Short summary

Some countries have enrolled the HPV vaccine in their vaccination programs for boys, because of the evidence-based effect of the vaccine. Denmark is not one of these countries. The PAPPIR-study is a prospective cohort designed to acquire more detailed knowledge of HPV infections from a high risk group of 81 MSM. The aims of this study are to investigate the prevalence and incidence of anal, penile and oral HPV infections, as well as the coherence between prevalence and HPV-related risk factors. Also the incidence and clearance of HPV infection will be investigated as well as whether the incidences of HPV are caused by new HPV infection or reactivation.

Rationale

It is estimated that oncogenic HPV strains are the cause of approximately 80% of all diagnosed anal cancers, 50% of all penis cancers and 24-36% of all oropharyngeal cancers. In particular, HPV16 and HPV18 are known to cause about 90% of all HPV caused cancers. Anal cancer is a rare disease in the general public of Denmark, but the incidences of anal cancers have still been increasing throughout the last 50 years. In 2003-2008 the incidence was approximately 1 diagnosed anal cancer per 100.000 person year. For comparison the incidence of anal cancer for men who have sex with men (MSM) is known as 100 per 100.000 person years, and a recent study conducted by The Danish HIV Cohort found that the incidence rate for HIV-positive MSM was 180 anal cancers per 100.000 person years. The development in Denmark is similar to countries like North America, Australia and the rest of Europe.

The results of a meta-analysis clarifies that HIV-positive MSM have the largest prevalence of HPV infections, including the oncogenic HPV16. The HPV16 prevalence are calculated as 35% and the anal intraepithelial neoplasia (AIN) which is a potentially pre-cancerous condition have the prevalence of 29%. A Canadian study investigated a population of 247 MSM and found that 98% had HPV DNA. The median was 5 types of HPV and the most common was HPV16 (38%). The HPV16 incidence rate was 1 per 1000 person months, and a median of 36 months for the period infected.

Knowledge of clearance, the incidence of reactivation and reinfection of HPV infections are limited. The consequences of multiple oncogenic HPV infections and the effect on clearance, reinfection and reactivation are still unknown. New methods like next-generation sequencing from the field of molecular biology, may contribute to new knowledge in the research of the HPV.

At some point the HPV can be prevented. The oncogenic HPV16 and 18, and the non-oncogenic HPV6 and HPV11 can be prevented through a vaccine. The vaccine will prevent anal HPV infections normally caused by the HPV types in the vaccine and AIN among younger MSM. AIN relapse among MSM will also be prevented. Knowledge of the effect of the vaccine considering reinfection and reactivation are still modest. USA, Canada and Australia have included the HPV vaccine in there national vaccination programs for boys, because of the proved effect of the vaccine. In spite of many recommendations, Denmark seems not to be following in their footsteps.    

In order to acquire more detailed knowledge of HPV infections among Danish HIV-positive and sexually active MSM, the aims of this study are to:

• Investigate the prevalence of HPV and subtypes of HPV in anus, oral cavity and urine
• Investigate the coherence between prevalence and potentially HPV-related risk factors. Including:

1. HIV plasma RNA
2. Immunsuppression
3. Antiretroviral (ART) treatment compliance
4. Co-infection with other sexually transmitted diseases
5. Sexual behavior
6. Use of alcohol, tobacco and other substances
7. Demographics

• Investigate the spontaneous clearance of HPV infection within a period of 12 month
• Investigate the incidence of HPV infection within a period of 12 month
• Investigate whether incidence of HPV infection are caused by new infection or reactivation.

Description of the cohort

The study population contains 81 HIV-positive MSM allocated at fire different hospitals in Denmark. The hospitals are;

• Odense University Hospital, Department of Infectious Diseases (20 participants)
• Aarhus University Hospital, Department of Infectious Diseases (7 participants)
• Hvidovre University Hospital, Department of Infectious Diseases (35 participants)
• Rigshospitalet, Department of Infectious Diseases (19 participants)

Inclusion criteria's are:

• MSM
• HIV-positive patients +/- HAART treatment (Highly Active Antiretroviral Treatment)
• Age over 18 years old and under 50 years old
• At least 1 sexual partner within the latest month
•  No previous diagnosis with anal, penis or oral/pharyngeal neoplasia
• Able to give informed consent
• Not previously vaccinated against HPV

Exclusion criteria's are:

• Alcohol or substance abuse, as well as other conditions that can affect the ability to attend the scheduled activities.

Data and biological material

The study is designed as a prospective cohort study with a total of 2 visits, baseline and 12 month after baseline (+/- 2 months). The following data will be collected at each visit:

• Targeted physical examination of the oral cavity, genitalia and perianal area. Questionnaire targeted demographic and lifestyle, including sexual behavior, smoking and alcohol consumption.
• Blood sample for biobank
• Samples taken from anus, pharynx and urine for PCR examination for:
• HPV and HPV subtypes
• Treponema pallidum, Neisseria gonorrhoea and Chlamydia trachomatis
• Mycoplasma genitalium Cytomegalovirus
• Herpes simplex virus type 1 and 2
• Human Herpes virus type 8
• Data such as antiretroviral treatment, CD-4 cell count and HIV RNA in plasma is being collected via the Electronic Patient Record.

Surveys:

• PCR surveys
• After Nucleic Acid Purification with MagNA Pure LC Total Nucleic Acids Isolation Kit (Roche, Mannheim, Germany) are micro-array analysis done by Microbial Detection Array (LLMDA).
• Next-generation sequencing (NGS):
• Performed on the MiSeq equipment (Illumina)
• This survey depends on the micro-array results, and the amount of performed analyzes are still unknown. The survey is capable of showing:

1. Whether the HPV infection are caused by activation of persistent infection or a new infection.
2. Whether a persistent HPV infection are caused by integration of the genetic material of the HPV in the genetic material of the participant.
3. Whether a participant is infected with other HPV subtypes, viruses or bacteria's, then those detected via the micro-array analyzes.

Collaborating researchers and departments

Department of infectious Diseases Q, Odense University Hospital

• Professor Court Pedersen, MD, DrSci
• Consultant Physician Olav Larsen, PhD
• Doctor Sandra Dröse

Department of Virological Analyzes, Statens Seruminstitut

• Consultant, Anders Fomsgaard, MSc
• Cand. Scient. Maiken Worsøe Rosenstierne, PhD,
• Student Majken L. Olesen, PhD

Department of Infectious Diseases, Hvidovre Hospital

• Consultant Physician Anne-Mette Kjær Lebech, DMSc
• Doctor Kristina Thorsteinsson, PhD

Department of Infectious Diseases and Rheumatology, Rigshospitalet

• Consultant Physician Terese Katzenstein, DMSc

Department of Infectious Diseases, Aarhus University Hospital

• Doctor Lars Toft Nielsen, PhD
• Consultant Physician Merete Storgaard, PhD

Departments of Infectious Diseases

• Permanent employees at the participating centers, including specialized study nurses