Genotypes and Risk Factors for Anal Cytological Abnormalities in People Living With HIV and High‐Risk Human Papillomavirus: The GAIA 2 Study Cytological Abnormalities in People Living With HIV and High‐Risk Human Papillomavirus

ABSTRACT

Background

Human papillomavirus (HPV) infection is a major risk factor for cervical, anal, and oropharyngeal cancers, particularly in people living with HIV (PWH), who face higher HPV prevalence, especially in men who have sex with men (MSM). Limited data on HPV in Mexican PWH highlight the need for targeted screening.

Objective

To assess the prevalence of anal cytological abnormalities in PWH and their association with high‐risk HPV genotypes.

Methods

A cross‐sectional study (2023–2025) at Hospital de Infectología ‘La Raza’, Mexico City, included 137 PWH with confirmed high‐risk HPV. Anal cytology and PCR based HPV genotyping for 28 genotypes were conducted. Multivariate logistic regression adjusted for confounders, including vaccination status.

Results

Multiple high‐risk HPV genotypes (> 3) were linked to abnormal cytology (OR 3.094, 95% CI 1.297–7.383, p = 0.009) and ASC‐US (OR 3.060, 95% CI 1.177–7.954, p = 0.018). HPV 16+59 increased ASC‐US risk (OR 5.450, 95% CI 1.259–23.600, p = 0.032). Vaccination reduced risks for abnormal cytology (OR 0.331, 95% CI 0.116–0.948, p = 0.039) and ASC‐US (OR 0.209, 95% CI 0.053–0.823, p = 0.025).

Conclusion

High‐risk HPV genotypes 16 and 59 and multiple HPV infections are risk factors for anal cytological abnormalities in PWH. Vaccination is protective, supporting enhanced screening and vaccination strategies to reduce HPV‐related cancer risks.

Trial Registration: ClinicalTrials.gov identifier: R‐2025‐3502‐052

Prevalence of abnormal anal cytology in young MSM living with HIV: 21.1%. Key associations: HPV 16 (OR 2.71), HPV 59 (OR 2.80), > 3 high‐risk HPV genotypes (OR 3.09).

In a study of people living with HIV (Hispanic, median age 30), abnormal anal cytology prevalence was 21.1% (mainly ASC‐US). Key associations with abnormalities included HPV 16 (OR 2.709), HPV 59 (OR 2.795), and > 3 high‐risk HPV genotypes (OR 3.094).

1. Background

Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections worldwide [1]. Persistent infection with high‐risk HPV types is associated with development of cervical, anal, and oropharyngeal cancers [2]. This association underscores the critical role of HPV in oncogenic processes, particularly in vulnerable populations where coinfections exacerbate risks [3]. Late detection of HPV‐related malignancies increases mortality rates, diminishes quality of life, and imposes substantial economic burdens on healthcare systems [4]. Vaccination and early diagnostic screening are essential for mitigating these adverse outcomes [5].

Among people living with HIV (PWH), HPV prevalence is markedly elevated compared to the general population, influenced by impaired viral clearance, antiretroviral therapy, and behavioural risk factors [6]. This heightened susceptibility significantly increases the risk of HPV‐associated neoplasia across sub‐Saharan Africa and other regions with high HIV burdens [7]. Notably, men who have sex with men (MSM) living with HIV face up to a 60‐fold greater likelihood of developing anal cancer, particularly in older individuals and those with advanced immunosuppression [8]. Screening in younger MSM cohorts also reveals high rates of abnormalities, emphasizing the need for targeted interventions [9]. Furthermore, as life expectancy improves due to effective HIV management, non‐AIDS‐defining cancers (NADCs) have emerged as a leading cause of mortality, contributing significantly to overall deaths in long‐term follow‐up cohorts [10, 11].

In high‐resource settings, screening for high‐risk HPV genotypes in PWH is often augmented by high‐resolution anoscopy (HRA) to identify precancerous lesions [12]. Consensus guidelines from international societies further endorse HRA as a key tool for anal cancer screening in high‐risk groups [13]. However, in Mexico, HRA availability is limited to select centres, necessitating alternative approaches such as PCR‐based HPV detection followed by anal cytology, with early resection for high‐grade lesions [14]. Despite the clinical importance of these findings, epidemiological studies on HPV prevalence in Mexican PWH remain limited, often involving small samples that underestimate cytological abnormalities [15, 16]. This gap highlights the urgent need for localized evidence to guide screening and prevention strategies in this high‐risk population. The objective of this study is to determine the prevalence of anal cytological abnormalities in PWH and to evaluate their correlation with the presence of high‐risk HPV genotypes, to identify risk factors associated with the development of anal intraepithelial lesions (AILs).

2. Materials and Methods

2.1. Design and Location

Clinical‐epidemiological, cross‐sectional, analytical study conducted at the HIV Clinic of the Hospital de Infectología National Medical Center ‘La Raza’, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico. The study period spanned from October 2023 to August 2024, with approval record R‐2025‐3502‐052.

2.2. Patients

The study population consisted of individuals aged ≥ 18 years living with HIV who had previously tested positive for high‐risk HPV genotypes in the anal region via polymerase chain reaction (PCR). Inclusion criteria were as follows: men living with HIV with prior detection of high‐risk HPV in the anal region by PCR and availability to take anal cytology. Exclusion criteria were incomplete information in the clinical record and loss of data during the study process. This distinction ensures that only eligible and complete cases were analysed, minimizing bias.

