size, using murine models and various treatment modalities: Phosphate buffer saline (PBS or vehicle, negative control), doxycycline, and two doses of A. baumannii phage mixtures. We uncovered that the interplay between bacterial bioburden and host immune system may be bidirectional, and that there is an interaction between host CD3+ T-cells and phage dosage, which significantly impacts bacterial bioburden. Furthermore, the bacterial bioburden and wound size association is significantly modulated by the host CD3+ T-cells. When the host CD3+ T-cells (x on log10 scale) are in the appropriate range (1.35 < x < = 1.5), we observed a strong association between colony forming units (CFU) and wound size, indicating a hallmark of wound healing. On the basis of the findings and our previous work, we proposed an integrated parallel systems biology model.

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds.

Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

To Scan or Not to Scan: Development of a Clinical Decision Support Tool to Determine if Imaging Would Aid in the Diagnosis of Appendicitis.

BACKGROUND: Appendicitis is one of the most common surgically treated diseases in the world. CT scans are often over-utilized and ordered before a surgeon has evaluated the patient. Our aim was to develop a tool using machine learning (ML) algorithms that would help determine if there would be benefit in obtaining a CT scan prior to surgeon consultation. METHODS: Retrospective chart review of 100 randomly selected cases who underwent appendectomy and 100 randomly selected controls was completed. Variables included components of the patient's history, laboratory values, CT readings, and pathology. Pathology was used as the gold standard for appendicitis diagnosis. All variables were then used to build the ML algorithms. Random Forest (RF), Support Vector Machine (SVM), and Bayesian Network Classifiers (BNC) models with and without CT scan results were trained and compared to CT scan results alone and the Alvarado score using area under the Receiver Operator Curve (ROC), sensitivity, and specificity measures as well as calibration indices from 500 bootstrapped samples. RESULTS: Among the cases that underwent appendectomy, 88% had pathology-confirmed appendicitis. All the ML algorithms had better sensitivity, specificity, and ROC than the Alvarado score. SVM with and without CT had the best indices and could predict if imaging would aid in appendicitis diagnosis. CONCLUSION: This study demonstrated that SVM with and without CT results can be used for selective imaging in the diagnosis of appendicitis. This study serves as the initial step and proof-of-concept to externally validate these results with larger and more diverse patient population.

Clinical risk factors and inflammatory biomarkers of post-traumatic acute kidney injury in combat patients.

BACKGROUND: Post-traumatic acute kidney injury has occurred in every major military conflict since its initial description during World War II. To ensure the proper treatment of combat casualties, early detection is critical. This study therefore aimed to investigate combat-related post-traumatic acute kidney injury in recent military conflicts, used machine learning algorithms to identify clinical and biomarker variables associated with the development of post-traumatic acute kidney injury, and evaluated the effects of post-traumatic acute kidney injury on wound healing and nosocomial infection. METHODS: We conducted a retrospective clinical cohort review of 73 critically injured US military service members who sustained major combat-related extremity wounds and had collected injury characteristics, assayed serum and tissue biopsy samples for the expression of protein and messenger ribonucleic acid biomarkers. Bivariate analyses and random forest recursive feature elimination classification algorithms were used to identify associated injury characteristics and biomarker variables. RESULTS: The incidence of post-traumatic acute kidney injury was 20.5%. Of that, 86% recovered baseline renal function and only 2 (15%) of the acute kidney injury group required renal replacement therapy. Random forest recursive feature elimination algorithms were able to estimate post-traumatic acute kidney injury with the area under the curve of 0.93, sensitivity of 0.91, and specificity of 0.91. Post-traumatic acute kidney injury was associated with injury severity score, serum epidermal growth factor, and tissue activin A type receptor 1, matrix metallopeptidase 10, and X-C motif chemokine ligand 1 expression. Patients with post-traumatic acute kidney injury exhibited poor wound healing and increased incidence of nosocomial infections. CONCLUSION: The occurrence of acute kidney injury in combat casualties may be estimated using injury characteristics and serum and tissue biomarkers. External validations of these models are necessary to generalize for all trauma patients.

