Abstract This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA-20-032. This is a supplement to U54 CA242646, “California-Mexico-Puerto Rico Partnership (CAMPO) Center for Prevention of HPV-related Cancer in HIV+ Populations”. The parent grant's focus is to perform research on reducing the risk of cervical and anal cancer in men and women living with HIV in Latin America. CAMPO Study 1 will enroll 4000 women and 1000 men living with HIV to optimize screening algorithms to detect cervical and anal high-grade squamous intraepithelial lesions (HSIL). The incidence of anal cancer is high among all groups of Hispanic people living with HIV (PLWH), especially among those in Puerto Rico (PR). HPV infection is necessary but insufficient for the development of anal HSIL and cancer. The goal of this supplement is to elucidate the role of the anal microbiome as a contributor to this increased risk of anal cancer risk in these high-risk populations. Specific aims are 1) Study the relationship between anal high- risk HPV (hr-HPV) infection and the anal microbiome among three distinct populations of Hispanic PLWH in California, Mexico and PR; and 2) Study the relationship between anal HSIL and the anal microbiome among three distinct populations of Hispanic PLWH with anal hr-HPV infection. These populations are of particular interest given their divergent lifestyles but similarity in genetic background. We hypothesize that among Hispanic PLWH without anal HSIL, the microbiome will be different among those with and without hr-HPV infection. We hypothesize that the proportion with pro-inflammatory-mediating taxa will be highest among the PR population compared with the Mexican and California populations given the higher incidence in anal cancer in PR. We also hypothesize that among Hispanic PLWH with anal hr-HPV at the 3 locations, detection of HSIL will be associated with a pro-inflammatory microbiome and a decrease in butyrate-producing signatures, with the proportion with pro-inflammatory changes highest in PR compared with the other populations. We will enroll 100 PLWH (50 men and 50 women) at each of the PR, Mexico City and San Francisco CAMPO sites, for a total of 300 Hispanic PLWH. Data on anal HPV, anal cytology and anal HSIL will be available through CAMPO Study 1 in PR and Mexico City, and from the San Francisco ANCRE Clinic, where PLWH are screened for anal HSIL as part of routine clinical care. A validated food frequency questionnaire in Spanish will be administered to assess dietary intake for the previous 3 and 6 months. Anal swab specimens will be retrieved from the CAMPO Biorepository and used for microbiome analysis. The 16S gene will be amplified from extracted anal swab genomic DNA and subjected to Illumina Miseq. Sequences of the 16S gene V4 region will be analyzed in QIIME2. Random forest classification will be performed to understand how our predictors, i.e. microbiome variables (dive...