The sample size was calculated to estimate the prevalence of anal cytological abnormalities among PWH and high‐risk HPV. Using the formula for estimating proportions, with a 95% confidence level, a margin of error of 5%, and a statistical power of 80%, the initial sample size was determined to be 131 participants. To account for an anticipated 5% loss to follow‐up, the sample size was adjusted to 138 participants. Recruitment was conducted no probabilistically, enrolling eligible patients attending routine visits.

2.3. Measurements

Data collection for this study was conducted prospectively at the HIV Clinic of the Hospital de Infectología ‘La Raza’ National Medical Cener in Mexico City. Anal cytology samples were collected using a Dacron swab, inserted approximately 4–5 cm into the anal canal. The swab was gently rotated 360° five times to ensure adequate cellular sampling, then carefully removed and immediately smeared onto a glass slide. The slide was fixed using a cytofixative spray to preserve cellular integrity for subsequent microscopic evaluation. This standardized technique enhances the accuracy of cytological analysis by minimizing contamination and ensuring a representative sample of the anal epithelium. All participants provided written informed consent prior to sample collection, with the process overseen by the institutional ethics committee to ensure compliance with ethical standards.

The anal brushing samples for HPV detection were performed using the commercial Allplex HPV28 Detection kit (Seegene Inc., Seoul, South Korea), a multiplex real‐time PCR assay that detects 28 HPV genotypes: 19 high‐risk types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low‐risk types (6, 11, 40, 42, 43, 44, 54, 61, 70) [17]. The assay was conducted following the manufacturer's standardized protocols, incorporating positive and negative controls to ensure accuracy. Results were reported as positive or negative for each genotype and categorized into high‐risk and low‐risk HPV types.

Additional variables included

• Sociodemographic: Age (years at cytology collection); sex assigned at birth (male/female, though the sample focused on men); sexual orientation (heterosexual, HSH, bisexual, other), educational level, occupation, and socioeconomic status.

• HIV‐related parameters: Time since HIV diagnosis (years); baseline CD4+ cell count and HIV‐RNA viral load (measured via PCR), maximum historical HIV viral load (copies/mL); nadir CD4+ cells count (cells/μL), CD4+ cell count (cells/μL, categorized as < 200 or ≥ 200); HIV viral load (copies/mL, categorized as undetectable [< 50] or detectable [≥ 50]); ART regimen (integrase inhibitors, protease inhibitors, non‐nucleoside reverse transcriptase inhibitors); time on ART (months); history of opportunistic infections.

• Clinical and behavioural: smoking status (yes/no, pack‐years); alcohol consumption (yes/no, frequency); drug use (marijuana, amphetamines, intravenous drugs); number of sexual partners; condom use (always/sometimes/never); receptive anal sex (yes/no); age at sexual debut (years); history of sexually transmitted infections (syphilis, gonorrhoea, hepatitis B/C).

• HPV vaccination status: prior receipt of HPV vaccination (yes/no, type: 4vHPV, 9vHPV, 2vHPV); age at HPV vaccination (years).

• Comorbidities: diabetes, hypertension.

2.4. Statistical Analysis

Descriptive statistics were used to summarize variables: frequencies and percentages for categorical variables; medians with interquartile ranges or means with standard deviations for continuous variables, depending on data distribution assessed via Kolmogorov–Smirnov tests.

Anal cytological abnormalities were categorized as Negative for Anal Lesions (NILM), Atypical Squamous Cells of Undetermined Significance (ASCUS), Low‐Grade Squamous Intraepithelial Lesion (LSIL), Atypical Squamous Cells, Cannot Exclude HSIL (ASC‐H), and High‐Grade Squamous Intraepithelial Lesion (HSIL). Associations between variables and cytological abnormalities were evaluated using chi‐square (χ ²) or Fisher's exact tests, as appropriate. Bivariate analyses were performed to identify factors associated with abnormalities, expressed as odds ratios (OR) with 95% confidence intervals (CI). Variables with p < 0.05 in bivariate analysis were included in a multivariate logistic regression model to estimate adjusted ORs, controlling for confounders. All analyses were conducted using SPSS version 25 (IBM Corp., Armonk, NY, USA).

2.5. Ethical Considerations

The study was approved by the Institutional Review Board of the National Medical Center ‘La Raza’, Health Research Committee 3502 (protocol number: R‐2025‐3502‐052). All procedures complied with the ethical principles outlined in the Declaration of Helsinki. Participants' confidentiality was ensured through anonymized data and secure storage of personal information.