Behavior Profiles at 2 Years for Children Born Extremely Preterm with Bronchopulmonary Dysplasia.

OBJECTIVE: To characterize behavior of 2-year-old children based on the severity of bronchopulmonary dysplasia (BPD). STUDY DESIGN: We studied children born at 22-26 weeks of gestation and assessed at 22-26 months of corrected age with the Child Behavior Checklist (CBCL). BPD was classified by the level of respiratory support at 36 weeks of postmenstrual age. CBCL syndrome scales were the primary outcomes. The relationship between BPD grade and behavior was evaluated, adjusting for perinatal confounders. Mediation analysis was performed to evaluate whether cognitive, language, or motor skills mediated the effect of BPD grade on behavior. RESULTS: Of 2310 children, 1208 (52%) had no BPD, 806 (35%) had grade 1 BPD, 177 (8%) had grade 2 BPD, and 119 (5%) had grade 3 BPD. Withdrawn behavior (P < .001) and pervasive developmental problems (P < .001) increased with worsening BPD grade. Sleep problems (P = .008) and aggressive behavior (P = .023) decreased with worsening BPD grade. Children with grade 3 BPD scored 2 points worse for withdrawn behavior and pervasive developmental problems and 2 points better for externalizing problems, sleep problems, and aggressive behavior than children without BPD. Cognitive, language, and motor skills mediated the effect of BPD grade on the attention problems, emotionally reactive, somatic complaints, and withdrawn CBCL syndrome scales (P values < .05). CONCLUSIONS: BPD grade was associated with increased risk of withdrawn behavior and pervasive developmental problems but with decreased risk of sleep problems and aggressive behavior. The relationship between BPD and behavior is complex. Cognitive, language, and motor skills mediate the effects of BPD grade on some problem behaviors.

Longitudinal evaluation of ventricular volume changes associated with mild traumatic brain injury in military service members.

PRIMARY OBJECTIVE: To investigate differences in longitudinal trajectories of ventricle-brain ratio (VBR), a general measure of brain atrophy, between Veterans with and without history of mild traumatic brain injury (mTBI). RESEARCH DESIGN: Structural magnetic resonance imaging (MRI) was used to calculate VBR in 70 Veterans with a history of mTBI and 34 Veterans without such history at two time points approximately 3 and 8 years after a combat deployment. MAIN OUTCOMES AND RESULTS: Both groups demonstrated a quadratic relationship between VBR and age that is consistent with normal developmental trajectories. Veterans with history of mTBI had larger total brain volume, but no interaction between mTBI and age was observed for brain volume, ventricular volume, or VBR. CONCLUSIONS: In our longitudinal sample of deployed Veterans, mTBI was not associated with gross brain atrophy as reflected by abnormally high VBR or abnormal increases in VBR over time.

Association of Anemia With Outcomes in Patients Undergoing Percutaneous Peripheral Vascular Intervention: Insights From the Blue Cross Blue Shield of Michigan Cardiovascular Consortium (BMC2 VIC).

