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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.

BackgroundMicrobiota 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.MethodsHuman 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.ResultsSamples 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.ConclusionOur 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.

Technical advances in next-generation sequencing (NGS) have made it more accessible to study the human microbiome, resulting in more available data and knowledge. As a result of this expansion of data, the need to obtain comparable and reproducible data has become one of the most important challenges facing microbiome research nowadays. In this study, we aim to contribute to existing knowledge to promote high quality microbiome data and minimize bias introduced by technical variation throughout studies, from sample collection, storage, to sequencing strategies. While immediate freezing upon sampling has been the "golden standard" in the field, this method is often logistically difficult and expensive, becoming a limiting factor when conducting large scale studies or in regions where maintenance of the cold-chain presents difficulties. Therefore, we compared the immediately frozen method to storage at room temperature for 3 - 5 days in two commercially available stabilization solutions (Omnigene gut and Zymo Research) as well as without buffer. Other important aspects were tested, such as DNA extraction, bacterial DNA input or number of PCR cycles. Method choice for cell disruption resulted in the biggest difference in compositional profiles. The changes observed in microbiome profiles in samples stored at RT without stabilization solution was prevented by the use of these. For library preparation and sequencing, we found the highest heterogeneity in the DNA extraction step, followed by the use of different Illumina barcodes, indicating that both of these steps have an impact during library preparation. We did not observe a batch effect between the different sequencing runs. Standardized methods are important to allow comparison of results between different research groups worldwide and reliably expand microbiome data to a broad range of diseases, ethnical backgrounds and geographic locations. A more global perspective will increase our understanding of the human microbiome around the world.