3. Results

3.1. Baseline Characteristics

The study consisted of 137 participants living with HIV, with a median age of 30 years (IQR: 24–36.5). Participants were predominantly of Mexican mestizo ethnicity (self‐reported as Hispanic/Latino of Mexican origin), reflecting the demographic composition of urban Mexico where mestizo heritage is most common (87.6%), with educational attainment distributed as elementary school (14.6%), high school (39.5%), college (34.3%), and postgraduate (11.7%). Clinically, the median time since HIV diagnosis was 37 months (IQR: 26–56), with a median CD4+ T‐cell count of 628 cells/mm³ (IQR: 429–876) and nadir CD4+ categorized as < 200 (4.4%), 200–500 (38%), and > 500 (57.7%). Virologic suppression (HIV‐1 RNA < 40 copies/mL) was achieved in 88.3% of participants. Antiretroviral therapy (ART) had a median duration of 20 months (IQR: 12–30), with 99.2% on their first regimen and 0.8% on optimized regimens due to prior failure. ART regimens included mostly DTG/3TC/ABC (19.7%) and BIC/TAF/FTC (29.9%), grouped pharmacologically as INSTI‐based (58.4%), PI‐based (38.7%), and NNRTI‐based (2.9%). Behaviourally, the median age at first sexual intercourse was 17 years (IQR: 15–19) and at first receptive anal intercourse 18 years (IQR: 16–21). Annual sexual partners were reported as < 1 (10.2%), 1–4 (56.2%), 5–9 (15.3%), 10–19 (8.0%), ≥ 20 (7.3%), and unknown (2.9%). Consistent condom use was reported by 85.4%, smoking by 25.5%, and alcohol consumption in the past 12 months by 81.8%. HPV vaccination coverage was 26.3%, with 13.1% receiving 1 dose, 6.6% 2 doses, and 6.6% 3 doses, before sampling (Table 1).

TABLE 1.

Baseline characteristics (n = 137).

Characteristics	n	%	Median (IQR)
Demographic characteristics
Age (years)	—	—	30 (24–36.5)
Race/Ethnicity
Hispanic	120	87.6	—
White	13	9.5	—
Other	4	3	—
Education
Elementary school	20	14.6	—
High school	54	39.5	—
College	47	34.3	—
Postgraduate	16	11.7	—
Clinical characteristics
Time since HIV diagnosis (months)	—	—	37 (26–56)
CD4+ T‐cell count (cells/mm3)	—	—	628 (429–876)
Nadir CD4+ T‐Cell Count (cells/mm3)
< 200	6	4.4	—
200–500	52	38	—
> 500	79	57.7	—
HIV‐1 RNA < 40 copies/mL	121	88.3	—
ART duration (months)	—	—	20 (12–30)
Treatment History
First treatment regimen	136	99.2	—
Optimized regimen due to previous treatment failure	1	0.8	—
ART regimen
DTG/3TC/ABC	27	19.7	—
BIC/TAF/FTC	41	29.9	—
DRV/c + TDF/FTC	37	27.0	—
DTG + TDF/FTC	9	6.5	—
DRV/c + 3TC	16	11.7	—
DOR/TDF/3TC	4	2.9	—
DTG/3TC	2	1.5	—
DRV/c + DTG + TDF/FTC	1	0.7	—
ART Regimen by Pharmacological Group
INSTI‐based	80	58.4	—
PI‐based	53	38.7	—
NNRTI‐based	4	2.9	—
Behavioural characteristics
Age at first sexual intercourse (years)	—	—	17 (15–19)
Age at first receptive anal intercourse (years)	—	—	18 (16–21)
Sexual partners per year
0	14	10.2	—
1–4	77	56.2	—
5–9	21	15.3	—
10–19	11	8.0	—
≥ 20	10	7.3	—
Unknown	4	2.9	—
Consistent condom use	117	85.4	—
Smoking	35	25.5	—
Alcohol consumption (past 12 months)	112	81.8	—
HPV vaccination
Not vaccinated	101	73.7	—
1 Dose	18	13.1	—
2 Doses	9	6.6	—
3 Doses	9	6.6	—

Abbreviations: 3TC, lamivudine; ABC, abacavir; ART, antiretroviral therapy; BIC, bictegravir; DOR, doravirine; DRV/c, darunavir/cobicistat; DTG, dolutegravir; FTC, emtricitabine; HIV, human immunodeficiency virus; HPV, human papillomavirus; INSTI, integrase strand transfer inhibitor; NNTRI, non‐nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate.

3.2. Prevalence of Abnormal Cytology and Associated Factors With Abnormal Anal Cytology in PWH

The prevalence of abnormal anal cytology was 29/137 (21.1%, 95% CI 15.1–28.7). Of these, 24/137 (17.5%, 95% CI 12.0–24.7) were ASC‐US, 4/137 (2.9%, 95% CI 1.1–7.2) were LSIL, and 1/137 (0.7%, 95% CI 0.01–4.0) was HSIL. In the bivariate analysis of risk factors for anal cytological abnormalities, undetectable HIV‐1 RNA viral load was significantly associated with a reduced likelihood of abnormal cytology (OR = 0.302, 95% CI: 0.102–0.895, p = 0.025) and ASC‐US (OR = 0.210, 95% CI: 0.069–0.640, p = 0.003), indicating a protective effect. Conversely, alcohol consumption in the past 12 months was linked to an increased risk of abnormal cytology (OR = 8.386, 95% CI: 1.085–64.785, p = 0.015). Smoking showed a borderline association with decreased ASC‐US (OR = 0.220, 95% CI: 0.049–0.991, p = 0.039), but not with overall abnormal cytology (p = 0.100). Additionally, the use of INSTI based ART for ≥ 12 months was associated with a reduced risk of abnormal cytology (OR = 0.437, 95% CI: 0.189–0.971, p = 0.049), though this did not reach statistical significance (Figure 1).

