Long‐term intraanal HPV16 infection in men having sex with men with HIV: Persistence or reinfection?

Dear Editor,

Persistent infections with high‐risk (HR) human papillomaviruses such as HPV16 can cause anal cancer. ¹ Men who have sex with men living with HIV (MSMLWH) have high anal HPV prevalence rates and a high risk for anal cancer. ¹ , ²

This retrospective study was undertaken to analyse whether the long‐term detection of intraanal HPV16 in MSMLWH reflects persistent infection with the same HPV16‐strain or repetitive new infections with different HPV16‐strains in a sexually active population. Of 794 MSMLWH who underwent anal cancer screening between 2003 and 2019, ² 153 were repeatedly HPV16‐positive. Extracted DNA was available from 155 intraanal swabs from 54 of the repeatedly HPV16‐positive MSMLWH for further analysis (median follow‐up 82 months, IQR 50.3–116.5). Twenty‐six of the 54 MSMLWH had continuously HPV16‐positive swabs (group‐1), and 28 had an HPV16‐negative interval (group‐2) (Figure 1). The median number of HPV16‐positive swabs/patient was 3 (IQR 2–3). Two variable non‐coding HPV16‐genome regions (LCR 815 bp, NCR 136 bp) were analysed by Sanger‐sequencing of both viral DNA strands. Phylogenetic analysis was performed for HPV16‐sublineage determination (Figure 2).

FIGURE 1.

Intra‐individual HPV16‐LCR/NCR‐sequence changes found by Sanger sequencing in patients from group‐1 and group‐2 during the study period. (a) Percentage distribution of the 26 patients from group‐1 (left pie chart) and the 28 patients from group‐2 (right pie chart) into those with identical intra‐individual sequences (blue) and with different sequences (group‐1 purple, group‐2 red or orange) during the study period. Group‐1 consisted of MSMLWH with continuously HPV16‐positive intraanal swabs during the study period. Group‐2 comprised MSMLWH who had ≥1 HPV16‐negative swabs between HPV16‐positive samples. The median length of the HPV16‐negative interval in group‐2 was 31 months (IQR 15–51). (b, c) The upper parts with the arrows show the follow‐up time (months) for the respective patients of group‐1 (b) and group‐2 (c) schematically on a timeline. The follow‐up period was defined as the time‐period in months between the first (baseline) and the last HPV16‐positive swab of a patient. Each circle represents one patient. The number in the circles after the ‘P’ is the patient‐ID, the number after the ‘#’ indicates the number of HPV16‐positive swabs sequenced per patient. Blue circles indicate patients with absolutely identical intra‐individual HPV16‐LCR/NCR‐sequences within the study period. In (b), purple circles and in (c), red, pink, orange and yellow circles indicate patients with intra‐individual sequence variations within the study period. The lower parts of (b) (group‐1, 2 patients) and (c) (group‐2, 8 patients) show schematic representations of intra‐individual sequence differences of HPV16‐LCR (left) and ‐NCR (right), respectively. A cut‐off of ≥1 nucleotide difference in LCR/NCR‐sequences (951 bp) was applied to discriminate between different HPV16‐isolates/strains. Experimental details of sequence analyses are available from the authors. HPV, human papillomavirus; LCR, long control region; NCR, non‐coding region.

FIGURE 2.

Neighbour‐joining tree of merged HPV16‐LCR/NCR Sanger sequences. The tree visualizes pairwise Levenshtein distances between 59 combined LCR + NCR Sanger‐sequences from 52 patients and three HPV reference sequences (sublineages A1, A2, A3), which were identified as the reference sequences most closely related to the shown patient sequences. Identical sequences from the same patient were collapsed into a single node; the coloured lines on the outer edge of the phylogenetic tree highlight instances in which intra‐individual differences were observed (the colouring corresponds to the classification of patients shown in Figure 1). The shading of the node labels shows the assignment of sequences to the next‐closest HPV reference sequence (cyan: HPV16‐A1, GenBank ID K02718.1; lime: HPV16‐A2, GenBank ID AF536179.1; red: HPV16‐A3, GenBank ID HQ644236.1). Two outliers (samples P02_2006, closest to reference HPV16‐C, GenBank ID AF472509.1, and P08_2011, closest to reference HPV16‐D3 GenBank ID AF402678.1) exhibited increased distances to the other samples and were removed for visual clarity. The coloured dots on the inner edge of the tree mark patients with inter‐individually identical LCR/NCR‐sequences. Identical inter‐individual LCR/NCR‐sequences were found in eight patient pairs (in different years) and in a group of three (P04, P24, P43) and four patients (P03, P12, P21, P34), respectively. The neighbour‐joining tree was constructed using Python 3.8.1040 and the resulting tree data (newick format) was visualized with the iTol online tool. Methodological details of phylogenetic analysis are available from the authors. The LCR‐ and NCR‐sequences obtained in this study were submitted to GenBank, accession numbers OR044448 to OR044529 and PP717891. LCR, long control region; NCR, non‐coding region.

