Sequential HPV16 variant infections in the genital epithelium and anal canal of men

Abstract

This study aimed to assess HPV16 variant period prevalence and sequential acquisition at genital and anal sites in men from the USA and Latin America, using data from the HPV Infection in Men (HIM) Study. Among 635 men with genital and 115 with anal HPV16, 53 men tested positive for any HPV16 variant in both sites, and HPV16 A1 was most prevalent at both sites, more so at the anal canal. Sequential acquisition of HPV16 A1 occurred in 30.4% (genital to anal) and 29.4% (anal to genital). These findings offer insight into HPV16 variant distribution across anatomic sites in men.

Introduction

Persistent infections with Human papillomavirus (HPV) lead to cancer¹. In 2018, HPV was attributed to 31% oropharyngeal, 100% anal, and 53% penile cancers among men globally². Together with HPV18, HPV16 is reported in 87% of anal and 85% of HPV-related head and neck cancers³, thus emphasizing the need for an improved understanding of HPV-related cancers and prevention strategies among men. Furthermore, research on HPV variants has mainly been performed in women with cervical cancer^4–7, and there is a paucity of data on intra-genotype variability in HPV-related diseases in men.

Over 200 HPVs have been taxonomically classified as genotypes based on at least 10% divergence of their respective L1 open reading frame (ORF) that encodes the viral capsid⁸. A 2% difference within the complete genome sequence of these genotypes further differentiates them into variants⁹. The HIM study has previously reported HPV16 as the most prevalent in the genitals¹⁰, anus¹¹ and oral cavity¹² of men. We also previously reported the prevalence and persistence of HPV16 molecular variants in the anal canal¹³ and genital region of these men¹⁴, independent of each anatomic site. However, the prevalence and sequential infections of HPV16 variants at lineage and sublineage level, in both the anal canal and genital epithelium have yet to be reported. Overall, the role of HPV variants in disease development across multiple anatomic sites of the same individual (intra-person) is still under-researched, leaving a significant knowledge gap regarding the dynamics of viral transmission and persistence. We aimed to estimate HPV16 variant period prevalence at both anal and genital regions of men from the USA and Latin America who were recruited in the HIM Study. We further described the sequential acquisition of the most common variant, HPV16 A1, from genital to anal, and vice versa.

Methods

Participants were drawn from the HIM study cohort, which included approximately 4,500 HIV-negative individuals aged 18 to 70. Recruitment took place between July 2005 and June 2009 in Cuernavaca, Mexico; São Paulo, Brazil; and Tampa, USA¹⁰. Participants were monitored for HPV acquisition every six months until 2016. During study visits, participants completed a computer-assisted self-interview and provided swabs from the genital and anal canal regions. At the H. Lee Moffitt Cancer Center (USA), DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, CA, USA) according to the manufacturer's instructions and analyzed using the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, CA, USA).^10,15 Genital HPV sampling, of the penile and scrotal regions, was conducted using three prewetted Dacron swabs, which were combined into a single specimen for HPV testing, as previously described¹⁰. Anal swabs were collected using a fourth prewetted Dacron swab, rotated along the anal orifice and dentate line, as previously described¹¹.

HPV16 positive samples from the genital epithelium (n=1,943) and anal canal (n=227) were sent to ICESP (Brazil) for variant analysis. A 364 bp fragment of the LCR region (nts 7482–7839) was amplified using AmpliTaq Gold^® polymerase (Applied Biosystems, CA, USA). This fragment was selected for its abundance of single nucleotide polymorphisms (SNPs) that differentiate all HPV16 lineages and sublineages (Ferreira et al., 2023). In cases of unsuccessful amplification, two additional primer sets were used to amplify 250 bp and 296 bp LCR fragments. Amplicons were purified using the Illustra ExoProStar 1-Step (Healthcare, Buckinghamshire, UK). Sanger sequencing was conducted with the BigDye^® Terminator v3.1 Cycle Sequencing kit in the ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, NY, USA). Sequences were classified into lineages (A, B, C, and D) and sublineages (A1–4; B1–4; C1–4; and D1–4) by comparing them to the HPV16 prototype (sublineage A1) (GenBank K02718.1). Descriptive statistics were calculated to summarize the participants’ characteristics. Continuous variables were summarized by median, range, mean, and standard deviation. Count and percentage of summarized categorical variables. Period prevalence was used to capture the proportion of individuals infected at any point during the study period to provide a broader picture of HPV exposure over time, rather than a snapshot at each visit. All analyses were performed using SAS version 9.4.