FIGURE 1.

Association between high‐risk HPV genotypes and anal cytological outcomes.

Other factors, including age > 35 years, consistent condom use, CD4 counts < 200 or < 500 cells/μL, vaccination status (overall or fewer doses), and ART classes (INSTI, PI, NNRTI), did not reach statistical significance for abnormal cytology (all p > 0.05) (Table 2).

TABLE 2.

Associated factors with abnormal anal cytology in PWH.

Risk factor	Cytology outcome	OR	95% CI	p
> 35 Years	Abnormal Cytology	0.436	0.164‐1.158	0.090
> 35 Years	ASC‐US	0.480	0.167‐1.383	0.232
> 35 Years	LSIL	0.674	0.068‐6.670	1.000
Smoking	Abnormal Cytology	0.377	0.122‐1.171	0.100
Smoking	ASC‐US	0.220	0.049‐0.991	0.039
Smoking	LSIL	0.971	0.098‐9.647	1.000
Consistent condom use	Abnormal Cytology	1.700	0.463‐6.241	0.564
Consistent condom use	ASC‐US	1.240	0.333‐4.617	1.000
Consistent condom use	LSIL	1.035	1.001‐1.071	1.000
CD4 > 200	Abnormal Cytology	0.404	0.064‐2.536	0.301
CD4 > 200	ASC‐US	0.300	0.047‐1.902	0.211
CD4 > 200	LSIL	0.093	0.008‐1.102	0.140
CD4 > 500	Abnormal Cytology	1.147	0.488‐2.697	0.753
CD4 > 500	ASC‐US	1.437	0.550‐3.750	0.458
CD4 > 500	LSIL	0.176	0.018‐1.743	0.131
HIV‐1 RNA undetectable	Abnormal Cytology	0.302	0.102‐0.895	0.025
HIV‐1 RNA undetectable	ASC‐US	0.210	0.069‐0.640	0.003
HIV‐1 RNA undetectable	LSIL	1.034	1.001‐1.069	1.000
HPV vaccinated	Abnormal Cytology	1.899	0.798‐4.520	0.143
HPV vaccinated	ASC‐US	1.518	0.587‐3.925	0.387
HPV vaccinated	LSIL	0.933	0.094‐9.271	1.000
1 Dose of HPV vaccine	Abnormal Cytology	1.979	0.674‐5.813	0.208
1 Dose of HPV vaccine	ASC‐US	1.414	0.421‐4.746	0.521
1 Dose of HPV vaccine	LSIL	2.275	0.224‐23.138	0.435
2 Doses of HPV vaccine	Abnormal Cytology	0.427	0.051‐3.554	0.683
2 Doses of HPV vaccine	ASC‐US	0.813	0.748‐0.883	0.360
2 Doses of HPV vaccine	LSIL	0.969	0.939‐0.999	1.000
3 Doses of HPV vaccine	Abnormal Cytology	3.138	0.787‐12.519	0.105
3 Doses of HPV vaccine	ASC‐US	4.320	1.067‐17.495	0.050
3 Doses of HPV vaccine	LSIL	0.969	0.939‐0.999	1.000
Alcohol (past 12 months)	Abnormal Cytology	8.386	1.085‐64.785	0.015
Alcohol (past 12 months)	ASC‐US	6.202	0.797‐48.287	0.077
Alcohol (past 12 months)	LSIL	0.661	0.066‐6.628	0.558
INSTI‐based ART	Abnormal Cytology	0.646	0.286‐1.458	0.291
INSTI‐based ART	ASC‐US	0.811	0.334‐1.969	0.644
INSTI‐based ART	LSIL	0.705	0.096‐5.159	1.000
PI‐based ART	Abnormal Cytology	1.526	0.673‐3.458	0.310
PI‐based ART	ASC‐US	1.163	0.475‐2.848	0.741
PI‐based ART	LSIL	1.608	0.220‐11.773	0.640
NNRTI‐based ART	Abnormal Cytology	1.195	0.120‐11.927	1.000
NNRTI‐based ART	ASC‐US	1.594	0.159‐16.018	0.542
NNRTI‐based ART	LSIL	0.970	0.941‐0.999	1.000
INSTI‐based ART > 12 months	Abnormal cytology	0.437	0.189‐0.971	0.049
INSTI‐based ART > 12 months	ASC‐US	0.588	0.241‐1.434	0.240
INSTI‐based ART > 12 months	LSIL	0.900	0.123‐6.579	1.000

Abbreviations: ASC‐US, atypical squamous cells of undetermined significance; HPV, human papillomavirus; INSTI, integrase strand transfer inhibitor; LSIL, low‐grade squamous intraepithelial lesion; NNTRI, non‐nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; PWH, people with HIV.

