Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

Detection of Neisseria meningitidis in saliva and oropharyngeal samples from college students.

Carriage of Neisseria meningitidis is an accepted endpoint in monitoring meningococcal vaccines effects. We have assessed N. meningitidis and vaccine-type genogroup carriage prevalence in college students at the time of MenACWY vaccine introduction in the Netherlands, and evaluated the feasibility of saliva sampling for the surveillance of carriage. For this, paired saliva and oropharyngeal samples collected from 299 students were cultured for meningococcus. The DNA extracted from all bacterial growth was subjected to qPCRs quantifying meningococcal and genogroup-specific genes presence. Samples negative by culture yet positive for qPCR were cultured again for meningococcus. Altogether 74 (25%) of students were identified as meningococcal carrier by any method. Sixty-one students (20%) were identified as carriers with qPCR. The difference between number of qPCR-positive oropharyngeal (n = 59) and saliva (n = 52) samples was not significant (McNemar's test, p = 0.07). Meningococci were cultured from 72 students (24%), with a significantly higher (p < 0.001) number of oropharyngeal (n = 70) compared with saliva (n = 54) samples. The prevalence of genogroups A, B, C, W, and Y was none, 9%, 1%, 1% and 6%, respectively, and 8% of students carried MenACWY vaccine-type genogroup meningococci. Saliva is easy to collect and when combined with qPCR detection can be considered for meningococcal carriage studies.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota.

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

Effect of the bivalent HPV vaccine on viral load of vaccine and non-vaccine HPV types in incident clearing and persistent infections in young Dutch females.

BACKGROUND: HPV vaccination with the bivalent vaccine is efficacious against HPV16 and 18 infections and cross-protection against non-vaccine HPV types has been demonstrated. Here, we assessed (cross-) protective effects of the bivalent HPV16/18 vaccine on incident and persistent infections and viral load (VL) of fifteen HPV types in an observational cohort study monitoring HPV vaccine effects. METHODS: Vaginal samples were obtained annually. Type-specific VL assays were developed for HPV6,11,31 33,35,39,45,51,52,56,58,59 and 66 and used in addition to existing HPV16 and 18 assays. Rate differences of incident clearing and persistent infections were correlated with differences in VL and vaccination status. RESULTS: HPV16/18 vaccination resulted in significantly lower incidence of HPV16/18 infections and significantly lower VL in breakthrough HPV16 (p<0.01) and 18 infections (p<0.01). The effects of vaccination on non-vaccine type VL were ambiguous. Incidence and/or persistence rates of HPV31, 33, 35 and 45 were reduced in the vaccinated group. However, no significant type specific VL effects were found against HPV31, 33, 45, 52 in the vaccinated group. For HPV 6, 59 and 66 no significant reductions in numbers of incident and persistent infections were found, however borderline) VL reductions following vaccination were observed for HPV6 (p = 0.01), 59 (p = 0.10) and 66 (p = 0.03), suggesting a minor effect of the vaccine on the VL level of these HPV types. Overall, vaccination resulted in infections with slightly lower VL, irrespective of HPV type. CONCLUSIONS: In conclusion, vaccination with the bivalent HPV16/18 vaccine results in significantly reduced numbers of HPV16 and 18 incidence rates and reduced VL in breakthrough infections. Significant reductions in incident and/or persistent HPV31, 33, 35 and 45 infections were found, but no significant effect was observed on the VL for infections with these types. For the other non-vaccine HPV types no reduction in incident and/or persistent infections were found, but overall the VL tended to be somewhat lower in vaccinated women.

Anal HPV 16 and 18 viral load: A comparison between HIV-negative and -positive MSM and association with persistence.

Does anal HPV viral load explain the difference in anal HPV persistence between HIV-negative and -positive men who have sex with men (MSM)? MSM ≥18 years were recruited in Amsterdam, the Netherlands, in 2010-2011. Anal self-swabs were collected every 6 months and genotyped (SPF10 -PCR-DEIA-LIPA25 -system). HPV16 and HPV18 load was determined with a type specific quantitative (q)PCR, and compared between HIV-negative and -positive men using ranksum test. Persistence was defined as ≥3 positive samples for the same HPV-type. Determinants of persistent HPV16/18 infection and its association with HPV16/18 load were assessed with logistic regression. Of 777 recruited MSM, 54 and 22 HIV negative men were HPV16 and HPV18 positive at baseline, and 64 and 39 HIV-positive MSM. The geometric mean titer (GMT) of HPV16 was 19.6 (95%CI 10.1-38.0) and of HPV18 8.6 (95%CI 2.7-27.5) DNA copies/human cell. HPV16 and HPV18 load did not differ significantly between HIV-negative and -positive MSM (P = 0.7; P = 0.8, respectively). In multivariable analyses HPV16 load was an independent determinant of HPV16 persistence (OR 1.8, 95%CI 1.3-2.4). No difference in anal HPV viral load was found between HIV-positive and HIV-negative MSM. HPV 16/18 viral load is an independent determinant of type-specific persistence.

Correlation between viral load, multiplicity of infection, and persistence of HPV16 and HPV18 infection in a Dutch cohort of young women.

BACKGROUND: Persistent high-risk human papillomavirus infection precedes the development of cervical cancer. Here we evaluated the contribution of HPV16/18 viral load and the presence of infections with multiple HPV types to persistence and clearance of HPV16/18 infections. METHODS: Vaginal self-swabs were obtained from young women (16-29 y) with one year interval. HPV genotyping was performed using the highly sensitive SPF10-DEIA-LiPA25 system. HPV16/18 DNA loads were quantified via an adapted, highly sensitive qPCR protocol targeting the L1 gene. RESULTS: We identified 227 HPV16 and 111 HPV18 infections with follow-up. For HPV16 132/227 (58%) were persistent and 95/227 cleared. For HPV18 49/111 (44%) infections were persistent and 62/111 cleared. Baseline viral load was significantly higher in persistent infections than in clearing infections for both HPV16 (p=0.022) and HPV18 (p=0.013). At baseline, only HPV16 viral load was significantly higher in multiple HPV infections compared with single infections (p=0.003). In logistic regression analysis HPV16 and HPV18 viral load were found to contribute to persistency with OR=1.279 (95%CI=1.074-1.524) and OR=1.256 (95%CI=1.028-1.533) per log-unit increase HPV16 and HPV18 viral load respectively. The presence of multiple HPV type infections was not associated with higher persistency. CONCLUSION: HPV16/18 viral load might be used as a marker for persisting infections and is affected by the presence of multiple HPV infections. Evaluation of these parameters at the population level may be of value to assess the presence of persistent or clearing HPV16/18 infections as an early marker, and may provide useful quantitative information in (epidemiological) vaccine monitoring studies.