OBJECTIVES: To evaluate the clinical features and outcomes of patients with anemia undergoing percutaneous peripheral vascular intervention (PVI) in a contemporary registry. METHODS: We evaluated the differences in the clinical features and outcomes of patients with and without anemia undergoing PVI in the Blue Cross Blue Shield of Michigan Cardiovascular Consortium (BMC2 VIC) registry. Anemia was defined using World Health Organization criteria. RESULTS: Baseline anemia was present in 42.3% of 15,683 patients undergoing PVI. Compared to patients without anemia, those with anemia were older (mean age, 67 years vs 71 years), were more often black (16% vs 29%), and had higher comorbidities. Anemic patients were twice as likely to present with acute limb ischemia (5% vs 11%) and undergo urgent PVI (6% vs 15%) or below-the-knee PVI (18% vs 35%). Many in-hospital adverse events were higher in anemic patients. In a propensity-matched cohort, any adverse outcome (3.4% vs 8.4%; odds ratio [OR], 2.58; 95% confidence interval [CI], 1.94-3.42) or major cardiovascular event, defined as death, myocardial infarction, stroke, or amputation (1.1% vs 3.2%; OR, 2.96; 95% CI, 1.83-4.79) was more likely in anemic patients. Of all adverse events, the highest odds were observed for post-PVI transfusions and amputations in anemic patients. Multivariable logistic regression showed that baseline hemoglobin (1 g/dL below the normal value) was associated with greater risk of any adverse event (OR, 1.57; 95% CI, 1.47-1.68). CONCLUSION: The prevalence of anemia was high among PVI patients and was associated with significantly greater likelihood of amputation, any adverse event, and major cardiovascular events. Whether preprocedure correction of anemia has the potential to decrease post-PVI adverse events remains to be studied.

The Clinical Impact of Cardiology Consultation Prior to Major Vascular Surgery.

OBJECTIVE: To understand statewide variation in preoperative cardiology consultation prior to major vascular surgery and to determine whether consultation was associated with differences in perioperative myocardial infarction (poMI). SUMMARY BACKGROUND DATA: Medical consultation prior to major vascular surgery is obtained to reduce perioperative risk. Despite perceived benefit of preoperative consultation, evidence is lacking specifically for major vascular surgery on the effect of preoperative cardiac consultation. METHODS: Patient and clinical data were obtained from a statewide vascular surgery registry between January 2012 and December 2014. Patients were risk stratified by revised cardiac risk index category and compared poMI between patients who did or did not receive a preoperative cardiology consultation. We then used logistic regression analysis to compare the rate of poMI across hospitals grouped into quartiles by rate of preoperative cardiology consultation. RESULTS: Our study population comprised 5191 patients undergoing open peripheral arterial bypass (n = 3037), open abdominal aortic aneurysm repair (n = 332), or endovascular aneurysm repair (n = 1822) at 29 hospitals. At the patient level, after risk-stratification by revised cardiac risk index category, there was no association between cardiac consultation and poMI. At the hospital level, preoperative cardiac consultation varied substantially between hospitals (6.9%-87.5%, P <0.001). High preoperative consulting hospitals (rate >66%) had a reduction in poMI (OR, 0.52; confidence interval: 0.28-0.98; P <0.05) compared with all other hospitals. These hospitals also had a statistically greater consultation rate with a variety of medical specialties. CONCLUSIONS: Preoperative cardiology consultation for vascular surgery varies greatly between institutions, and does not appear to impact poMI at the patient level. However, reduction of poMI was noted at the hospitals with the highest rate of preoperative cardiology consultation as well as a variety of medical services, suggesting that other hospital culture effects play a role.

Heterogeneity of Ankle-Brachial Indices in Patients Undergoing Revascularization for Critical Limb Ischemia.

OBJECTIVES: This study sought to describe the distribution of pre-intervention treated-limb ankle-brachial indices (ABIs) among patients with critical limb ischemia (CLI) undergoing percutaneous vascular intervention (PVI) or surgical revascularization (SR). BACKGROUND: CLI is diagnosed by the presence of rest pain, tissue ulceration, or gangrene due to chronic arterial insufficiency. It is unclear what fraction of patients with suspected CLI have severe peripheral artery disease (PAD) on noninvasive functional testing. METHODS: The study included patients who underwent lower extremity revascularization for CLI in a multicenter registry in Michigan from January 2012 through June 2015. ABIs were classified as normal (ABI: 0.91 to 1.40), mild-moderate (ABI: 0.41 to 0.90), and severe (ABI: ≤0.40). Pre- and post-intervention Peripheral Artery Questionnaire summary scores were assessed in a subset of patients. RESULTS: Among 10,756 patients with signs or symptoms of CLI, 9,113 (84.7%) underwent PVI and 1,643 (15.3%) underwent SR. ABIs were recorded in 4,972 (54.6%) PVI and 1,012 (61.6%) SR patients. Patients undergoing PVI had higher ABIs than those undergoing SR, with substantial variation in both groups (PVI: 0.72 ± 0.29 vs. SR: 0.61 ± 0.29; p < 0.001). Nearly a quarter of patients with compressible arteries had normal ABIs (24.0%), whereas severe PAD was uncommon (16.5%). A significant improvement in Peripheral Artery Questionnaire scores was noted after intervention across all ABI categories. CONCLUSIONS: Among patients undergoing revascularization for CLI in contemporary practice, the authors found substantial heterogeneity in pre-intervention ABIs. The disconnect between ABI results and clinical diagnosis calls into question the utility of ABIs in this population and suggests the need for standardization of functional PAD testing.