3.3. Association Between HR‐HPV and Abnormal Anal Cytology in PWH

In the bivariate analysis, significant associations were observed between specific high‐risk HPV genotypes and anal cytological outcomes. HPV 16 infection was associated with increased likelihood of abnormal cytology (OR = 2.382, 95% CI: 1.022–5.555, p = 0.041) and ASC‐US (OR = 2.569, 95% CI: 1.035–6.378, p = 0.038). Similarly, HPV 59 showed a strong association with abnormal cytology (OR = 3.527, 95% CI: 1.249–9.965, p = 0.013), and ASC‐US (OR = 3.818, 95% CI: 1.300–11.219, p = 0.010). No significant associations (p < 0.05) were found for LSIL across any genotypes, and other HPV types (e.g., 18, 26, 33, 35, 39, 45, 51, 52, 53, 56, 58, 66, 68, 69, 72, 82) did not reach statistical significance for any cytological category (Table 3, Figure 2).

TABLE 3.

Association between high‐risk HPV genotypes and anal cytological outcomes.

HPV genotype	Cytology outcome	OR	95% CI	p
16	Abnormal cytology	2.382	1.022–5.555	0.041
16	ASC‐US	2.569	1.035–6.378	0.038
16	LSIL	2.595	0.352–19.101	0.320
18	Abnormal cytology	1.158	0.416–3.220	0.779
18	ASC‐US	0.876	0.271–2.833	1.000
18	LSIL	1.514	0.151–15.189	0.558
26	Abnormal cytology	0.213	0.154–0.294	0.219
26	ASC‐US	0.169	0.177–0.245	0.175
26	LSIL	0.971	0.943–0.999	1.000
31	Abnormal cytology	0.738	0.197–2.759	0.764
31	ASC‐US	0.800	0.732–0.875	0.043
31	LSIL	2.438	0.239–24.870	0.415
33	Abnormal cytology	1.940	0.608–6.189	0.319
33	ASC‐US	1.855	0.536–6.413	0.300
33	LSIL	2.833	0.276–29.119	0.375
35	Abnormal cytology	1.343	0.395–4.565	0.741
35	ASC‐US	1.202	0.312–4.636	0.727
35	LSIL	2.833	0.276–29.119	0.375
39	Abnormal cytology	0.870	0.297–2.552	1.000
39	ASC‐US	0.876	0.271–2.833	1.000
39	LSIL	1.514	0.151–15.189	0.558
45	Abnormal cytology	2.267	0.509–10.087	0.372
45	ASC‐US	0.145	3.086–13.909	0.145
45	LSIL	0.969	0.940–0.999	1.000
51	Abnormal cytology	1.024	0.371–2.822	0.964
51	ASC‐US	0.783	0.244–2.514	0.785
51	LSIL	0.964	0.929–0.999	0.585
52	Abnormal cytology	0.758	0.686–0.837	0.071
52	ASC‐US	0.806	0.740–0.879	0.124
52	LSIL	0.968	0.937–0.999	1.000
53	Abnormal cytology	2.656	0.928–7.599	0.061
53	ASC‐US	2.806	0.933–8.435	0.058
53	LSIL	2.275	0.224–23.138	0.435
56	Abnormal cytology	1.329	0.437–4.041	0.565
56	ASC‐US	1.307	0.392–4.352	0.745
56	LSIL	2.130	0.210–21.607	0.454
58	Abnormal cytology	1.324	0.471–3.722	0.594
58	ASC‐US	0.989	0.304–3.224	1.000
58	LSIL	5.333	0.711–39.989	0.131
59	Abnormal cytology	3.527	1.249–9.965	0.013
59	ASC‐US	3.818	1.300–11.219	0.010
59	LSIL	2.275	0.224–23.138	0.435
66	Abnormal cytology	1.533	0.538–4.373	0.422
66	ASC‐US	2.178	0.746–6.361	0.148
66	LSIL	1.883	0.186–19.022	0.490
68	Abnormal cytology	0.273	0.034–2.189	0.297
68	ASC‐US	0.366	0.045–2.958	0.465
68	LSIL	3.361	0.324–34.879	0.332
69	Abnormal cytology	2.267	0.509–10.087	0.372
69	ASC‐US	1.621	0.307–8.567	0.629
69	LSIL	0.969	0.940–0.999	1.000
72	Abnormal cytology	1.210	0.306–4.782	0.725
72	ASC‐US	0.403	0.050–3.281	0.692
72	LSIL	3.697	0.354–38.601	0.310
82	Abnormal cytology	3.655	0.222–60.233	0.391
82	ASC‐US	4.870	0.294–80.712	0.321
82	LSIL	0.970	0.942–0.999	1.000

Abbreviations: ASC‐US, atypical squamous cells of undetermined significance; HPV, human papillomavirus; LSIL: low‐grade squamous intraepithelial lesion.

FIGURE 2.

Photographs of cytological abnormalities. (a) ASC‐US: Slightly increased nuclear‐to‐cytoplasmic (N/C) ratio, moderate nuclear enlargement (> 2× normal intermediate cell nucleus size), mild hyperchromasia, irregular chromatin distribution, and slightly irregular nuclear contours. Associated dense orangeophilic cytoplasm (atypical parakeratosis). (b) LSIL: Marked nuclear enlargement (> 3× the area of normal intermediate nuclei), mildly increased N/C ratio, variable hyperchromasia (may be normochromatic), coarse/granular or densely opaque chromatin, irregular nuclear contours (ranging from smooth to markedly notched), frequent binucleation and multinucleation. Perinuclear halos (koilocytosis) and other human papillomavirus (HPV)‐related changes are commonly present. (c) HSIL: High N/C ratio, variable but often substantial nuclear enlargement (≈3× or greater), pronounced hyperchromasia, evenly distributed coarse/granular chromatin, irregular nuclear membranes with prominent indentations and grooves, and inconspicuous or absent nucleoli. Cells appear singly, in sheets, or in syncytial‐like crowded/hyperchromatic groups (All images captured at 40× magnification using Papanicolaou stain). ASC‐US, atypical squamous cells of undetermined significance; HPV, Human papillomavirus; HSIL, high‐grade squamous intraepithelial lesion; LSIL, low‐grade squamous intraepithelial lesion. Photographs courtesy of Dr. Adrián Reyes Ayuso.

3.4. Association Between Combinations of HR‐HPV and Abnormal Anal Cytology in PWH

The most relevant risk factors for abnormal cytology associated with combinations of HPV genotypes were:

• HPV 16+59: Notable risk is seen with ASC‐US (OR 5.450, IC 95% 1.259–23.600, p = 0.032), suggesting a statistically significant association with abnormal cytology in this subgroup.

• > 3 HPV high‐risk genotypes: This shows the strongest and most significant association with abnormal cytology (OR 3.094, IC 95% 1.297–7.383, p = 0.009), particularly with ASC‐US (OR 3.060, p = 0.018), highlighting a clear risk factor when multiple high‐risk HPV types are present.

These findings suggest that the presence of multiple high‐risk HPV genotypes, especially exceeding three, is a critical factor in increasing the likelihood of abnormal cytology (Table 4).

TABLE 4.

Associated factors for HR HPV combination with abnormal anal cytology in PWH.

Risk factor	Cytology outcome	OR	95% CI	p
HPV 16+18	Abnormal Cytology	1.870	0.439–7.790	0.410
HPV 16+18	ASC‐US	1.377	0.268–1.383	0.657
HPV 16+18	LSIL	5.208	0.485–55.903	0.240
HPV 16+59	Abnormal Cytology	2.962	0.928–16.908	0.069
HPV 16+59	ASC‐US	5.450	1.259–23.600	0.032
HPV 16+59	LSIL	0.969	0.940–1.999	1.000
≥ 2 HR HPV	Abnormal Cytology	1.558	0.580–4.190	0.377
≥ 2 HR HPV	ASC‐US	1.437	0.494–4.181	0.615
≥ 2 HR HPV	LSIL	1.041	0.101–1.083	0.573
≥ 3 HR HPV	Abnormal Cytology	3.094	1.297–7.383	0.009
≥ 3 HR HPV	ASC‐US	3.060	1.177–31.894	0.018
≥ 3 HR HPV	LSIL	3.234	0.328–1.102	0.359

Abbreviations: ASC‐US: atypical squamous cells of undetermined significance; HPV: human papillomavirus; HR HPV: High‐risk human papilomavirus; LSIL: low‐grade squamous intraepithelial lesion.

3.5. Multivariate Analysis to Identify Association of Abnormal Anal Cytology and ASC‐US in PWH

The analysis of associated factors adjusted for abnormal anal cytology, ASC‐US, and LSIL in people living with HIV (PWH) reveals several key findings from Table 4. HPV vaccination emerged as a significant protective factor, with an odds ratio (OR) of 0.331 (95% CI 0.1160.948, p = 0.039) for abnormal cytology, indicating a reduced risk. For ASC‐US, the protective effect was even more pronounced, with an OR of 0.209 (95% CI 0.053–0.823, p = 0.025), while the association with LSIL showed a non‐significant trend (OR 0.554, 95% CI 0.052–5.917, p = 0.625). These results highlight the potential of HPV vaccination to mitigate cytological abnormalities in this population.

Regarding specific HPV genotypes, HPV 16 was strongly associated with abnormal cytology (OR 2.709, 95% CI 1.120–6.555, p = 0.027), with a notable trend for ASC‐US (OR 2.943, 95% CI 1.080–8.020, p = 0.035), though the link to LSIL was not significant (OR 2.930, 95% CI 0.356–24.153, p = 0.318). Similarly, HPV 59 showed a significant association with abnormal cytology (OR 2.795, 95% CI 1.001–7.902, p = 0.050), with a trend toward ASC‐US (OR 2.885, 95% CI 0.927–8.983, p = 0.067), but no significant association with LSIL (OR 0.816, 95% CI 0.076–8.714, p = 0.866). These findings underscore the oncogenic potential of HPV 16 and 59 in PWH (Table 5).

TABLE 5.

Associated factors adjusted for abnormal anal cytology, ASC‐US and LSIL in PWH (Multivariate analysis ^a ).

Risk factor	Cytology outcome	OR	95% CI	p
HPV vaccinated	Abnormal Cytology	0.331	0.116–0.948	0.039
HPV vaccinated	ASC‐US	0.209	0.053–0.823	0.025
HPV vaccinated	LSIL	0.554	0.052–5.917	0.625
HPV 16	Abnormal Cytology	2.709	1.120–6.555	0.027
HPV 16	ASC‐US	2.943	1.080–8.020	0.035
HPV 16	LSIL	2.930	0.356–24.153	0.318
HPV 59	Abnormal Cytology	2.795	1.001–7.902	0.050
HPV 59	ASC‐US	2.885	0.927–8983	0.067
HPV 59	LSIL	0.816	0.076–8.714	0.866

Abbreviations: ASC‐US: atypical squamous cells of undetermined significance; HPV: human papillomavirus; LSIL: low‐grade squamous intraepithelial lesion.

