Reducing bias in microbiome research: Comparing methods from sample collection to sequencing.

Background: Microbiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially available mock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples. Methods: Human fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls. Results: Samples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs. Conclusion: Our study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecal microbiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation.

Digital Health Interventions for Musculoskeletal Pain Conditions: Systematic Review and Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Digital health solutions can provide populations with musculoskeletal pain with high-reach, low-cost, easily accessible, and scalable patient education and self-management interventions that meet the time and resource restrictions. OBJECTIVE: The main objective of this study was to determine the effectiveness of digital health interventions for people with musculoskeletal pain conditions (ie, low back pain, neck pain, shoulder pain, knee pain, elbow pain, ankle pain, and whiplash). METHODS: A systematic review and meta-analysis was conducted. We searched PubMed and Cochrane Central Register of Controlled Trials (from 1974 to August 2021) and selected randomized controlled trials of digital health interventions in the target population of patients with musculoskeletal pain with a minimum follow-up of 1 month. A total of 2 researchers independently screened and extracted the data. RESULTS: A total of 56 eligible studies were included covering 9359 participants, with a mean follow-up of 25 (SD 15.48) weeks. In moderate-quality evidence, digital health interventions had a small effect on pain (standardized mean difference [SMD] 0.19, 95% CI 0.06-0.32), disability (SMD 0.14, 95% CI 0.03-0.25), quality of life (SMD 0.22, 95% CI 0.07-0.36), emotional functioning (SMD 0.24, 95% CI 0.12-0.35), and self-management (SMD 0.14, 95% CI 0.05-0.24). CONCLUSIONS: Moderate-quality evidence supports the conclusion that digital health interventions are effective in reducing pain and improving functioning and self-management of musculoskeletal pain conditions. Low-quality evidence indicates that digital health interventions can improve the quality of life and global treatment. Little research has been conducted on the influence of digital health on expenses, knowledge, overall improvement, range of motion, muscle strength, and implementation fidelity. TRIAL REGISTRATION: PROSPERO CRD42022307504; https://tinyurl.com/2cd25hus.

Changes in HPV Seroprevalence from an Unvaccinated toward a Girls-Only Vaccinated Population in the Netherlands.

BACKGROUND: In the Netherlands, bivalent human papillomavirus (HPV) vaccination was included in the National Immunization Program for 12-year-old girls in 2010 (vaccination coverage, 45%-60%). We examined possible changes in HPV seroprevalence in the HPV-unvaccinated Dutch population aged 0-89 years, comparing prevaccination data with data of approximately 6 years after implementation of national vaccination. METHODS: Serum samples of men and women were used from two cross-sectional population-based serosurveillance studies performed before (2006-07, n = 6,384) and after (2016-17, n = 5,645) implementation of HPV vaccination in the Netherlands. Seven high-risk HPV-specific antibodies (HPV16, 18, 31, 33, 45, 52, and 58) were tested in a virus-like particle-based multiplex immunoassay. RESULTS: Type-specific HPV seroprevalence increased in women between 2006-07 and 2016-17. Also, a higher seroprevalence for at least one type in women >15 years was found in 2016-17 (31.7%) compared with 2006-07 (25.2%). In men, overall HPV seroprevalence remained similar; however, a lower seroprevalence was found for HPV16 in 2016-17 (7.5%) compared with 2006-07 (10.6%). CONCLUSIONS: Our results indicate an increase in high-risk HPV types in women and a rather stable exposure in men. No clear effects of the strategy of girls-only vaccination were observed in men, probably because of the short time after introduction combined with suboptimal coverage. IMPACT: No herd immunity has been observed yet in a population with suboptimal HPV vaccination coverage.

High seroprevalence of multiple high-risk human papillomavirus types among the general population of Bonaire, St. Eustatius and Saba, Caribbean Netherlands.

BACKGROUND: Incidence and mortality of human papillomavirus (HPV)-related cancers differs geographically, with high rates in Caribbean countries. Seroepidemiological data provide information on lifetime cumulative HPV exposure and contributing risk factors, but has not been available yet for Caribbean Netherlands (CN), comprising the islands Bonaire, St. Eustatius and Saba. Therefore, a cross-sectional population-based serosurveillance study was performed in this (recently girls-only HPV-vaccinated) population in 2017. METHODS: Blood samples from participants (n = 1,823, 0-90 years) were tested for seven high-risk (hr)-HPV-specific IgG-antibodies using a VLP-based multiplex-immunoassay. Risk factors for HPV-seropositivity were analysed among persons unvaccinated aged ≥ 15 years who ever had sex (n = 1,080). RESULTS: Among unvaccinated individuals aged ≥ 15 years, overall seropositivity was high (34%), with over half of them being seropositive for ≥ 2 hr-HPV types, and HPV16 and 52 being most prevalent (13%). Seroprevalence was substantial higher in unvaccinated women (51%) than men (18%), predominantly peaking in women aged 20-59 years, and was highest on St. Eustatius (38%). Besides age and sex, sexual risk factors were associated with HPV-seropositivity. CONCLUSIONS: In accordance with the Caribbean region, seroprevalence of multiple hr-HPV types was high in CN. These data corroborate the decision regarding introduction of a sex-neutral HPV-vaccination program and the relevance for considering a population-based cervical cancer screening program.