Predictors of surgical site infection after open lower extremity revascularization.

OBJECTIVE: Surgical site infection (SSI) after open lower extremity bypass (LEB) is a serious complication leading to an increased rate of graft failure, hospital readmission, and health care costs. This study sought to identify predictors of SSI after LEB for arterial occlusive disease and also potential modifiable factors to improve outcomes. METHODS: Data from a statewide cardiovascular consortium of 35 hospitals were used to obtain demographic, procedural, and hospital risk factors for patients undergoing elective or urgent open LEB between January 2012 and June 2015. Bivariate comparisons and targeted maximum likelihood estimation were used to identify independent risk factors of SSI. Adjusted odds ratios (ORs) were calculated for patient demographics, comorbidities, operative details, and hospital-level factors. RESULTS: Our study population included 3033 patients who underwent 703 femoral-femoral bypasses, 1431 femoral-popliteal bypasses, and 899 femoral-distal vessel bypasses. An SSI was diagnosed in 320 patients (10.6%) ≤30 days after the index operation. Adjusted patient and procedural predictors of SSI included renal failure currently requiring dialysis (OR, 4.35; 95% confidence interval [CI], 3.45-5.47; P < .001), hypertension (OR, 4.29; 95% CI, 2.74-6.72; P < .001), body mass index ≥25 kg/m2 (OR, 1.78; 95% CI, 1.23-2.57; P = .002), procedural time >240 minutes (OR, 2.95; 95% CI, 1.89-4.62; P < .001), and iodine-only skin preparation (OR, 1.73; 95% CI, 1.02-2.91; P = .04). Hospital factors associated with increased SSI included hospital size <500 beds (OR, 2.22; 95% CI, 1.09-4.55; P = .028) and major teaching hospital (OR, 1.66; 95% CI, 1.07-2.58; P = .024). SSI resulted in increased risk of major amputation and surgical reoperation (P < .01), but did not affect 30-day mortality. CONCLUSIONS: SSI after LEB is associated with an increase in rate of amputation and reoperation. Several patient, operative, and hospital-related risk factors that predict postoperative SSI were identified, suggesting that targeted improvements in perioperative care may decrease complications and improve vascular patient outcomes.

Influenza Vaccine Uptake, Hand Hygiene Practices, and Perceived Barriers in Decision Making.

The annual costs of influenza are in the billions of dollars, with employers bearing substantial burdens. Yet, influenza vaccine uptake is sub-optimal. A random survey was administered to employees at a Midwestern public university using mixed quantitative and qualitative methods to identify the rate, characteristics, and barriers of self-reported flu vaccine uptake during March-April of 2012. The lowest uptake was among adults, ages 18 to 49 (29.8%), even though they are included in universal recommendations. Multiple regression analysis adjusted for demographic confounders showed an increase in self-identified protective hand hygiene behavior among those who reported influenza vaccine uptake compared with those who did not. Qualitative thematic analysis revealed contextual accounts of why vaccine uptake was declined including structural, perceptual, and knowledge barriers. Implementation and evaluation of novel multicomponent worksite vaccine interventions tailored to reach young and middle-aged employees including utilization of risk communication is needed to facilitate increased uptake.

