Oral microbiome and risk of incident head and neck cancer: A nested case-control study.

OBJECTIVES: This nested case-control study in the NIH-AARP Diet and Health Study was carried out to prospectively investigate the relationship of oral microbiome with head and neck cancer (HNC). MATERIALS AND METHODS: 56 incident HNC cases were identified, and 112 controls were incidence-density matched to cases. DNA extracted from pre-diagnostic oral wash samples was whole-genome shotgun metagenomic sequenced to measure the overall oral microbiome. ITS2 gene qPCR was used to measure the presence of fungi. ITS2 gene sequencing was performed on ITS2 gene qPCR positive samples. We computed taxonomic and functional alpha-diversity and beta-diversity metrics. The presence and relative abundance of groups of red-complex (e.g., Porphyromonas gingivalis) and/or orange-complex (e.g., Fusobacterium nucleatum) periodontal pathogens were compared between cases and controls using conditional logistic regression models and MiRKAT. RESULTS: Participants with higher taxonomic microbial alpha-diversity had a non-statistically significant decreased risk of HNC. No case-control differences were found for beta diversity by MiRKAT model (all p > 0.05). A greater relative abundance of red-complex periodontal pathogens (OR = 0.51, 95 % CI = 0.26-1.00), orange-complex (OR = 0.38, 95 % CI = 0.18-0.83), and both complexes' pathogens (OR = 0.32, 95 % CI = 0.14-0.75), were associated with reduced risk of HNC. The presence of oral fungi was also strongly associated with reduced risk of HNC compared with controls (OR = 0.39, 95 % CI = 0.17-0.92). CONCLUSION: Greater taxonomic alpha-diversity, the presence of oral fungi, and the presence or relative abundance of multiple microbial species, including the red- and orange-complex periodontal pathogens, were associated with reduced risk of HNC. Future studies with larger sample sizes are needed to evaluate these associations.

Evaluation of alcohol-free mouthwash for studies of the oral microbiome.

Oral bacteria play important roles in human health and disease. Oral samples collected using ethanol-containing mouthwash are widely used for oral microbiome studies. However, ethanol is flammable and not ideal for transportation/storage in large quantities, and some individuals may avoid ethanol due to the burning sensation or due to various personal, medical, religious, and/or cultural factors. Here, we compared ethanol-free and ethanol-containing mouthwashes using multiple microbiome metrics and assessed the stability of the mouthwash samples stored up to 10 days before processing. Forty volunteers provided oral wash samples collected using ethanol-free and ethanol-containing mouthwashes. From each sample, one aliquot was immediately frozen, one was stored at 4°C for 5 days and frozen, while the third aliquot was stored for 5 days at 4°C and 5 days at ambient temperature to mimic shipping delays and then frozen. DNA was extracted, the 16S rRNA gene V4 region was amplified and sequenced, and bioinformatic processing was performed using QIIME 2. Microbiome metrics measured in the two mouthwash types were very similar, with intraclass correlation coefficients (ICCs) for alpha and beta diversity metrics greater than 0.85. Relative abundances of some taxa were significantly different, but ICCs of the top four most abundant phyla and genera were high (> 0.75) for the comparability of the mouthwashes. Stability during delayed processing was also high for both mouthwashes based on alpha and beta diversity measures and relative abundances of the top four phyla and genera (ICCs ≥ 0.90). These results demonstrate ethanol-free mouthwash performs similarly to ethanol-containing mouthwash for microbial analyses, and both mouthwashes are stable for at least 10 days without freezing prior to laboratory processing. Ethanol-free mouthwash is suitable for collecting and shipping oral wash samples, and these results have important implications for planning future epidemiologic studies of the oral microbiome.

Cigarette Smoking and Opium Use in Relation to the Oral Microbiota in Iran.

