Albeit uncommon in the general population, there is increasing awareness of marked (60–130 fold) increased risk of anal cancer in people living with HIV, specially among MSM [1,2]. For reasons that are poorly understood, HIV infection facilitates persistence of mucosal human papillomavirus (HPV), and increases both the risk of anal squamous intraepithelial neoplasia (AIN) and the progression from low-grade (LSIL) to high-grade intraepithelial lesions (HSIL). In women, cervical microbiota has been implicated in the development of HPV-associated precancerous lesions and linked with proinflammatory species [3,4]. Outside the HPV and HIV fields, emerging evidence supports that intestinal bacteria might amplify or mitigate carcinogenesis [5,6]. Hence, the altered composition  and function  of the so-called HIV-associated microbiome might explain the altered course of HPV anal disease during HIV infection.
We performed a cross-sectional study in a high-resolution anoscopy (HRA) clinic. Participants were asymptomatic study participants undergoing screening for anal HPV-associated lesions with anal cytology, HRA, and biopsy of acetowhite lugol-negative lesions or blindly if no lesions were identified. Supra-adjacent biopsies (1–2 of 2–3 mm of length) proximally to the squamocolumnar junction were sampled with baby Tischler forceps for 16SrRNA gene sequencing. We used V3 MiSeq (Illumina, San Diego, California, USA) for sequencing of the V3-V4 16SrRNA gene amplification from feces and biopsies. DNA purification procedures, bacterial 16SrRNA gene analysis, biodiversity, clustering, and biomarker discovery analysis are described in the supplementary materials, https://links.lww.com/QAD/B22.
We calculated the diagnostic accuracy of the bacterial biomarkers with a linear discriminant analysis (LDA) score at least three using logistic regression models and the area under the receiver operating characteristic (AUCROC). The Ethics Committee ([email protected]) approved the study and all patients signed the informed consent.
We studied 42 MSM HIV infected undergoing screening for AIN with HRA. Mean age was 39 ± 9 years, 95% on antiretroviral therapy, and median CD4+ T cells 605/μl (interquartile range 475–819), and median CD4/CD8 ratio 0.90 (interquartile range 0.51–1.08). DNA was successfully extracted from all samples, yet the bacterial 16S rRNA gene could only be amplified in 23(55%) biopsies, given the relative excess amount of human DNA and the low volume of tissue samples. Study participants were classified according to the presence of histologic lesions: HSIL (N = 14), LSIL (N = 13), and normal (N = 15), of whom the bacterial 16SrRNA gene could be amplified in tissue in 10, eight, and five study participants, respectively. α diversity was higher in rectal mucosa compared with feces (Shannon diversity index, 2.4 ± 0.6 vs. 2.1 ± 0.6, P = 0.028 and Chao1 richness estimator, 76.8 ± 17.8 vs. 63.3 ± 13.0, P = 0.010, respectively). The principal coordinates analysis of weighted UniFrac distances showed a 34% of variability between paired fecal and tissue samples (P = 0.001), indicating a distinct microbiota composition in mucosa compared with feces (figure S1, https://links.lww.com/QAD/B22). None of these gross community metrics significantly differed in patients with AIN (LSIL or HSIL) (table S1, https://links.lww.com/QAD/B22). Prevotella was the most abundant genus in mucosa and feces (figure S2, https://links.lww.com/QAD/B22).
To investigate whether specific bacteria might serve as biomarkers of AIN, we first used the LEfSe biomarker discovery tool (Fig. 1). In mucosa, Peptostrepcoccus, together with Anerovibrio, were enriched in study participants with AIN. Campylobacter was found to be predictive of LSIL, and Gardnerella and Catenibacterium discriminated study participants with HSIL. In feces, patients with AIN showed depletion of Bifidobacterium, and enrichment for Peptostreptococcus. Campylobacter discriminated study participants with LSIL, whereas Ruminococcus and Pseudomonas were predictive of HSIL.
Then, we calculated the diagnostic accuracy of those bacterial biomarkers with a LDA score at least 3 (Fig. 1 and table S2, https://links.lww.com/QAD/B22). In mucosa, a number of taxa showed an AUCROC at least 0.800, including Ruminococcus, Peptostreptococcus, and Clostridium for detection of AIN, Campylobacter and Gardnerella for LSIL, and Catenibacterium for HSIL. In feces, only Ruminococcus was predictive of HSIL. Demonstrative cut-offs optimizing either sensitivity or specificity are shown in table S2, https://links.lww.com/QAD/B22.
Here, we studied the rectal and fecal microbiota associated with AIN in a cohort of HIV-infected individuals. The taxa identified as predictive of AIN might prove relevant for HPV pathogenesis. In mucosa, Campylobacter, which produces the cytolethal distendin toxin  with DNAse activity , was found to be predictive of LSIL, suggesting that colonization by this pathogenic genus might initiate carcinogenesis. Peptostreptococcus, previously linked with colon cancer , was enriched in study participants with AIN. Gardnerella, for which the abundance progressively decreases in the vaginal microbiota during the development of precancerous cervical lesions , was predictive of HSIL. A member of the Lachnospiraceae family, Catenibacterium, which has been defined as a component of the HIV-associated microbiota , was also here a biomarker of HSIL.
