Catharina J Alberts

Isabelle Heard

Ana Canestri

Lucie Marchand

Jean-François Fléjou

Lionel Piroth

Tristan Ferry

Jean-Michel Didelot

Laurent Siproudhis

Sebastien Henno

Sébastien Henno

Isabelle Poizot-Martin

Teresa M Darragh

Dr Gary M Clifford
email: cliffordg@iarc.fr

Jean- Damien Combes

Isabelle Etienney

Determinants, Among Human Immunodeficiency Virus-Infected Men Who Have Sex

Keywords: incidence, clearance, anal HPV infection, men who have sex with men, human immunodeficiency virus, France

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A c c e p t e d M a n u s c r i p t 4

Background

The incidence of anal cancer has increased in the past decades [START_REF] Kang | Anal cancer in high-income countries: Increasing burden of disease[END_REF], for which the main riskfactor is anal exposure to high-risk human papillomavirus (hrHPV) [START_REF] De Martel | Worldwide burden of cancer attributable to HPV by site, country and HPV type[END_REF] especially HPV-16 [START_REF] Machalek | Anal human papillomavirus infection and associated neoplastic lesions in men who have sex with men: a systematic review and meta-analysis[END_REF][START_REF] Lin | Human papillomavirus types from infection to cancer in the anus, according to sex and HIV status: a systematic review and meta-analysis[END_REF].

The high burden of anal cancer among men who have sex with men (MSM), especially among HIV-infected MSM, is well characterized [START_REF] Machalek | Anal human papillomavirus infection and associated neoplastic lesions in men who have sex with men: a systematic review and meta-analysis[END_REF][START_REF] Lin | Human papillomavirus types from infection to cancer in the anus, according to sex and HIV status: a systematic review and meta-analysis[END_REF][START_REF] Marra | Type-Specific Anal Human Papillomavirus Prevalence Among Men, According to Sexual Preference and HIV Status: A Systematic Literature Review and Meta-Analysis[END_REF]. However, data on the natural history of anal HPV infections among HIV-infected MSM are scarce.

Among MSM, anal incidence rates of HPV-16 can be 2 times higher in HIV-positive MSM compared to HIV-negative MSM, while no significant difference in clearance has been found [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Phanuphak | Anal human papillomavirus infection among Thai men who have sex with men with and without HIV infection: prevalence, incidence, and persistence[END_REF]. Among HIV-infected MSM, sexual risk behavior is an important predictor of HPV incidence [START_REF] Phanuphak | Anal human papillomavirus infection among Thai men who have sex with men with and without HIV infection: prevalence, incidence, and persistence[END_REF][START_REF] Hernandez | Incidence of and risk factors for type-specific anal human papillomavirus infection among HIV-positive MSM[END_REF], while smoking has been found to be a predictor of HPV persistence [START_REF] Phanuphak | Anal human papillomavirus infection among Thai men who have sex with men with and without HIV infection: prevalence, incidence, and persistence[END_REF]. It is unclear whether HIV-related factors are predictors of HPV incidence and/or clearance [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Geskus | Incidence and clearance of anal high-risk human papillomavirus in HIV-positive men who have sex with men: estimates and risk factors[END_REF].

In a prospective multicenter study of HIV-infected MSM in France, in which we have previously shown hrHPV and HPV-16 to be highly prevalent at baseline [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF], we explore the incidence and clearance of HPV-16 and HPV-18 infection as well as the determinants thereof.

Methods

Study participants

As described previously [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF][START_REF] Clifford | Determinants of high-grade anal intraepithelial lesions in HIV-positive MSM[END_REF], the ANRS EP57 APACHES study concerns an HIV-infected MSM population aged ≥35 years followed up in infectious disease units in six hospitals across

Data collection

All participants completed a questionnaire on tobacco use and sexual behavior. Participants' medical records were used to retrieve HIV-related data. The number of new recent sexual partners was asked at every subsequent visit.

Biological samples

Two anal swabs (Anex brush) were taken at each visit by a proctologist. Swabs were transferred to PreservCyt medium (Hologic, Boxbourough, Massachusetts, USA) to be used for cytology and for HPV DNA testing. After specimen collection, patients received a standard proctologic examination (including digital anal-rectal examination [DARE]) and highresolution anoscopy as described before [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF][START_REF] Clifford | Determinants of high-grade anal intraepithelial lesions in HIV-positive MSM[END_REF]. Lesions were classified based on the highest diagnostic category found using either cytology or histology [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF]. Currently, (consensus) cytology and histology is only available for the baseline visits. Anal swabs were tested for HPV DNA using the Cobas 4800 system (Roche Molecular Systems, Alameda). This assay provides partial genotyping: HPV-16, HPV-18 and other hrHPV aggregated (-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66 and -68).

