Critical Relevance of Stochastic Effects on Low-Bacterial-Biomass 16S rRNA Gene Analysis.

The bacterial microbiome of human body sites, previously considered sterile, remains highly controversial because it can be challenging to isolate signal from noise when low-biomass samples are being analyzed. We tested the hypothesis that stochastic sequencing noise, separable from reagent contamination, is generated during sequencing on the Illumina MiSeq platform when DNA input is below a critical threshold. We first purified DNA from serial dilutions of Pseudomonas aeruginosa and from negative controls using three DNA purification kits, quantified input using droplet digital PCR, and then sequenced the 16S rRNA gene in four technical replicates. This process identified reproducible contaminant signal that was separable from an irreproducible stochastic noise, which occurred as bacterial biomass of samples decreased. This approach was then applied to authentic respiratory samples from healthy individuals (n = 22) that ranged from high to ultralow bacterial biomass. Using oral rinse, bronchoalveolar lavage (BAL) fluid, and exhaled breath condensate (EBC) samples and matched controls, we were able to demonstrate (i) that stochastic noise dominates sequencing in real-world low-bacterial-biomass samples that contain fewer than 104 copies of the 16S rRNA gene per sample, (ii) that critical examination of the community composition of technical replicates can be used to separate signal from noise, and (iii) that EBC is an irreproducible sampling modality for sampling the microbiome of the lower airways. We anticipate that these results combined with suggested methods for identifying and dealing with noisy communities will facilitate increased reproducibility while simultaneously permitting characterization of potentially important low-biomass communities.IMPORTANCE DNA contamination from external sources (reagents, environment, operator, etc.) has long been assumed to be the main cause of spurious signals that appear under low-bacterial-biomass conditions. Here, we demonstrate that contamination can be separated from another, random signal generated during low-biomass-sample sequencing. This stochastic noise is not reproduced between technical replicates; however, results for any one replicate taken alone could look like a microbial community different from the controls. Using this information, we investigated respiratory samples from healthy humans and determined the narrow range of bacterial biomass where samples transition from producing reproducible microbial sequences to ones dominated by noise. We present a rigorous approach to studies involving low-bacterial-biomass samples to detect this source of noise and provide a framework for deciding if a sample is likely to be dominated by noise. We anticipate that this work will facilitate increased reproducibility in the characterization of potentially important low-biomass communities.

Longitudinal Analysis of Long-Term Air Pollution Levels and Blood Pressure: A Cautionary Tale from the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Air pollution exposures are hypothesized to impact blood pressure, yet few longitudinal studies exist, their findings are inconsistent, and different adjustments have been made for potentially distinct confounding by calendar time and age. OBJECTIVE: We aimed to investigate the associations of long- and short-term [Formula: see text] and [Formula: see text] concentrations with systolic and diastolic blood pressures and incident hypertension while also accounting for potential confounding by age and time. METHODS: Between 2000 and 2012, Multi-Ethnic Study of Atherosclerosis participants were measured for systolic and diastolic blood pressure at five exams. We estimated annual average and daily [Formula: see text] and [Formula: see text] concentrations for 6,569 participants using spatiotemporal models and measurements, respectively. Associations of exposures with blood pressure corrected for medication were studied using mixed-effects models. Incident hypertension was examined with Cox regression. We adjusted all models for sex, race/ethnicity, socioeconomic status, smoking, physical activity, diet, season, and site. We compared associations from models adjusting for time-varying age with those that adjusted for both time-varying age and calendar time. RESULTS: We observed decreases in pollution and blood pressures (adjusted for age and medication) over time. Strong, positive associations of long- and short-term exposures with blood pressure were found only in models with adjustment for time-varying age but not adjustment for both time-varying age and calendar time. For example, [Formula: see text] higher annual average [Formula: see text] concentrations were associated with 2.7 (95% CI: 1.5, 4.0) and [Formula: see text] (95% CI: [Formula: see text] 1.0) mmHg in systolic blood pressure with and without additional adjustment for time, respectively. Associations with incident hypertension were similarly weakened by additional adjustment for time. Sensitivity analyses indicated that air pollution did not likely cause the temporal trends in blood pressure. CONCLUSIONS: In contrast to experimental evidence, we found no associations between long- or short-term exposures to air pollution and blood pressure after accounting for both time-varying age and calendar time. This research suggests that careful consideration of both age and time is needed in longitudinal studies with trending exposures. https://doi.org/10.1289/EHP2966.

Ambient Coarse Particulate Matter and the Right Ventricle: The Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Coarse particulate matter (P10-2.5) is primarily mechanically generated and includes crustal material, brake and tire wear, and biological particles. PM10-2.5 is associated with pulmonary disease, which can lead to right ventricular (RV) dysfunction. Although RV characteristics have been associated with combustion-related pollutants, relationships with PM10-2.5 remain unknown. OBJECTIVES: To quantify cross-sectional associations between RV dysfunction and PM10-2.5 mass and components among older adults and susceptible populations. METHODS: We used baseline cardiac magnetic resonance images from 1,490 participants (45-84 y old) from the Multi-Ethnic Study of Atherosclerosis and assigned 5-y residential concentrations of PM10-2.5 mass, copper, zinc, phosphorus, silicon, and endotoxin, using land-use regression models. We quantified associations with RV mass, end-diastolic volume, and ejection fraction after control for risk factors and copollutants using linear regression. We further examined personal susceptibility. RESULTS: We found positive associations of RV mass and, to a lesser extent, end diastolic volume with PM10-2.5 mass among susceptible populations including smokers and persons with emphysema. After adjustment for copollutants, an interquartile range increase in PM10-2.5 mass (2.2 µg/m3) was associated with 0.5 g (95% CI: 0.0, 1.0), 0.9 g (95% CI: 0.1, 1.7), and 1.4 g (95% CI: 0.4, 2.5) larger RV mass among former smokers, current smokers, and persons with emphysema, respectively. No associations were found with healthy individuals or with ejection fraction. CONCLUSIONS: Alterations to RV structure may represent a mechanism by which long-term PM10-2.5 exposure increases risks for adverse respiratory and cardiovascular outcomes, especially among certain susceptible populations. https://doi.org/10.1289/EHP658.