Results

HPV16 DNA positivity was reported among 867 men for the genital epithelium and among 124 men for the anal canal. After removing samples with insufficient volume for variant analyses, 635 men and 115 men, for the genital epithelium and anal canal, respectively, were analyzed for HPV16 variants. A total of 53 men tested positive for any HPV16 variant DNA at both anatomic sites from at least one visit. Of the 53 men, 60% were from Brazil, with the highest proportion aged 25–32 years (Table 1). Fifteen (31%) were men who have sex with men (MSM), while 16 (33%) were heterosexual (33%) and 18 (37%) were bi-sexual (Table 1). The median age of the study participants was 27 years, with an interquartile range (IQR) of 19–52 years. Many of the men were white (62%), Hispanic (51%), and had completed 12 years or less of education (43%). The predominant marital status among participants was single (68%). A total of 39 men were infected with HPV16 A1, while 14 men were infected with other HPV16 variants. No demographic or sexual behavioral variables differed significantly between men infected with HPV16 A1 and those infected with other HPV16 variants, except for the lifetime number of female sex partners (Table 1).

Table 1:

Descriptive demographics of men infected with HPV 16 variants in both genital epithelium and anal canal.

	All Men N=53 (%)	Men infected with HPV16 A1 variants N=40 (%)	Men infected with other HPV16 variants N=13 (%)	p-value*
Country of residence
US	11 (20.8)	10 (25)	1 (7.69)	0.332
BZ	32 (60.4)	22 (55)	10 (76.92)
MX	10 (18.9)	8 (20)	2 (15.38)
Age
Median (range)	27 (19–52)	26.5 (19–52)	30 (23–49)	0.089
Mean (SD)	29.7 (8.4)	28.83 (8.43)	32.23 (7.89)
18–24	15 (28.3)	13 (32.5)	2 (15.38)	0.635
25–32	22 (41.5)	16 (40)	6 (46.15)
33–41	9 (17.0)	6 (15)	3 (23.08)
42+	7 (13.2)	5 (12.5)	2 (15.38)
Marital Status
Divorced/Separated/Widowed	4 (7.5)	3 (7.5)	1 (7.69)	0.769
Married/Cohabiting	13 (24.5)	11 (27.5)	2 (15.38)
Single	36 (67.9)	26 (65)	10 (76.92)
Race
White	33 (62.3)	26 (65)	7 (53.85)	0.359
Black	8 (15.1)	4 (10)	4 (30.77)
Asian/Pacific Islander	1 (1.9)	1 (2.5)	0 (0)
Other	11 (20.8)	9 (22.5)	2 (15.38)
Ethnicity
Hispanic	26 (51.0)	21 (55.26)	5 (38.46)	0.296
Non-Hispanic	25 (49.0)	17 (44.74)	8 (61.54)
Missing	2 (−)	2 (−)	0 (0)
Education duration
Completed 12 Years or Less	23 (43.4)	16 (40)	7 (53.85)	0.657
13–15 Years	14 (26.4)	12 (30)	2 (15.38)
Completed at Least 16 Years	16 (30.2)	12 (30)	4 (30.77)
Number of drinks of any alcoholic beverage consumed in the past one month
0	10 (18.9)	9 (22.5)	1 (7.69)	0.071
1–30	22 (41.5)	14 (35)	8 (61.54)
31–60	6 (11.3)	4 (10)	2 (15.38)
>60	14 (26.4)	13 (32.5)	1 (7.69)
Refused	1 (1.9)	0 (0)	1 (7.69)
Smoking status
Current	18 (34.6)	15 (38.46)	3 (23.08)	0.115
Former	12 (23.1)	6 (15.38)	6 (46.15)
Never	22 (42.3)	18 (46.15)	4 (30.77)
Missing	1 (−)	1 (−)	0 (0)
Sexual orientation
MSW	16 (32.7)	13 (36.11)	3 (23.08)	0.662
MSWM	18 (36.7)	13 (36.11)	5 (38.46)
MSM	15 (30.6)	10 (27.78)	5 (38.46)
Missing	4 (−)	4 (−)	0 (0)
Lifetime number of female sex partners
Median (range)	5 (0–120)	5 (0–120)	2 (0–52)	0.679
Mean (SD)	10.6 (19.2)	10.13 (19.56)	12.27 (18.66)
0	10 (18.9)	8 (20)	2 (15.38)	0.035
1–2	9 (17.0)	5 (12.5)	4 (30.77)
3–7	15 (28.3)	13 (32.5)	2 (15.38)
8–19	8 (15.1)	8 (20)	0 (0)
>19	9 (17.0)	6 (15)	3 (23.08)
Refused	2 (3.8)	0 (0)	2 (15.38)
Number of female sex partners in past 6 months
Median (range)	0 (0–15)	0 (0–15)	0 (0–15)	0.893
Mean (SD)	1.6 (3.3)	1.20 (2.68)	2.75 (4.77)
0	31 (58.5)	23 (57.5)	8 (61.54)	0.070
1	9 (17.0)	9 (22.5)	0 (0)
2+	12 (22.6)	8 (20)	4 (30.77)
Refused	1 (1.9)	0 (0)	1 (7.69)
Lifetime number of male sex partners
Median (range)	3.5 (0–500)	5.00 (0–500)	1.00 (0–100)	0.415
Mean (SD)	32.6 (83.7)	38.31 (94.88)	15.46 (29.24)
0	16 (30.2)	11 (27.5)	5 (38.46)	0.884
1–2	6 (11.3)	4 (10)	2 (15.38)
3–7	8 (15.1)	7 (17.5)	1 (7.69)
8–19	7 (13.2)	5 (12.5)	2 (15.38)
>19	15 (28.3)	12 (30)	3 (23.08)
Refused	1 (1.9)	1 (2.5)	0 (0)
Number of male sex partners in past 6 months
Median (range)	1 (0–40)	0.50 (0–40)	2 (0–13)	0.293
Mean (SD)	2.6 (6.0)	2.58 (6.64)	2.69 (3.64)
0	25 (47.2)	20 (50)	5 (38.46)	0.328
1	9 (17.0)	8 (20)	1 (7.69)
2+	19 (35.8)	12 (30)	7 (53.85)