In keeping with the findings in mucosa, Ruminococcus, a genus which appears to be a central component of the healthy microbiota [8,12], was found in feces predictive of HSIL. This suggests that this taxa might increase as a compensatory response to a chronic perturbation (i.e. HPV infection) to mitigate the harm in its habitat. In feces, study participants with HSIL also showed enrichment for the pathogenic Pseudomonas aeruginosa, which might influence HIV/HPV disease via induction of the kynurenine pathway . Last, Bifidobacterium, which enhance antitumor immunity and anti-PD-L1 efficacy , was depleted in feces in study participants with AIN, suggesting that loss of this taxa could promote HPV disease .
To further explore the diagnostic accuracy of the biomarkers identified in the LEfSe analysis, we calculated the predictive values (AUCROC, sensitivity, and specificity) of those taxa with higher LDA scores. Some, but not all, of these biomarkers proved an adequate diagnostic accuracy (AUCROC ≥0.800). In mucosa, the taxa highlighted in Fig. 1 yielded diagnostic scores that invite to consider the microbiota for the diagnosis of HSIL as a complementary diagnostic tool to anal citology. In feces, however, only Ruminococcus showed an adequate AUCROC, indicating that the mucosal-adherent microbiota might be preferable to the fecal microbiota for future studies in this field.
In summary, components of the fecal and rectal microbiota are able to predict the presence of precancerous anal lesions. Further, while our study cannot prove causality, we found preliminary evidence suggesting that some taxa of the HIV-associated dysbiosis might fuel HPV persistence and pathogenesis. These bacteria could be exploited as diagnostic tools for the screening of precancerous anal lesions in HIV-infected MSM and might eventually become targets for interventions.
The authors would like to particularly thank the study participants for their commitment to participate in research studies. This study was supported by the Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016, projects PIE14/00045, PI15/00345 and EE) and cofinanced by the European Development Regional Fund ‘A way to achieve Europe’ (ERDF) and by the Spanish Ministry of Economy and Competitiveness (SAF2015–65878-R). S.S.V. is supported by a grant from the Spanish Ministry of Science and Innovation (Contratos Juan Rodés, ECC/1051/2013), and T.S. by a grant from the European Society of Pediatric Infectious Diseases (ESPID).
S.S.V., J.P.M., M.J.G, and S.M. designed the study, S.S.V. obtained institutional review board approval, S.S.V. and J.P.M managed study participant recruitment, performed the anoscopic procedures and sampled biopsies, A.B. performed the histologic studies. E.V-D., A.L., A.M., and M.J.G. performed the metagenomic experiments and bioinformatic analyses, S.S.V and M.J.G. drafted the first version of the manuscript, all authors reviewed and approved the manuscript.
Conflicts of interest
There are no conflicts of interest.
1. van Leeuwen MT, Vajdic CM, Middleton MG, McDonald AM, Law M, Kaldor JM, et al. Continuing declines in some but not all HIV-associated cancers in Australia after widespread use of antiretroviral therapy
2. Silverberg MJ, Lau B, Achenbach CJ, Jing Y, Althoff KN, D'Souza G, et al. Cumulative incidence of cancer among persons with HIV in North America: a cohort study
. Ann Intern Med
3. Audirac-Chalifour A, Torres-Poveda K, Bahena-Román M, Téllez-Sosa J, Martínez-Barnetche J, Cortina-Ceballos B, et al. Cervical microbiome and cytokine profile at various stages of cervical cancer: a pilot study
. PLoS One
4. Lee JE, Lee S, Lee H, Song Y-M, Lee K, Han MJ, et al. Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort
. PLoS One
5. Garrett WS. Cancer and the microbiota
6. Tjalsma H, Boleij A, Marchesi JR, Dutilh BE. A bacterial driver-passenger model for colorectal cancer: beyond the usual suspects
. Nat Rev Microbiol
7. Harper KN. HIV-altered gut microbiome may be driving disease progression
8. Serrano-Villar S, Rojo D, Martínez-Martínez M, Deusch S, Vázquez-Castellanos JF, Bargiela R, et al. Gut bacteria metabolism impacts immune recovery in HIV-infected individuals
9. Martínez I, Mateo E, Churruca E, Girbau C, Alonso R, Fernández-Astorga A. Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR
. Int J Med Microbiol
10. Frisan T, Cortes-Bratti X, Chaves-Olarte E, Stenerlöw B, Thelestam M. The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA
. Cell Microbiol
11. Candela M, Turroni S, Biagi E, Carbonero F, Rampelli S, Fiorentini C, Brigidi P. Inflammation and colorectal cancer, when microbiota-host mutualism breaks
. World J Gastroenterol
12. Vázquez-Castellanos JF, Serrano-Villar S, Latorre A, Artacho A, Ferrús ML, Madrid N, et al. Altered metabolism of gut microbiota contributes to chronic immune activation in HIV-infected individuals
. Mucosal Immunol
13. Favre D, Mold J, Hunt PW, Kanwar B, Loke P, Seu L, et al. Tryptophan catabolism by indoleamine 2, 3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease
. Sci Transl Med
14. Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy