The effect of low-abundance OTU filtering methods on the reliability and variability of microbial composition assessed by 16S rRNA amplicon sequencing

PCR amplicon sequencing may lead to detection of spurious operational taxonomic units (OTUs), inflating estimates of gut microbial diversity. There is no consensus in the analytical approach as to what filtering methods should be applied to remove low-abundance OTUs; moreover, few studies have investigated the reliability of OTU detection within replicates. Here, we investigated the reliability of OTU detection (% agreement in detecting OTU in triplicates) and accuracy of their quantification (assessed by coefficient of variation (CV)) in human stool specimens. Stool samples were collected from 12 participants 22–55 years old. We applied several methods for filtering low-abundance OTUs and determined their impact on alpha-diversity and beta-diversity metrics. The reliability of OTU detection without any filtering was only 44.1% (SE=0.9) but increased after filtering low-abundance OTUs. After filtering OTUs with <0.1% abundance in the dataset, the reliability increased to 87.7% (SE=0.6) but at the expense of removing 6.97% reads from the dataset. When filtering was based on individual sample, the reliability increased to 73.1% after filtering OTUs with <10 copies while removing only 1.12% of reads. High abundance OTUs (>10 copies in sample) had lower CV, indicating better accuracy of quantification than low-abundance OTUs. Excluding very low-abundance OTUs had a significant impact on alpha-diversity metrics sensitive to the presence of rare species (observed OTUs, Chao1) but had little impact on relative abundance of major phyla and families and alpha-diversity metrics accounting for both richness and evenness (Shannon, Inverse Simpson). To increase the reliability of microbial composition, we advise removing OTUs with <10 copies in individual samples, particularly in studies where only one subsample per specimen is available for analysis

Treatment of Obstructive Sleep Apnea in Young and Middle-Aged Adults: Effects of Positive Airway Pressure and Compliance on Arterial Stiffness, Endothelial Function, and Cardiac Hemodynamics.

BackgroundThe cardiovascular effects of positive airway pressure (PAP) therapy in obstructive sleep apnea (OSA) patients are not clear because of confounding by comorbid conditions.Methods and resultsProspective interventional study of PAP therapy and withdrawal. Apnea Hypopnea Index (AHI; events/hour of sleep) was determined from polysomnography. Central aortic blood pressures (BPs), Aortic Augmentation Index (AAIx), and central (PWVc-f) and peripheral pulse wave (PWVc-r) velocities were determined by applanation tonometry. Echocardiography and brachial artery reactivity testing were performed at baseline, after 4 and 12 weeks of PAP therapy, and 1 week after PAP withdrawal. The 84 participants were mean (SD) 41.1 (7.6) years old and had 39.8 (24.5) AHI events/hour. After 4 weeks post-PAP initiation and sustained after 12 weeks, subjects experienced decreases in central systolic BP (P=0.008), diastolic BP, mean BP, AAIx, and PWVc-r, and brachial artery dilation (all P<0.001), as well as improvements in left ventricular diastolic function and systemic and pulmonary vascular resistance. In adjusted models, PAP use (hours/night) predicted reductions in diastolic BP (β=-0.65 [SE, 0.32] mm Hg/hour; P=0.045), AAIx (β=-0.53 [0.27] %/hour; P=0.049) and PWVc-r (β=-0.13 [0.05] m·s(-1)/hour; P=0.007), and improved brachial artery flow-mediated dilation (β=0.31 [0.14] %/hour use; P=0.015). After 1 week of PAP withdrawal, brachial diameter, diastolic BP, mean BP, AAIx, and heart rate increased (P≤0.05).ConclusionsPAP therapy reduces arterial tone and improves endothelial and diastolic function in young to middle-aged adults. This positive effect is observed after 4 weeks and depends on hours of use, but reverts quickly with PAP withdrawal.Clinical trial registrationURL: https://clinicaltrials.gov/. Unique identifier: NCT01317329