^*The p-value is calculated by ANOVA or Kruskal-Wallis Test for numerical covariates; and chi square test or Fisher's exact for categorical covariates, where appropriate.

Comparing HPV16 variant period prevalence between genital epithelium and anal canal (Table 2), there was no significant difference between the two sites (p=0.782). HPV16 A1 was the most prevalent at both sites, with the same prevalence at the anal canal and at the genital epithelium (86.8%) as summarized in Table 2. Variants A2 and B1 were detected exclusively in the anal canal, whereas variants A3, D2, D3, and A1/D3 were found only in the genital epithelium. In the context of sequential HPV16 A1 variant infections, among 32 men with an incident anal HPV16 A1 variant acquisition, 9 were excluded because they had concurrent acquisition on both genital epithelium and anal canal sites. A total of 16 had no prior genital HPV16 A1 variant infections, and 7 (30.4%) had prior genital HPV16 variant A1 infection (Supplementary Fig 1 A). Similarly, among 26 men with incident genital HPV16 A1 variant infections, 9 were excluded because they had concurrent acquisition on both genital epithelium and the anal canal. Therefore 12 had no prior anal infections, and 5 (29.4%) had prior anal HPV16 A1 infections (Supplementary Fig 1B).

Table 2:

Period prevalence of HPV 16 variants detected in the Genital Epithelium and Anal Canal.

HPV 16 variant	Genital Epithelium (GE)		Anal Canal (AC)
	Count	Period Prevalence (%)	Count	Period Prevalence (%)
A1	46	81.5	46	86.8
A2	0	0	1	1.9
A3	1	1.9	0	0
B1	0	0	1	1.9
C1	4	7.4	2	3.8
C2	0	0	1	1.9
D2	1	1.9	0	0
D3	2	3.7	0	0
A1/A2	2	3.7	2	3.8
A1/D3	1	1.9	0	0
TOTAL	57 *		53

^*There were 53 unique participants included. Three patients had different HPV16 variants at different visits, thus giving a total number 57. (One patient had A1, A3; one patient had A1, C1 and A1/A2; another patient had D3, A1/A2).