Statistical analyses

Incidence and clearance rates were estimated for HPV-16 and HPV-18 using Poisson regression analyses, assuming a constant rate over time. Cumulative hazard estimates for incidence and clearance were deducted from Kaplan-Meier survival analysis. We included all participants completing ≥2 visits.

Incidence was defined as a positive HPV test result preceded by a negative HPV test. Time at risk for incidence started from the first HPV negative visit of the HPV type under consideration, and ended, either at the midpoint between the last negative test and the first positive test, or at the date of the last HPV negative sample (if the HPV outcome was not A c c e p t e d M a n u s c r i p t 6 detected). Incident infection with the same HPV type, after clearance of a first infection, was ignored in this analysis.

Clearance was defined as one negative test result that was preceded by a positive test result. People entered risk-set for clearance from the first HPV positive visit and they left the risk-set either at the midpoint between the last positive test and the first negative test, or at the date of the last HPV positive sample (if the HPV infection had not cleared).

The incidence and clearance rate and rate ratios were estimated using Poisson regression. Poisson regression was also used to estimate the incidence/clearance-ratio within one HPV type.

Results

Description of study population

In this study, 438 HIV-infected MSM who had ≥2 visits were included. Baseline HPV-16, HPV-18, other hrHPV, and any hrHPV prevalence were 29%, 12%, 64%, and 71%, respectively; this was similar to the full cohort of participants (n=490) [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF]. The median number of visits was 3 (interquartile range, [IQR]:2-4), the median time interval between two visits was 12 (IQR:12-13) months. Median age at entry was 51 years (IQR:46-57) and median baseline CD4+ cell count was 682 cells/µl (IQR:499-884). Most individuals were on cART (95%) and had an undetectable HIV viral-load (93%). 18% had a high-grade lesion detected at baseline (Supplementary Table 1).

Incidence and clearance of HPV-16 and HPV-18

Anal incidence rates (IRs) and clearance rates (CRs) are presented in Table 1. IRs of HPV-16 and HPV-18 were 4.4 (95%C=3.15-6.17) and 4.3 (95%C=3.12-5.81) per 1,000 person-months, respectively. The incidence of HPV-16 and HPV-18 was similar (the incidence rate ratio of HPV- HPV-16 cleared 2.0 (95%CI: 1.30-2.99) times slower than HPV-18. The incidence/clearance ratio was 0.3 (95%CI=0.18-0.43) for HPV-16 and 0.1 (95%CI=0.09-0.21) for HPV-18.

Determinants of HPV-16 and HPV-18 incidence

In Table 2, determinants of anal HPV-16 incidence are presented. Significant determinants of HPV-16 incidence were 'current' versus 'never' tobacco use (adjusted-IRR [aIRR]=3.2; 95%CI=1.14-8.73), having a new receptive ano-genital partner (aIRR=2.3; 95%CI=1.06-4.85), and having a high-grade lesion detected at baseline (aIRR=3.0; 95%CI=1.07-8.18). Of the 75 individuals with a high-grade lesion, 57% (n=43) were already HPV-16 positive at baseline, and among the 32 who were HPV-16 negative at baseline, 7 were HPV16-positive at their next 12 month visit and 1 at his 36 month visit (Table 2). Of note, the 8 individuals with an incident HPV-16 event also tested HPV16-negative by PapilloCheck at baseline. Anal HPV-16 incidence was not associated with age, nor with most HIV-related variables (cART use, AIDS-defining condition, HIV-viral load at baseline, or CD4+ at baseline and nadir). However, participants with CD4 + /CD8 + -ratio ≥1 had lower incidence than those with CD4 + /CD8 + -ratio <0.5 (aIRR=0.3; 95%CI:0.10-0.87).

The only significant determinant for anal HPV-18 incidence, in either bivariable or multivariable analyses, was number of lifetime receptive-anal sexual partners (for 10-39 versus 0-9, aIRR=5.2; 95%CI=1.19-22.85) (Supplementary Table 2).

Determinants of HPV-16 and HPV-18 clearance

Determinants of anal HPV-16 and HPV-18 clearance are presented in Supplementary Tables 3 and4, respectively. Clearance of HPV-16 happened less often after a persistent infection compared to an incident infection (aIRR=0.3; 95%CI=0.11-1.01). This association was significant for HPV-18 (aIRR=0.3; 95%CI=0.10-0.67). Participants with a high-grade lesion A c c e p t e d M a n u s c r i p t 8 cleared HPV-18 infection less often than participants without a lesion (aIRR=0.2; 95%CI=0.06-0.60), but a similar significant effect was not seen for HPV16 (aIRR=0.7; 95%CI=0.31-1.60).