Hard times: the effects of financial strain on home care services use and participant outcomes in Michigan.

PURPOSE: When government funding for long-term care is reduced, participant outcomes may be adversely affected. We investigated the effect of program resources on individuals enrolled in the Michigan Home- and Community-Based Services (HCBS) waiver program for elderly and disabled adults. DESIGN AND METHODS: Using dates of major policy and budget changes, we defined 4 distinct time periods between October 2001 and December 2005. Minimum Data Set for Home Care assessment records for HCBS participants (n = 112,182) were used to examine temporal trends in formal care hours and 6 outcomes: emergency room (ER) use, hospitalization, caregiver burden, death, nursing facility (NF) use, and permanent NF placement. Controlling for demographics, functional status, and cognitive status, adjusted odds of outcomes were obtained using discrete-time survival analysis. RESULTS: As resources diminished, mean formal care hours decreased, declining most for persons with moderate functional or cognitive impairment, for up to an approximately 30% decrease. In the most financially restricted period, 3 adverse outcomes increased relative to baseline: hospitalization (odds ratio [OR] = 1.10; 95% confidence interval [CI] = 1.03-1.18), ER use (OR = 1.13; 95% CI = 1.03-1.24), and permanent NF placement (OR = 1.20; 95% CI = 1.00-1.42). IMPLICATIONS: Reductions in resources for home care were associated with increased probability of adverse outcomes. Cutting funds to home care programs can increase utilization of other more costly services, thus offsetting potential health care savings. Policymakers must consider all ways in which budget reductions and policy changes can affect participants.

Analysis of the Third National Health and Nutrition Examination Survey (NHANES III) using expert ratings of job categories.

BACKGROUND: Few epidemiologic studies have addressed the exposure-response relationships between work activities and symptomatic knee osteoarthritis (OA). This analysis used data from a national survey and ergonomists' ratings to address this issue. METHODS: Interview and knee X-ray data were obtained from the Third National Health and Nutrition Examination Survey. Occupational ratings were obtained using ergonomists. A weighted logistic regression was used. RESULTS: Among men, a significant exposure-response relationship was found between symptomatic knee OA and kneeling. In both genders, there was a significant trend in heavy lifting and severe symptomatic knee OA. Approximately 20.7% of knee OA can be attributed to kneeling >14% of the workday among men. CONCLUSIONS: The significant exposure-response relationships suggest that modest reductions in certain occupational activities can reduce the burden of knee OA. The study was limited by unvalidated expert ratings. Research is needed to identify hazardous characteristics of work activities and to clarify exposure-response relationships.

Expert consensus ratings of job categories from the Third National Health and Nutrition Examination Survey (NHANES III).

BACKGROUND: A method of occupational physical exposure assessment is needed to improve analyses using large data sets (e.g., national surveys) that provide only job title/category information as a proxy for exposure. METHODS: Five ergonomic experts rated and arrived at consensus ratings for job categories used in the Third National Health and Nutrition Examination Survey. Interrater agreement was examined for initial (pre-consensus) ratings. Correlation between consensus ratings and an independent source of ratings (US Department of Labor (DOL)) was used as a basis of comparison. RESULTS: Interrater agreements for the initial ratings were weak. Highest interrater agreement was for sitting (weighted kappa (kappa(w)) = 0.56). Lowest agreement was for standing (kappa(w) = 0.07). Consensus ratings were well correlated with DOL ratings (correlation coefficients >or=0.6). CONCLUSIONS: The correlation between consensus ratings and DOL ratings support the use of expert consensus to assess physical exposures in national data sets. However, validation of expert consensus ratings is required.

Review of epidemiologic studies on occupational factors and lower extremity musculoskeletal and vascular disorders and symptoms.

The epidemiologic literature on lower extremity musculoskeletal disorders (MSDs), vascular disorders, and occupational mechanical factors is relatively sparse, compared to the low back and upper extremities. The present literature review aims to summarize and evaluate the current literature on lower extremity vascular disorders, MSDs, and symptoms, and to update previous literature reviews. A search was conducted in PubMed. Articles were included if they had a lower extremity musculoskeletal outcome, and a workplace factor(s). They were evaluated based upon their generalizability, exposure, and outcome assessments, study design, and controlling of confounders. Most of the literature has focused on osteoarthritis of the hip and knee, whereas not much research has been done examining the feet/ankles and lower legs. Overall, better exposure assessment is needed to examine the causal pathway between occupational factors and MSDs.