Cigarettes and opium contain chemicals and particulate matter that may modify the oral microbiota. This study aimed to investigate the association between cigarette and opium use with the oral microbiota. A total of 558 participants were recruited from Iran between 2011 and 2015. Individuals were categorized as never cigarette nor opium users, ever cigarette-only smokers, ever opium-only users, and ever both cigarette and opium users. Participants provided saliva samples for 16S rRNA gene sequencing. Logistic regression, microbiome regression-based kernel association test (MiRKAT), and zero-inflated beta regression models were calculated. For every increase in 10 observed amplicon sequence variants (ASVs), the odds for being a cigarette-only smoker, opium-only user, and both user compared to never users decreased by 9% (odds ratio [OR] = 0.91; 95% confidence interval [95% CI] = 0.86 to 0.97), 13% (OR = 0.87; 95% CI = 0.75 to 1.01), and 12% (OR = 0.88; 95% CI = 0.80 to 0.96), respectively. The microbial communities differed by cigarette and opium use as indicated by MiRKAT models testing the three beta-diversity matrices (P < 0.05 for all). Three genera were less likely and one genus was more likely to be detected in cigarette-only smokers or opium-only users than in never users. The relative abundance of the phylum Actinobacteria (never, 14.78%; both, 21.20%) was higher and the phyla Bacteroidetes (never, 17.63%; both, 11.62%) and Proteobacteria (never, 9.06%; both, 3.70%) were lower in users of both cigarettes and opium, while the phylum Firmicutes (never, 54.29%; opium, 65.49%) was higher in opium-only users. Cigarette and opium use was associated with lower alpha-diversity, overall oral microbiota community composition, and both the presence and relative abundance of multiple taxa. IMPORTANCE Cigarette smoking and opium use are associated with periodontal disease caused by specific bacteria such as Porphyromonas gingivalis, which suggests a link between cigarette smoking and opium use and the oral microbiota. Alterations of the oral microbiota in cigarette smokers compared to nonsmokers have been reported, but this has not been studied across diverse populations. Additionally, the association of opium use with the oral microbiota has not been investigated to date. We conducted this study to investigate differences in the oral microbiota between ever users of cigarettes only, opium only, and both cigarettes and opium and never users of cigarettes and opium in Iran. Lower alpha-diversity, distinct overall oral microbial communities, and the presence and relative abundance of multiple taxa have been found for users of cigarettes and/or opium.

Variants in interferon-alpha pathway genes and response to pegylated interferon-Alpha2a plus ribavirin for treatment of chronic hepatitis C virus infection in the hepatitis C antiviral long-term treatment against cirrhosis trial.

UNLABELLED: Combination treatment with pegylated-interferon-alpha (PEG IFN-alpha) and ribavirin, the current recommended therapy for chronic hepatitis C virus (HCV) infection, results in a sustained virological response (SVR) in only about half of patients. Because genes involved in the interferon-alpha pathway may affect antiviral responses, we analyzed the relationship between variants in these genes and SVR among participants in the Hepatitis C Antiviral Long-Term treatment Against Cirrhosis (HALT-C) trial. Patients had advanced chronic hepatitis C that had previously failed to respond to interferon-based treatment. Participants were treated with peginterferon-alpha2a and ribavirin during the trial. Subjects with undetectable HCV RNA at week 72 were considered to have had an SVR. Subjects with detectable HCV RNA at week 20 were considered nonresponders. We used TaqMan assays to genotype 56 polymorphisms found in 13 genes in the interferon-alpha pathway. This analysis compares genotypes for participants with an SVR to nonresponders. The primary analysis was restricted to European American participants because a priori statistical power was low among the small number (n = 131) of African American patients. We used logistic regression to control the effect of other variables that are associated with treatment response. Among 581 European American patients, SVR was associated with IFNAR1 IVS1-22G (adjusted odds ratio, 0.57; P = 0.02); IFNAR2 Ex2-33C (adjusted odds ratio, 2.09; P = 0.02); JAK1 IVS22+112T (adjusted odds ratio, 1.66; P = 0.04); and ADAR Ex9+14A (adjusted odds ratio, 1.67; P = 0.03). For the TYK2-2256A promoter region variant, a borderline association was present among European American participants (OR, 1.51; P = 0.05) and a strong relationship among African American patients; all 10 with SVR who were genotyped for TYK2 -2256 carried the A variant compared with 68 of 120 (57%) nonresponders (P = 0.006). CONCLUSION: Genetic polymorphisms in the interferon-alpha pathway may affect responses to antiviral therapy of chronic hepatitis C.