^a Logistic regression model.

4. Discussion

The GAIA2 study highlights the significant association between multiple HR‐HPV genotypes and anal cytological abnormalities in PWH already infected with HR‐HPV. Specifically, the presence of more than three HR‐HPV genotypes was strongly linked to abnormal cytology (OR 3.094, 95% CI 1.297–7.383, p = 0.009) and ASC‐US (OR 3.060, 95% CI 1.177–7.954, p = 0.018). Additionally, coinfection with HPV genotypes 16 and 59 demonstrated a notable risk for ASC‐US (OR 5.450, 95% CI 1.259–23.600, p = 0.032). These findings underscore the role of genotypic multiplicity and specific combinations in exacerbating cytological changes, providing crucial insights for targeted screening in this vulnerable population.

Secondary findings from the study include non‐significant associations for other combinations, such as HPV 16+18 with LSIL. Coinfection with more than two HR‐HPV genotypes showed no significant link to abnormal cytology, indicating a threshold effect where risks escalate beyond three genotypes. These observations also extend to normal cytology, where inverse associations were noted for multiple infections, reinforcing the protective or neutral role in non‐abnormal states.

The observed rates of anal cytological abnormalities in our cohort were relatively low overall (21.1%, 95% CI 14.6%–28.9%), with ASC‐US predominant (17.5%, 95% CI 11.7%–24.7%) compared to LSIL (2.9%, 95% CI 0.8%–7.3%) and HSIL (0.7%, 95% CI 0.01%–4.0%), resulting in a high ASC:SIL ratio (approximately 4.8:1). These rates are lower than those commonly reported in the literature for MSM living with HIV, which may be partly explained by the young median age of our participants (30 years, IQR 25–37). Younger age is associated with lower progression to high‐grade lesions, as persistent HPV infections often require time to develop into HSIL. For comparison, a 2024 study by Liu et al. evaluated 18–34‐year‐old MSM living with HIV (median age 28 years) and reported a higher abnormal cytology rate of 65%, with abnormalities increasing with age and a significantly elevated prevalence of AIN 3 (HSIL equivalent) in the 30–34 age group (up to 19% in stratified analyses) [9]. A key difference was their low HPV vaccination coverage (only 19% received at least one dose), contrasting with our findings of vaccination's protective effect (OR 0.331 for abnormal cytology). Additional factors in our study, such as high ART adherence (> 95% with undetectable viral loads) and early HPV detection via PCR, may have contributed to reduced high‐grade abnormalities. These comparisons highlight the importance of age‐ and vaccination‐tailored screening strategies in PWH.

The elevated risk associated with more than three HR‐HPV genotypes aligns with the understanding that polygenic infections amplify oncogenic potential through synergistic viral interactions and immune evasion in immunocompromised hosts. In our cohort of 137 PWH, this multiplicity likely contributes to persistent infection and cytological progression, consistent with international literature showing that multiple HPV types correlate with higher grades of anal intraepithelial neoplasia (AIN) in HIV‐infected individuals. For instance, a study in China reported high prevalence of multiple HPV infections linked to abnormal anal cytology in HIV‐positive men who have sex with men (MSM), with ORs indicating compounded risks [18]. Similarly, global analyses reveal that HIV coinfection exacerbates HPV persistence, leading to a twofold to fivefold increase in cytological abnormalities compared to HIV‐negative populations [19].

Specific genotype combinations, such as HPV 16+59, emerged as predictors of ASC‐US, reflecting the oncogenic synergy between well‐established (HPV16) and emerging (HPV59) HR types. HPV16 is a predominant driver of anal cancers worldwide, often associated with high‐grade lesions, while HPV59's role in anal pathology is less documented but increasingly recognized in HIV contexts. This contrasts with cervical studies where HPV16/18 dominate, but anal sites in PWH show broader genotypic diversity [20]. A French cohort identified HPV53 and 39 alongside 16 as key in cytological abnormalities [21], paralleling our findings and suggesting site‐specific viral tropisms. However, our non‐significant association for HPV16 + 18 with LSIL may indicate slower progression in anal versus cervical epithelia, as supported by meta‐analyses showing variable risks across anatomical sites [22].

The threshold effect observed for > 3 HR‐HPV genotypes highlights a dose–response relationship, where cumulative viral load overwhelms host defenses in PWH. This is corroborated by longitudinal data from HIV‐positive MSM, where incident high‐risk HPV infections predict cytological progression, with risks amplified by low CD4+ counts—though our study focused on genotypes, implying indirect immunosuppression effects [23]. In contrast, studies in sub‐Saharan Africa report even higher multiplicity rates (up to 4–5 types) linked to 60‐fold anal cancer risks in MSM [24], exceeding our ORs possibly due to regional differences in ART access and viral strains. Antiretroviral therapy (ART) may mitigate some risks, as evidenced by reduced AIN incidence in treated cohorts [25], yet our findings suggest persistent threats from genotypic diversity.