Discussion

This short report describes the period prevalence of HPV16 variants and their sequential acquisition from genital to anal, and vice-versa, among men. This is one of the first studies to focus on HPV variants in more than one anatomic site among men. The proportion of men who had HPV16 variants in both anatomic sites was relatively low; 53 of the 867 and 124 men were HPV16 positive in the genital epithelium and anal canal, respectively. HPV16 A1 variant was the most common in both anatomic sites, with the exact prevalence in both anatomic sites. However, the overall distribution of HPV16 variants A2, A3, B1, D2, D3 differed by anatomic site. HPV16 variant lineage/sublineage A1 is widely studied, and our data agree with global data showing HPV16 A1 as the most prevalent¹⁶. However, the biological properties that may give HPV16 variants a competitive advantage in establishing and maintaining infections in different epithelial tissues are yet to be determined. Previously, we reported a predominance of HPV16 A1 variant in the anal canal among the HIM study cohort, independent of other anatomic sites¹³. Non-A lineage variants were mostly observed among men from Brazil in both anatomic regions^{13, 14}. In our current report, sub analyses of HPV16 A1 period prevalence by demographic variables were not possible due to the small proportion of men with HPV16 variants on both the anal canal and genital epithelium. The few published studies on HPV16 variants in the anal canal, which included men living with HIV¹⁷ and those with histologically confirmed anal lesions¹⁸, also consistently report HPV16 A1 as the most common variant. In our current study, the high period prevalence of the HPV A1 variant in both anatomic sites may partly signify similarities in the transmission and replication efficiency of these variants regardless of the specific anatomic site. Given that ~60% of the 53 men reported having at least three-lifetime female and male sex partners, we attribute the high prevalence to risky sexual activity (Table 1). A notably higher proportion of those men having at least three-lifetime female sex partners were infected with HPV16 A1 variants (67.5% of 40 men) compared to those who were not infected with that variant (38.5% of 13 men). The number of female sex partners impacts our understanding of whether these HPV16 A1 detected are due to reinfection or persistent infections. Although we cannot fully answer this question without data from the partners, this sets a baseline to inform future studies that will focus on intra-person HPV diversity and risk of specific variants on carcinogenesis.

Of paramount importance to this short report, we observed proportions of about 30% of men who have genital HPV16 A1 variant infections after an anal infection and vice versa. Although our findings are descriptive, we set a baseline for investigating the role of HPV16 A1 variant infections in multiple anatomic sites of men. Previously, we reported sequential genital-to-oral infections of HPV genotypes in the HIM study and concluded that men with HPV at one site face an increased risk of subsequent genital or oral infections of the same type, underscoring the importance of HPV vaccination¹⁹. Our current report on sequential HPV16 variant infection generates hypotheses on the role of intra-host HPV16 A1 variant infections on multi-anatomic site neoplasia. To increase the sample size, we recommend future pooled multinational studies to investigate the factors associated with sequential genital to anal, and vice versa, HPV16 A1 variants infection. These data may inform early detection and personalized prevention of HPV-related cancers among men.

Some limitations to our study are worth noting. Despite starting with a relatively large sample of HPV16-infected men at either anatomic site, the total number of men with HPV16 variant infections at both anatomic sites was relatively small. Therefore, analyses of factors associated with sequential infections were not possible. However, the HIM cohort is one of the few with enough numbers to describe these data. We, therefore, provide a brief descriptive report to accompany previous publications and provide a different perspective on HPV variants.

In conclusion, the HPV16 A1 variant had the highest period prevalence among men with HPV16 infection at the anal canal and genital epithelium. The proportion of sequential HPV16 A1 infections in both anatomical sites was comparable (29.4% vs. 30.4%), indicating that contracting an HPV16 A1 infection in either the anal or genital site is likely reciprocal, regardless of which site is initially infected.

Novelty Impact Statement.

This study reveals notable HPV16 A1 sequential acquisition rates between anal and genital sites. The findings highlight potential variant-specific transmission dynamics, informing HPV prevention strategies.

List of abbreviations

DNA

Deoxyribonucleic acid

HIM

HPV Infection in Men (Study)

HPV

Human papillomavirus

IQR

Interquartile range

NCI

National Cancer Institute

NIH

National Institutes of Health

SNPs

Single nucleotide polymorphisms

USA

United States of America

Data Availability Statement

All relevant data to this manuscript are included. Further details and other data that support the findings of this study are available from the corresponding authors upon request.