Discussion

Given the limited prospective data on the natural history of anal HPV infections among HIVinfected MSM, we provide a detailed analysis of the incidence and clearance of anal HPV-16 and HPV-18. Previously, we showed that one third of the APACHES study population were HPV-16 positive and 12% HPV18-positive at baseline [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF]. Here we show that after 24 months, about 10% of all HIV-infected MSM have new HPV-16 or HPV-18 infection detected, and that 32% versus 54% subsequently cleared their HPV-16 or HPV-18 infection, respectively. Furthermore, we found that HPV-16 incidence was significantly higher among individuals with a high-grade lesion at baseline, hinting at the possibility of a small number of HPV-16 infections missed in baseline anal swabs. Furthermore, HIV-related parameters did not show associations with anal HPV clearance, at least not among this contemporary virally-controlled population of HIV-infected MSM.

IRs and CRs of this study are within the range of previous studies [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Phanuphak | Anal human papillomavirus infection among Thai men who have sex with men with and without HIV infection: prevalence, incidence, and persistence[END_REF][START_REF] Hernandez | Incidence of and risk factors for type-specific anal human papillomavirus infection among HIV-positive MSM[END_REF][START_REF] Geskus | Incidence and clearance of anal high-risk human papillomavirus in HIV-positive men who have sex with men: estimates and risk factors[END_REF]. Note that studies use different sampling and laboratory techniques, and different definitions of incidence and clearance, hampering direct comparisons between studies. HPV-16 is found to have the highest incidence in most studies and to have the lowest clearance in all studies [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Phanuphak | Anal human papillomavirus infection among Thai men who have sex with men with and without HIV infection: prevalence, incidence, and persistence[END_REF][START_REF] Hernandez | Incidence of and risk factors for type-specific anal human papillomavirus infection among HIV-positive MSM[END_REF][START_REF] Geskus | Incidence and clearance of anal high-risk human papillomavirus in HIV-positive men who have sex with men: estimates and risk factors[END_REF]. Only a few studies estimate the incidence/clearance-ratio of individual HPV types [START_REF] Geskus | Incidence and clearance of anal high-risk human papillomavirus in HIV-positive men who have sex with men: estimates and risk factors[END_REF][START_REF] Marra | Incidence and clearance of anal high-risk HPV infection and their determinants among HIV-negative men who have sex with men over a period up to five-years[END_REF],

showing --in agreement with our study --the highest incidence/clearance-ratio for HPV-16, highlighting the unique carcinogenic potential of HPV-16 [START_REF] Lin | Human papillomavirus types from infection to cancer in the anus, according to sex and HIV status: a systematic review and meta-analysis[END_REF].

We found a higher incidence of anal HPV-16 among individuals with high-grade lesions at baseline. This association remained strong even after adjustment for sexual activity, and given that 22% (7/32) of these incident infections were detected at the next visit, this suggests A c c e p t e d M a n u s c r i p t 9 a small number of missed HPV-16 infections at baseline. Hence, despite the already high correlation found between HPV-16 and high-grade lesions [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF], our results suggest that HPV-16 positivity in high-grade lesions might be even higher than that estimated based on a single concurrent anal swab. Although we excluded a small proportion of swabs with inadequate human DNA, sampling procedures of anal swab may need further standardization to ensure quality control given the complexity of sampling the entire anal canal.

Tobacco use was a borderline significant predictor for HPV-16 incidence and showed a positive trend with HPV-18 incidence. This association might be due to residual confounding, but it has also been shown that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers [START_REF] Phillips | Smoking-related DNA adducts in anal epithelium[END_REF].

Despite the fact that prevalence/incidence and the sequelae related to HPV infection are more often present among HIV-infected MSM [START_REF] Machalek | Anal human papillomavirus infection and associated neoplastic lesions in men who have sex with men: a systematic review and meta-analysis[END_REF][START_REF] Marra | Type-Specific Anal Human Papillomavirus Prevalence Among Men, According to Sexual Preference and HIV Status: A Systematic Literature Review and Meta-Analysis[END_REF][START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF], our study, in agreement with others [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Geskus | Incidence and clearance of anal high-risk human papillomavirus in HIV-positive men who have sex with men: estimates and risk factors[END_REF], supports the finding that HIV-related parameters are not strongly associated with anal HPV incidence/clearance. Further research needs to clarify whether this is because studies are underpowered and/or other unmeasured factors play a role among HIV-infected MSM. Higher CD4 + /CD8 + -ratio was associated with lower HPV-16 incidence; given that it was not consistent with other HIV-related variables, nor with the finding for HPV18, we do not have an explanation for this.