Classifying Patients with Chronic Pelvic Pain into Levels of Biopsychosocial Dysfunction Using Latent Class Modeling of Patient Reported Outcome Measures.

Chronic pelvic pain affects multiple aspects of a patient's physical, social, and emotional functioning. Latent class analysis (LCA) of Patient Reported Outcome Measures Information System (PROMIS) domains has the potential to improve clinical insight into these patients' pain. Based on the 11 PROMIS domains applied to n=613 patients referred for evaluation in a chronic pelvic pain specialty center, exploratory factor analysis (EFA) was used to identify unidimensional superdomains. Latent profile analysis (LPA) was performed to identify the number of homogeneous classes present and to further define the pain classification system. The EFA combined the 11 PROMIS domains into four unidimensional superdomains of biopsychosocial dysfunction: Pain, Negative Affect, Fatigue, and Social Function. Based on multiple fit criteria, a latent class model revealed four distinct classes of CPP: No dysfunction (3.2%); Low Dysfunction (17.8%); Moderate Dysfunction (53.2%); and High Dysfunction (25.8%). This study is the first description of a novel approach to the complex disease process such as chronic pelvic pain and was validated by demographic, medical, and psychosocial variables. In addition to an essentially normal class, three classes of increasing biopsychosocial dysfunction were identified. The LCA approach has the potential for application to other complex multifactorial disease processes.

Outcomes of a pilot hand hygiene randomized cluster trial to reduce communicable infections among US office-based employees.

OBJECTIVE: To determine the effectiveness of an office-based multimodal hand hygiene improvement intervention in reducing self-reported communicable infections and work-related absence. METHODS: A randomized cluster trial including an electronic training video, hand sanitizer, and educational posters (n = 131, intervention; n = 193, control). Primary outcomes include (1) self-reported acute respiratory infections (ARIs)/influenza-like illness (ILI) and/or gastrointestinal (GI) infections during the prior 30 days; and (2) related lost work days. Incidence rate ratios calculated using generalized linear mixed models with a Poisson distribution, adjusted for confounders and random cluster effects. RESULTS: A 31% relative reduction in self-reported combined ARI-ILI/GI infections (incidence rate ratio: 0.69; 95% confidence interval, 0.49 to 0.98). A 21% nonsignificant relative reduction in lost work days. CONCLUSIONS: An office-based multimodal hand hygiene improvement intervention demonstrated a substantive reduction in self-reported combined ARI-ILI/GI infections.

Hand hygiene performance and beliefs among public university employees.

The workplace is an important location to access community members, and employers have a direct interest in employee well-being. A survey administered to a random sample of employees at a Midwestern US university tested the ability of a model informed by the theory of planned behavior to predict hand hygiene practices and beliefs using structural equation modeling. Questions demonstrated acceptable validity and reliability. Constructs predicted self-reported hand hygiene behaviors, and hand hygiene behaviors reduced the odds of reporting sickness from respiratory tract and gastrointestinal infections. The findings support multi-modal hand hygiene improvement interventions.

Poly-traumatization and harmful behaviors in a sample of emergency department Psychiatric Intake Response Center youth.

The purpose of this study is to examine risk factors for poly-traumatization, and the impact of poly-traumatization on harmful behaviors (suicidal, self-harm, and violent), among a group of pediatric patients presenting at an emergency department's psychiatric intake response center. We employed a retrospective medical chart review in a children's hospital for a 2-year span (N=260). The study employed 2 statistical analyses. The first analysis used multinomial logistic regression to model the odds of harmful behaviors comparing increasing numbers of co-occurring traumatization types. The second analysis employed latent class modeling techniques in three ways to (a) define different poly-traumatization populations, (b) examine the relationship between predictors and class assignment, and (c) examine the relationship between class assignment and harmful behavioral outcomes. About 62% of the sample presented with at least 1 traumatization type and about 50% one harmful behavior type. Compared to those with 1, 2, or 3 traumatization types, patients with 4 or more traumatization types have higher odds of harmful behaviors. The latent class analysis revealed 2 populations: High serious victimization and minimal traumatization. History of family mental health issues was the only significant predictor of class membership. Class membership was associated with all of the harmful behavioral outcome categories. These findings support consideration of poly-traumatization as a risk factor for the high occurrence of harmful behaviors in this sample of pediatric psychiatric patients and that history of family mental health issues may contribute to the high co-occurrence of poly-traumatization.

