HPV Infection of the Oropharyngeal, Genital and Anal Mucosa and Associated Dysplasia in People Living with HIV

Background: The main objectives were to describe the prevalence of HPV, its genotypes and HPV-associated dysplastic lesions in the oropharyngeal mucosa of PLHIV and related factors. Material and Methods: This cross-sectional prospective study consecutively enrolled PLHIV attending our specialist outpatient units. At visit, HIV-related clinical and analytical variables were gathered, and oropharyngeal mucosa exudates were taken to detect HPV and other STIs by polymerase chain reaction. Samples were also taken from the anal canal of all participants and from the genital mucosa of the women for HPV detection/genotyping and cytological study. Results: The 300 participants had a mean age of 45.1 years; 78.7% were MSM and 21.3% women; 25.3% had a history of AIDS; 99.7% were taking ART; and 27.3% had received an HPV vaccine. HPV infection prevalence in the oropharynx was 13%, with genotype 16 being the most frequent (2.3%), and none had dysplasia. Simultaneous infection with Treponema pallidum (HR: 4.02 (95% CI: 1.06–15.24)) and a history of anal HSIL or SCCA (HR: 21.52 (95% CI: 1.59–291.6)) were risk factors for oropharyngeal HPV infection, whereas ART duration (8.8 vs. 7.4 years) was a protective factor (HR: 0.989 (95% CI: 0.98–0.99)). Conclusions: The prevalence of HPV infection and dysplasia was low in the oropharyngeal mucosae. A higher exposure to ART was protective against oral HPV infection.


Introduction
The survival of people living with human immunodeficiency virus (HIV) (PLHIV) has improved since the introduction of antiretroviral therapy (ART) [1][2][3][4]. HIV infection has become a chronic disease associated with a persistent inflammatory status that increases the probability of complications not related to acquired immunodeficiency syndrome (AIDS), such as non-AIDS defining neoplasms or cardiovascular disease [5][6][7]. Anal squamous cell carcinoma (ASCC) is one of the most frequent non-AIDS-defining neoplasms, with an incidence ranging from 85 × 100,000 p-y in MSM to 22 × 100,000 p-y in women [8]. ASCC is associated with a chronic infection caused by HPV high-risk genotypes, whose prevalence is around 50% in the anal mucosa of women living with HIV (WLHIV) and 74% in men who have sex with men living with HIV (MSMLHIV) [9]. The most frequent AIDS-defining neoplasm in women is HPV-associated cervical carcinoma. Notably, 63.8% of women with this disease in Southern Africa and 27.4% of those in Eastern Africa are HIVpositive, with 9200 and 14,000 new cases per year, respectively [10]. Besides persistent HIV infection, risk factors for the development of cervical, anal and head and neck squamous cell cancers include immunosuppression [11], tobacco [12] and sexual behavior, among others [13]. The risk of oropharyngeal mucosal HPV infection and its complications is greater among PLHIV than in the general population [14,15], but this relationship has been little studied in our country. The study objectives were to determine the prevalence of oropharyngeal mucosal HPV infection, the genotypes involved, infection-related factors and the prevalence of dysplasia in a cohort of PLHIV recruited from participants in a program of anal and cervical lesion screening, diagnosis, treatment and prophylaxis and to compare these data with the prevalence of HPV infection and dysplasia in anal and cervical mucosa.

Materials and Methods
This prospective cross-sectional study consecutively enrolled 300 persons living with confirmed HIV attended by infectious disease units in the "Hospital Universitario Virgen de las Nieves" (HUVN) and "Hospital Universitario San Cecilio" of Granada and "Complejo Hospitalario" of Jaen and included in a screening program for HPV infection-related anal and cervical dysplasia lesions.
Study inclusion criteria were age ≥18 years and participation in the anal and/or genital cancer screening program of the HUVN. The exclusion criteria included being a pregnant WLHIV.
During the first visit, data were gathered on sex, age and nationality; number of different sexual partners over past 12 months (NP12m); total number of sexual partners since onset of sexual relations (NPT); time in months since the onset of sexual relations; utilization of condoms and percentage utilization (during oral, anal and genital sex); employment situation (active or retired); educational level (illiterate, primary, secondary or university studies); smoker or ex-smoker (packs/year); alcohol consumption (standard drink units (SDUs)); intravenous drug user (IDU) or ex-IDU; HIV acquisition mechanism (MSM, heterosexual or IDU); HPV vaccination status (completed vaccination regimen or not, time since last dose and type of vaccine); months with HIV diagnosis; CDC-classified HIV stage; on-ART or ART-naïve patient; duration of ART (in months); number of ART regimen before first visit; current ART line and months under treatment with this line; virological failure; polypharmacy; presence/history of other infections (chronic liver disease or infection by hepatitis B virus (HBV), chronic liver disease or infection by hepatitis C virus (HCV), and syphilis); HBV vaccination status; presence and history of other sexually transmitted infections (STIs) in anal, female genital and oropharyngeal mucosae; presence and history of condylomas in any localization and their treatment; and history of anal, cervical/vaginal/vulvar and/or oropharyngeal dysplasia and its degree.
Analytical variables considered were nadir CD4+ T cell count, CD4 and CD8 lymphocyte counts and viral load at diagnosis and at baseline. At the same visit, cotton swabs impregnated with physiological saline were used to take anal canal and female genital samples for HPV detection and genotyping using the polymerase chain reaction (PCR) technique (GeneAmp PCR System 9700, Applied Biosystems, Roche, Foster City, CA, USA) and the ThinPrep Pap Test (Thin Prep Processor 2000, Hologic Corp., Marlborough, MA, USA) for cytology. Both samples were analyzed in the hospital pathology laboratory by the same pathologist who validated the HPV PCR results and cytology study. A cotton swab was introduced into the oral cavity and rotated over the internal and external surfaces of the upper and lower lips, the gingival region and the anterior and posterior surfaces of the tongue and the oropharynx; it was then placed in an individual vial with physiological solution. Oropharyngeal mucosa samples were sent to the Microbiology Department for DNA extraction and PCR study of HPV and other STIs (N. gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis).
The cytological study used the Bethesda classification to categorize lesions as lowgrade (LSIL) or high-grade (HSIL) anal squamous intraepithelial lesions (ASILs), atypical squamous cells of uncertain significance (ASCUS) or uncertain lesions whose high grade cannot be ruled out (ASCUS-H). The Reagan classification was used to categorize cases of ASCUS, LSIL or HSIL as dysplasia, with ASCUS and LSIL being low grade and HSIL high grade [16].
After rectal inspection and digital anorectal examination, all participants underwent high-resolution anoscopy (HRA) with a Carl Zeiss 150 fc © colposcope (Carl Zeiss, Oberkochen, Germany). In brief, 5 mL acetic acid was instilled through a transparent disposable anoscope and left in place for around 3 min before its removal for examination of the mucosa; 5% Lugol's iodine was then instilled for 1 min before repetition of the anoscopic examination. Samples were selected from quadrants of apparently normal mucosa and from areas with Lugol-negative aceto-white lesions. The biopsies were performed with an endoscopic retrograde cholangiopancreatography (ERCP) catheter.
Women with abnormal cervical cytology or genital HPV infection were referred to the Gynecology Department for evaluation.
All participants underwent oropharyngeal mucosa inspection before the sampling for HPV PCR, and those with a positive oropharyngeal HPV PCR and/or the presence of symptoms (e.g., chronic voice changes(hoarseness)) and/or visible lesions in the oropharyngeal mucosa were referred to the Otorhinolaryngology Department for examination.

Definition of Variables
The category HSIL-plus includes high-grade lesions (HSILs) and invasive ASCC.

Statistical Analysis
The sample size was calculated using Ene 2.0 statistical software. It was estimated that at least 123 PLHIV were needed to expect a frequency of 25% of oropharyngeal high-risk HPV prevalence in PLHIV, considering previously published global data, with a precision of 5% and a confidence interval of 95% [19].
Descriptive analysis: The means, standard deviations, medians and percentiles were calculated for quantitative variables and the absolute and relative frequencies for qualitative variables. The prevalence of HPV infection was calculated with 95% CI.
A bivariate analysis was conducted on the relationship between possible risk factors for HPV infection and the presence of dysplasia in oropharyngeal mucosa. The Kolmogorov-Smirnov test was used to check the normality of variable distribution. Student's t-test for independent samples was used for quantitative variables when normally distributed; the Mann-Whitney U test was used when non-normally distributed; and the Wilcoxon test was applied for related quantitative variables. Qualitative variables were analyzed with Pearson's chi-square test or, when application criteria were not met, Fisher's test. Finally, a multivariate logistic regression analysis was performed using a stepwise procedure, entering variables found to be significant in bivariate analyses and other factors considered relevant in the literature (sex, age, number of sexual partners throughout life and over previous 12 months, smoking habit, ART experience, STIs in oral mucosa, age at onset of sexual relations, utilization of condom in oral sex, time since HIV diagnosis and receipt of HPV vaccine). SPSS 21.0 (IBM SPSS, Armonk, NY, USA) was used for data analyses, and the level of significance was 0.05 in all tests.

Ethics Approval and Consent to Participate
All participants gave their informed consent to participate in the study, which was approved by the Research Ethics Committee of the hospital (Ref: OROHPV, 0098-N-22).
All data were treated in compliance with national data protection legislation (Organic Law 3/2018, 5 December).

Epidemiological Characteristics
The study included 300 PLHIV (78.7% male) with a mean age of 45.1 years; 87% held Spanish nationality. They had a median of 1 sexual partner over the previous 12 months and a median of 20.5 sexual partners since the onset of sexual relations; 2.7% used condoms during oral sex, 34.3% during vaginal sex and 34.7% during anal sex; and 35.9% of participants were active smokers. HPV vaccination had been received by 31% (tetravalent vaccine by 9% and nonavalent vaccine by 22.7%), and 27.3% had completed the vaccine regimen; 1.7% had chronic HBV infection and 6.3% chronic HCV infection; and 64.3% were vaccinated against HBV. At the first visit (baseline), 5% had active syphilis and 4% anogenital condylomas/warts. Table 1 lists the remaining epidemiological characteristics of the cohort.

History of Oral, Genital and Anal Mucosa Dysplasia
One MSM-LHIV (0.3%) had had a history of HPV-associated oropharyngeal epidermoid carcinoma five years earlier; 25% of women had a history of genital dysplasia (12.5% CIN1 and 18.8% CIN 2/3); none had a history of cervical squamous carcinoma, while 82% of PLHIV had a history of anal dysplasia (62.3% AIN1, 18% AIN2/3, and 1.7% ASCC), as shown in Table 3. Table 3. History of oral, genital and anal mucosa dysplasia.

Current HPV Infection and Oral Dysplasia
Among all participants, 39 (13.0%) had HPV infection in oropharyngeal mucosa, 29 (9.7%) with HR serotypes, 15 (5%) with LR serotypes and 5 (1.7%) with simultaneous infection. The most frequently isolated genotypes in patients with oropharyngeal HPV were HR genotypes 16 (2.3%) and 68 (1.7%) and LR genotype 6 (1%). The examination of all participants by the otorhinolaryngologist detected no symptoms or visible lesions in the oropharyngeal area, and no cases of dysplasia were observed in the oropharyngeal mucosa. The presence of STIs other than HPV in the oropharyngeal area was observed in 22 (7.4%) participants: N. gonorrhoeae in 20 (90.9%) and Chlamydia trachomatis in 2 (9.1). Table 4 lists results for the other variables.
Cervical cytology was normal in 55 women (88.7%), ASCUS in 4 (1.3%) and LSIL in 3 (1.0%). Gynecological evaluation detected CIN1 in one woman (0.7%). Table S1 lists results for the other variables.  Table S2 displays the results for the other variables.

Discussion
HPV infection of the oropharyngeal mucosa was infrequent in this cohort of PLHIV and less prevalent than anal or cervical HPV infections. Genotype 16 was the most frequently isolated genotype (2.3%), habitually as a monoinfection with no associated dysplasia. The most frequent genotypes in female genital mucosa were LR, including genotype 44/55, while the most frequent in the anus were HR-HPV, with a predominance of genotypes 16 and 68. A South American study of eight PLHIV with HPV-associated oropharyngeal lesions found that patients had multiple HPV infections, largely involving HR genotypes 16, 52 and 56 [20]. In a European study, 69% of a series of MSM living with HIV had at least one of the seven HR genotypes studied (16,18,31,33,45,52,58) in anal, oral and penile mucosae [21].
Among the mucosae examined in the present investigation, the highest prevalence and degree of dysplasia was observed in anal mucosa samples, with almost 30% diagnosed by HRA with LSIL/AIN1 and 1.7% with HSIL. No participant presented oropharyngeal dysplasia, and only 1.7% of the women had CIN1. The risk of HPV-related anal cancer is higher in PLHIV than in the general population [22]. Its greater frequency in comparison to oropharyngeal-laryngeal squamous cancer may be attributable to the higher incidence and longer persistence of this infection in the anal canal [23]. This is possibly due to the protective effect of metalloproteinases (MMP-8) in saliva against HPV infection [24] In contrast, the mucosa of the anus does not present any lubricating or protective substance. The common risk factors and acquisition mechanisms of oropharyngeal HPV infection were a lifetime history of more than six sex partners, tobacco consumption, age >50 and oral sex, among others [25].
In the present study, the prevalence of HPV was up to 6-fold higher in anal versus oral mucosae. A recent study of 103 MSM-LHIV described a prevalence of oral HPV infection of 14% and no associated dysplastic lesions, similar to the present findings, with an anal HPV infection prevalence of 88.3% and an HSIL prevalence of 24.3% [26].
In this cohort of PLHIV, concomitant infection by Treponema pallidum and a history of HSIL-plus in the anal canal emerged as risk factors for oropharyngeal HPV infection. Simultaneous infection with HPV and other STIs is frequent in PLHIV because they share common risk factors and acquisition mechanisms [27]. Studies in the general population have associated the prevalence of oropharyngeal mucosal HPV with male sex, older age, more sexual partners since starting sexual relations and an active smoking habit [28]. Another study of 170 MSM (including 72 PLHIV) found risk factors for oral HPV infection to be the commencement of oral sexual relations at the age of 18 years and a lifetime experience of more than 50 receptive oral sex partners [29].
A longer time with ART proved to be a protective factor against oropharyngeal HPV infection in the present study, in line with previous findings of a lower oral mucosal infection rate after 12 months of ART [30]. This is consistent with reports associating HPV infection in oropharyngeal [23], anal [31] and cervical [32] mucosae of PLHIV with low nadir CD4 [31] and current CD4+ T cell count [32]. Hence, ART appears to protect against HPV infection at all of these mucosal sites.
The main limitations of this study are derived from its design and the specific population, hampering the extrapolation of data to other types of populations. Its strengths include the largest sample size to date for this type of study, the methodology and systematic analysis applied and its novelty: it is one of the few prospective investigations of the characteristics of HPV infection in the oropharyngeal mucosa of PLHIV.

Conclusions
HPV infection has a low prevalence in the oropharyngeal mucosa of PLHIV in our setting, with a predominance of high-risk genotypes; it is not associated with dysplasia. Simultaneous infection by Treponema pallidum and a history of anal cancer precursor lesions or ASCC emerged as risk factors for this infection, while greater exposure to ART was a protective factor.   Informed Consent Statement: Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement:
The authors declare that the database will be made available on request.