One of the study's limitations is the number of events, which should be kept in mind when interpreting the analysis of the determinants of HPV incidence and clearance. The data did not enable stricter definitions to be applied for incidence (e.g., a positive visit preceded by two negative visits [0-0-1]) and clearance (e.g., a negative visit preceded by two positive visits [1-1-0]). Stricter definitions might avoid counting a deposition as an incident infection, rather than an active infection of the anal epithelium [START_REF] Mooij | The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM[END_REF][START_REF] Marra | Incidence and clearance of anal high-risk HPV infection and their determinants among HIV-negative men who have sex with men over a period up to five-years[END_REF][START_REF] Twisk | Detection of Incident Anal High-Risk Human Papillomavirus DNA in Men Who Have Sex With Men: Incidence or Reactivation?[END_REF]. Furthermore, only A c c e p t e d M a n u s c r i p t 10 aggregated data were available for 'other hrHPV', hampering a more detailed analysis of hrHPV types beyond HPV-16 and -18. Approximately 70% of this population were hrHPV positive at baseline [START_REF] Combes | Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus-Positive Men Who Have Sex with Men[END_REF]. Using the aggregated hrHPV-positivity, we observe that after 24 months, approximately half of the 30% hrHPV negative acquire a new hrHPV infection, and that one fifth of the hrHPV positive individuals become hrHPV negative. This high prevalence and turnover highlights how aggregated hrHPV testing (i.e. without genotyping) is not a useful marker for triage in this high-risk population.

Although MSM are clearly an important target group for primary prevention, caution is needed when translating epidemiological data on HPV among older MSM to implications for HPV vaccination. Around 30% of the men in this population were already HPV-16 positive [START_REF] Clifford | Determinants of high-grade anal intraepithelial lesions in HIV-positive MSM[END_REF], 68% still had their HPV-16 infection after 24 months, and 10% acquired a new HPV-16 infection within 24 months. Furthermore, a recent study showed that incident infections may be caused by reactivated latent HPV infections rather than new incident infections [START_REF] Twisk | Detection of Incident Anal High-Risk Human Papillomavirus DNA in Men Who Have Sex With Men: Incidence or Reactivation?[END_REF],

suggesting that HPV-infection was already present. We might expect that a large proportion of any future anal cancer in this MSM population arise from pre-existing infections.

In conclusion, incidence of HPV-16 and HPV-18 were similar, HPV-16 clearance was significantly lower, correlating with its known higher carcinogenic potential. In addition to the possibility of high-grade lesions going undetected in HPV-16 positive individuals through the limitations of high-resolution anoscopy [START_REF] Clifford | Determinants of high-grade anal intraepithelial lesions in HIV-positive MSM[END_REF], our data suggest that the reverse might also be possible, i.e. that a small proportion of HPV-16 infections in high-grade lesions may go undetected in a single anal swab. APACHES is currently finalizing study exit outcomes of cytology and histology diagnoses, future analyses of which, will provide more detailed insights into how HPV(-16) incidence and persistence predict the prospective risk for high-grade anal lesions.

A c c e p t e d M a n u s c r i p t

France.

  Participants were included between December 2014 and June 2016, with the last visit date in June 2018. Here, we analyzed annual follow-up visits [10]. Exclusion criteria were anal cancer or histologic high-grade anal intraepithelial lesions (hHSIL) treated in the preceding 12 months. The study was approved by the International Agency for Research on Cancer (IARC) Ethics Committee and the Comité de Protection des Personnes de Paris Ile de France VI in accordance with the Declaration of Helsinki.

7 (

 7 A c c e p t e d M a n u s c r i p t 95%CI: 12.04-21.19) and 31.4 (95%CI: 23.14-42.68) per 1,000 person-months, respectively.

Acknowledgements. The ANRS EP57 APACHES study group would like to thank Cécile Blot, Sophie Circosta, Claude Crisol, Anne Dumont, Damien Georges, Sandrine Gohier, Sophie Guillot, Alena Ivanova, Hélène Laroche, Lydia Leger, Karine Lelandais, Emmanuelle Norguet, Sadia Medaouri, Stanislas Ogoudjobi, Enora Ouamara-Digue, Christelle Paul, Maja Ratajczak, Anne-Suzel Ritleng, Jennifer Rohan, Typhaine Thomas and members of TRT-5. They are extremely grateful to all participants.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes