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Background. People with HIV (PWH) exhibit a markedly increased risk of anal cancer, especially men who have sex with men (MSM), with specific microbiota signatures possibly contributing to greater human papillomavirus (HPV) oncogenic potential. Low specificity of the current screening strategy for detecting high-grade squamous intraepithelial lesions (HSIL) hinders anal cancer prevention. We investigated microbiome derivatives associated with HSIL in anal cytology samples. Methods. We recruited a discovery and a validation cohort at four clinical sites in Spain and Italy. Study participants were mostly MSM undergoing HSIL screening with high-resolution anoscopy and anal biopsies to confirm HSIL. We extracted the bacterial DNA, proteins, and metabolites from anal cytology samples, and we performed 16SrRNA gene sequencing, mass spectrometry, and targeted metabolite quantification. Results. We included 213 participants, 167 in the discovery cohort (70 with confirmed HSIL) and 46 in the validation cohort (25 with confirmed HSIL). While we did not find clear microbiome composition signatures associated with HSIL, the microbiome associated with HSIL overexpressed proteins involved in the production of succinyl-CoA and cobalamin, with levels consistently increased in subjects with HSIL in the discovery and validation cohorts. Combined measurement of succinyl-CoA and cobalamin overperformed anal cytology, improving sensitivity from 91.2% to 96.6%, specificity from 34.1% to 81.8%, positive predictive value from 48.1% to 77.8%, and negative predictive value from 85.3% to 97.3%. While anal cytology correctly classified only 59.9% of individuals, combined measurement of both metabolites improved the classification to 87.7%. This test overcame internal (adjusted AUC 0.877) and external validation. From 98 false-positive cytologic results, succinyl-CoA and cobalamin level measurement reclassified 49 (50%) to true negatives. Finally, we demonstrate greater in vitro production of succinyl-CoA and cobalamin in bacteria associated with HSIL or cancer vs. those presumably protective. Conclusions. Cobalamin and succinyl-CoA are overexpressed in the anal microbiome of patients with HSIL and show excellent diagnostic capacity. Their measurement overperforms anal cytology to screen for patients with HSIL. Hence, we discovered two powerful biomarkers for which detection methods can readily be established, that could improve the current strategy of anal cancer screening.

The ingestion of mycotoxins can cause adverse health effects and represents a severe health risk to humans and livestock. Even though several acute and chronic effects have been described, the effect on the gut metaproteome is scarcely known. For that reason, we used metaproteomics to evaluate the effect of the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) on the gut microbiome of 15 weaned piglets. Animals were fed for 28 days with feed contaminated with different concentrations of DON (DONlow: 870 μg DON/kg feed, DONhigh: 2493 μg DON/kg feed) or ZEN (ZENlow: 679 μg ZEN/kg feed, ZENhigh: 1623 μg ZEN/kg feed). Animals in the control group received uncontaminated feed. The gut metaproteome composition in the high toxin groups shifted compared to the control and low mycotoxin groups, and it was also more similar among high toxin groups. These changes were accompanied by the increase in peptides belonging to Actinobacteria and a decrease in peptides belonging to Firmicutes. Additionally, DONhigh and ZENhigh increased the abundance of proteins associated with the ribosomes and pentose-phosphate pathways, while decreasing glycolysis and other carbohydrate metabolism pathways. Moreover, DONhigh and ZENhigh increased the abundance of the antioxidant enzyme thioredoxin-dependent peroxiredoxin. In summary, the ingestion of DON and ZEN altered the abundance of different proteins associated with microbial metabolism, genetic processing, and oxidative stress response, triggering a disruption in the gut microbiome structure.