Broader epidemiological correlates in our Mexican cohort, including MSM predominance and behavioural factors, mirror global patterns where receptive anal intercourse and smoking heighten HPV‐related abnormalities [26]. Unlike high‐resource settings with routine high resolution anoscopy (HRA), our PCR‐cytology approach addresses resource limitations, aligning with recommendations for low‐middle income countries [27]. However, discrepancies with U.S. studies, where HPV16/18 vaccines reduce abnormalities by 50%–70% [28], emphasize the need for expanded vaccination covering types like 59 in PWH.

The importance of HPV vaccination is underscored by our multivariate analysis, where it served as a protective factor against abnormal cytology and ASC‐US, highlighting its potential to mitigate cytological abnormalities even in PWH with established HR‐HPV infections. Prophylactic HPV vaccines, including quadrivalent and nonavalent formulations, have demonstrated efficacy in preventing persistent anal HPV infections and precancerous lesions in HIV‐infected populations, despite potential immunocompromise [29]. Systematic reviews confirm the safety and immunogenicity of these vaccines in PWH, with reduced incidence of vaccine‐targeted HPV types and associated dysplasia [30]. Recent evidence further supports vaccination's role in reducing incident anal HPV infections among homosexual, bisexual, and other MSM living with HIV, advocating for increased access and uptake to prevent progression to anal cancer in high‐risk groups [31].

A key weakness of this study is its cross‐sectional design, which limits causal inferences regarding genotype combinations and cytological progression, potentially confounding temporal relationships. The sample size, while calculated for prevalence estimation, may lack power for subgroup analyses, as seen in wide CIs for LSIL associations. Reliance on cytology without confirmatory HRA or histology introduces misclassification bias, and the single centre setting in Mexico City restricts generalizability to rural or diverse ethnic groups.

Strengths include the prospective data collection from 2023 to 2025, ensuring standardized PCR detection of 28 HPV genotypes, which provides granular insights into understudied combinations like 16+59. The focus on PWH with confirmed HR‐HPV fills a gap in Mexican epidemiology, where prior studies underestimated anal abnormalities due to small samples [32].

Future perspectives involve longitudinal follow‐up to track progression to high‐grade AIN or cancer, integrating HRA in expanded multicentre trials. Incorporating viral load quantification and immune markers could elucidate mechanisms, while evaluating vaccine efficacy against multiple genotypes in PWH may inform policy.

In conclusion, the GAIA2 study demonstrates that multiple HR‐HPV genotypes, particularly exceeding three, and specific combinations like 16+59, are critical risk factors for anal cytological abnormalities in PWH. The multivariate analysis further emphasizes the protective role of HPV vaccination, which significantly reduces the odds of abnormal cytology and ASC‐US, highlighting its potential to decrease epithelial cell alterations associated with HR‐HPV and cancer progression. These findings advocate for enhanced screening and vaccination strategies to mitigate HPV‐related cancers in this high‐risk group.

Author Contributions

Conceptualisation: Ana Luz Cano‐Díaz and José Antonio Mata‐Marín. Material and methods: Ana Luz Cano‐Díaz and José Antonio Mata‐Marín. Validation: Ana Luz Cano‐Díaz and José Antonio Mata‐Marín. Formal analysis: Omar Hernández‐López, Ana Luz Cano‐Díaz, and José Antonio Mata‐Marín. Investigation: Omar Hernández‐López, Brenda Clara González‐Contreras, Ana Luz Cano‐Díaz, José Antonio Mata‐Marín, Javier Vicente Noyola‐Gómez, Paola Edith Padilla‐Noguera, Alberto Chaparro‐Sánchez, Sócrates Alberto García‐Gutiérrez, and Jesús Enrique Gaytan‐Martínez. Resources: Omar Hernández‐López, Ana Luz Cano‐Díaz, José Antonio Mata‐Marín, Javier Vicente Noyola‐Gómez, Alberto Chaparro‐Sánchez, Sócrates Alberto García‐Gutiérrez, and Jesús Enrique Gaytan‐Martínez. Data curation: Omar Hernández‐López, Brenda Clara González‐Contreras, Ana Luz Cano‐Díaz, Javier Vicente Noyola‐Gómez, Alberto Chaparro‐Sánchez, Sócrates Alberto García‐Gutiérrez, and José Antonio Mata‐Marín. Writing – original draft preparation: Omar Hernández‐López, Ana Luz Cano‐Díaz, and José Antonio Mata‐Marín. Writing – review and editing: Omar Hernández‐López, Ana Luz Cano‐Díaz, and José Antonio Mata‐Marín. Supervision: Ana Luz Cano‐Díaz and José Antonio Mata‐Marín. Project administration: Ericka Nelly Pompa‐Mera, Alberto Chaparro‐Sánchez, Ana Luz Cano‐Díaz, José Antonio Mata‐Marín, and Jesús Enrique Gaytan‐Martínez. Visualisation: Ana Luz Cano‐Díaz, and José Antonio Mata‐Marín. Funding acquisition: Omar Hernández‐López, Ana Luz Cano‐Díaz, José Antonio Mata‐Marín, Jesús Enrique Gaytan‐Martínez, and Alberto Chaparro‐Sánchez. All authors have read and agreed to the published version of the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

This protocol was approved by the local health research committee 3502 and the research ethics committee 35028.

Consent

All participants provided written informed consent prior to sample collection.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.