Antimicrobial effects of virgin coconut oil and its medium-chain fatty acids on Clostridium difficile.

Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide; in addition, the proliferation of antibiotic-resistant C. difficile is becoming a significant problem. Virgin coconut oil (VCO) has been shown previously to have the antimicrobial activity. This study evaluates the lipid components of VCO for the control of C. difficile. VCO and its most active individual fatty acids were tested to evaluate their antimicrobial effect on C. difficile in vitro. The data indicate that exposure to lauric acid (C12) was the most inhibitory to growth (P<.001), as determined by a reduction in colony-forming units per milliliter. Capric acid (C10) and caprylic acid (C8) were inhibitory to growth, but to a lesser degree. VCO did not inhibit the growth of C. difficile; however, growth was inhibited when bacterial cells were exposed to 0.15-1.2% lipolyzed coconut oil. Transmission electron microscopy (TEM) showed the disruption of both the cell membrane and the cytoplasm of cells exposed to 2 mg/mL of lauric acid. Changes in bacterial cell membrane integrity were additionally confirmed for VCO and select fatty acids using Live/Dead staining. This study demonstrates the growth inhibition of C. difficile mediated by medium-chain fatty acids derived from VCO.

Pulse-based non-thermal plasma (NTP) disrupts the structural characteristics of bacterial biofilms.

Bacterial biofilms were constructed in vitro with two pathogenic strains of Pseudomonas aeruginosa and Staphylococcus aureus using a modified, novel sequential bioreactor system. The structure and stability of bacterial biofilms were evaluated following exposure to non-thermal plasma (NTP) discharge. Mathematical software was used to determine structural changes as biofilms grew over the course of 7 days. Statistical modeling was also performed to assess the ability of NTP to affect the development of the biofilms over different periods of time. Several structural characteristics were significantly affected by NTP discharge whereas others were unaffected. Changes in the three-dimensional structure of the biofilm following introduction of NTP was not limited to one period of development. The mechanism for this phenomenon is not understood but is likely to be a dual, synergistic effect due to the composition of the reactive species and other plasma-associated molecules isolated previously in the NTP discharge used in this study.

The influence of program mediators on eleventh grade outcomes for seventh grade substance users and nonusers.

OBJECTIVES: In their work examining the effects of the Take Charge of Your Life (TCYL) program, Sloboda and colleagues (This Issue) found that the TCYL program had significant positive effects on baseline marijuana users and significant negative effects on baseline nonusers of cigarettes and alcohol. METHODS: Mediational analyses were used to understand why the program had these differential impacts on baseline users and nonusers. RESULTS: Path models for binary outcomes revealed significant program impacts on marijuana normative beliefs and refusal skills. The treatment impacts were between 1.5 and 3 times larger for the baseline users than for nonusers. These direct effects of the program on normative beliefs and refusal skills mediated the treatment impact on use for baseline marijuana users. In contrast, the negative treatment effects on alcohol and cigarette use could not be explained by the program's targeted mediators (normative beliefs, refusal skills, consequences, attitudes and intentions). The direct effects of treatment on use for the baseline nonusers of cigarettes and alcohol remain unexplained. CONCLUSIONS: Possible explanations for this pattern and implications for strengthening universal prevention programs that are delivered to both users and nonusers are discussed. The importance of mediational analyses for programs that show negative impacts, as well as for those that show positive impacts is stressed.