One hundred and fifty LCR‐ and 150 NCR‐sequences were obtained from 155 HPV16‐positive swabs. 92.3% (24/26) of group‐1 and 71.4% (20/28) of group‐2 (p = 0.079) had absolutely identical intra‐individual HPV16‐LCR/NCR‐sequences within the study period of several years. In 7.7% (2/26) of group‐1 and 28.6% (8/28) of group‐2, different HPV16‐strains were newly detected (Figure 1). 70.4% of all 54 individuals had intra‐individual LCR/NCR‐sequences unique to them. Whole‐genome sequencing (WGS) with high‐accuracy long‐read sequencing (PacBio‐HiFi) of the complete HPV16‐genome of 12 samples from five patients corroborated the Sanger‐sequencing results. In phylogenetic analysis of merged LCR/NCR‐sequences from 52 MSMLWH, the majority had HPV16‐sequences that grouped with HPV16‐sublineage A3 (69.2%, 36/52), followed by A1 (19.2%, 10/52) and A1 + A3 (7.7%, 4/52). Sublineages A2, C and D3 were only found in one patient each. The sublineage distribution did not change during the investigation period (Figure 2). The presence of HPV16‐sublineage A3 was confirmed by WGS of 11 samples.

Limitations of our study are the retrospective design, the non‐uniform follow‐up period, the small sample size, the sole inclusion of MSMLWH and the investigation of only one HPV type. Strengths of our study are the length of the follow‐up period up to 13 years and corroboration of Sanger‐sequencing results by WGS.

More than 80% of the MSMLWH had long‐term anal infections with the identical HPV16‐strain, respectively. The repeated detection of HPV16 in MSMLWH most likely represents persistence of the initially detected strain in the majority of patients. Redetection of the identical HPV16‐strain after an HPV16‐negative interval suggests reactivation of persistent latent infection or redetection of low‐level chronic infection rather than clearance. ³ , ⁴ This could explain the limited efficacy of prophylactic HPV vaccination in MSMLWH and underlines the importance of early HPV vaccination. ¹ , ⁵ , ⁶ Guidance on anal cancer screening with HR‐HPV‐testing should consider the possible long‐term persistence of previously detected HR‐HPV‐types, even if they are not detectable at a given point in time. ³ , ⁴

HPV16 can be divided into four phylogenetic lineages (A–D) and 16 sublineages with different geographical distribution. ⁷ A1‐A3 are traditionally considered ‘European’ variants, but in contrast to A1 and A2, sublineage A3 is more frequent in East‐Asia. ⁷ The sublineage distribution in our study differed markedly from that reported for anal or cervical/vaginal samples from Europe and America, where A1 predominated (>70%), followed by A2 (17%–23%), while A3 was rarely found. ⁷ , ⁸ , ⁹ , ¹⁰ In contrast, 79% of the MSMLWH in our study carried sublineage A3 in all or part of their samples. This indicates that different HPV16‐sublineages prevail in different sexual networks. An HPV16‐sublineage associated with an increased risk of cervical cancer, which is rare in Europe, ⁷ is prevalent among MSMLWH in Germany.

FUNDING INFORMATION

This work was funded by the German Center for Infection Research (DZIF, grant no. TI 07.001) and by the German Federal Ministry of Health (grant No. 1369‐401). LB was supported by a clinical leave stipend from the German Centre for Infection Research (DZIF) and by the Koeln Fortune Program/Faculty of Medicine, University of Cologne. SS and UW were supported by the German Federal Ministry of Health (German National Reference Centre for Papilloma‐ and Polyomaviruses, grant No. 1369‐401). MS was supported by the Jürgen Manchot Foundation. Computational support of the Zentrum für Informations‐ und Medientechnologie, especially the HPC team (High Performance Computing) at the Heinrich‐Heine University Duesseldorf is acknowledged. The funders had no role in study design, data collection and analysis, decision to publish or manuscript preparation.

CONFLICT OF INTEREST STATEMENT

None declared.

ETHICAL APPROVAL

The study was performed according to the Declaration of Helsinki principles and was approved by the ethics review board of the Ruhr University Bochum (No. 3936‐1) and the University of Witten/Herdecke (No. 166/2017).

ETHICS STATEMENT

Patients gave written informed consent to participate in the study.

DATA AVAILABILITY STATEMENT

All data generated or analysed during this study are available in the article or from the corresponding author upon reasonable request.