Polysomnographic Indicators of Restorative Sleep and Body Mass Trajectories in the Wisconsin Sleep Cohort Study

Study Objectives Previous research suggests that reductions in restorative, slow-wave (N3), and rapid eye movement (REM) sleep are associated with weight gain and obesity in mid-to-late life. We extend prior work by examining how within-person (WP) changes and between-person (BP) differences in restorative sleep over several years are associated with body mass trajectories among participants in the Wisconsin Sleep Cohort Study (WSCS). Methods We used data from 4,862 polysomnographic (PSG) sleep studies and physical exams collected from 1,187 WSCS participants over an average duration of 14.9 years. Primary measures of interest included body mass index (BMI = kg/m2) and the percentages of time spent in N3 and REM sleep. We estimated a series of linear mixed regression models to examine how WP changes and BP differences in N3 and REM sleep affected BMI trajectories, controlling for other sleep measures, demographic characteristics, and health behaviors as potential confounders. Results Women in the WSCS experienced more rapid BMI gain than men. With some variation by sex, we found that (1) below-average N3 and REM sleep is associated with above-average BMI, and (2) within-person decreases in N3 and REM sleep over time are associated with gains in BMI. These findings persisted after adjustment for sleep duration and other potential confounders. Conclusions Our findings highlight the importance of PSG indices of restorative sleep in mid-to-late life, suggesting that future clinical treatments and public health policies will benefit from heightened attention to sleep quality

Meditation or exercise for preventing acute respiratory infection (MEPARI-2): A randomized controlled trial

<div><p>Background</p><p>Practice of meditation or exercise may enhance health to protect against acute infectious illness.</p><p>Objective</p><p>To assess preventive effects of meditation and exercise on acute respiratory infection (ARI) illness.</p><p>Design</p><p>Randomized controlled prevention trial with three parallel groups.</p><p>Setting</p><p>Madison, Wisconsin, USA.</p><p>Participants</p><p>Community-recruited adults who did not regularly exercise or meditate.</p><p>Methods</p><p>1) 8-week behavioral training in mindfulness-based stress reduction (MBSR); 2) matched 8-week training in moderate intensity sustained exercise (EX); or 3) observational waitlist control. Training classes occurred in September and October, with weekly ARI surveillance through May. Incidence, duration, and area-under-curve ARI global severity were measured using daily reports on the WURSS-24 during ARI illness. Viruses were identified multiplex PCR. Absenteeism, health care utilization, and psychosocial health self-report assessments were also employed.</p><p>Results</p><p>Of 413 participants randomized, 390 completed the trial. In the MBSR group, 74 experienced 112 ARI episodes with 1045 days of ARI illness. Among exercisers, 84 had 120 episodes totaling 1010 illness days. Eighty-two of the controls had 134 episodes with 1210 days of ARI illness. Mean global severity was 315 for MBSR (95% confidence interval 244, 386), 256 (193, 318) for EX, and 336 (268, 403) for controls. A prespecified multivariate zero-inflated regression model suggested reduced incidence for MBSR (p = 0.036) and lower global severity for EX (p = 0.042), compared to control, not quite attaining the p<0.025 prespecified cut-off for null hypothesis rejection. There were 73 ARI-related missed-work days and 22 ARI-related health care visits in the MBSR group, 82 days and 21 visits for exercisers, and 105 days and 24 visits among controls. Viruses were identified in 63 ARI episodes in the MBSR group, compared to 64 for EX and 72 for control. Statistically significant (p<0.05) improvements in general mental health, self-efficacy, mindful attention, sleep quality, perceived stress, and depressive symptoms were observed in the MBSR and/or EX groups, compared to control.</p><p>Conclusions</p><p>Training in mindfulness meditation or exercise may help protect against ARI illness.</p><p>Limitations</p><p>This trial was likely underpowered.</p><p>Trial registration</p><p>Clinicaltrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01654289" target="_blank">NCT01654289</a></p></div

Main ARI-related outcomes.

<p>Main ARI-related outcomes.</p