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Abstract

Background

A persistent infection by high-risk human papillomavirus (HR-HPV) is a prerequisite for the development of cervical neoplasms; however, most studies have focused on risk factors associated with HPV-16 and HPV-18 only.

Objectives

We assessed the association of risk factors with the prevalence of HPV-16, HPV-18, and non-16/18 HR-HPV infection and with the occurrence of cervical lesions in the baseline of a cohort study of HPV persistence in a Mexican population.

Methods

Cross-sectional study within the baseline of a 5-year dynamic cohort study of HR-HPV persistence in women with an abnormal cytology study result from 2015 to 2021. HPV DNA was detected using the Anyplex II HPV 28 kit. Data on lifestyle, sociodemographic, and reproductive factors were assessed using bivariate and multivariate analyses to determine the association of risk factors with HR-HPV infection status and histopathologic diagnosis.

Results

A total of 373 women were included in the study. The overall prevalence of HR-HPV infection was 69.97%. The most prevalent HR-HPV genotypes, including single and multiple infections, were HPV-53 (13.4%), HPV-16 (11.8%), HPV-58 (10.9%), HPV-31 (10.9%), and HPV-66 (10.7%). We found 90 multiple HR-HPV infection patterns, all of them with α-6 and -9 species. Significant associations of multiple HPV-16 and non-16/18 HR-HPV infections were found with marital status, number of lifetime sexual partners, and smoking history. The most prevalent genotype in CIN1 and CIN2 patients was HPV-16. No association was found between biological plausibility risk factors and cervical lesions.

Conclusions

The risk factors for non-16/18 HR-HPV multiple infections are no different than those linked to HPV-16 multiple infections.

Keywords

risk factors human papillomavirus infection squamous intraepithelial lesions cervical neoplasm

Introduction

A persistent cervical infection with high-risk human papillomavirus (HR-HPV) is the main etiological factor for the development of precancerous lesions and their progression to cervical cancer.¹ HPV is one of the most common viral agents of sexually transmitted infections in women worldwide, and sexually active women have a 60%–70% lifetime risk of acquiring an HPV infection, without necessarily developing any symptoms.² About 90% of HPV infection cases will clear spontaneously within 12–24 months, and only a small fraction of those infections will persist or progress to malignancy.³

The prevalence of HPV infection in cervical tissues varies depending on whether it is estimated in the open population or in sites with case concentration, such as dysplasia clinics. The worldwide prevalence of HPV infection in women with no cervical abnormalities is 11%–12%.⁴ In Mexico, the prevalence reported in two recent studies based on large populations is 11%–13%,^5,6 while the prevalence reported in dysplasia clinics is about 25%;⁷ however, recent studies have reported a high unusual prevalence of up to 79.2%.⁸

A woman’s age at first intercourse,⁹ the number of lifetime sexual partners,¹⁰ history of sexually transmitted infections (STIs), and other factors related to sexual activity are associated with exposure to the HPV infection.¹¹ Older age, high HPV viral load,^12,13 and lower socioeconomic and educational status^14-16 have been associated with an increased risk of disease in HPV-seropositive women.

Cofactors such as parity, use of hormonal contraceptives for more than 5 years—mainly diethylstilbestrol—^17,18 smoking,¹⁹ and concomitant STIs^20,21 have been associated with a higher probability of HPV persistence and the appearance of invasive cervical cancer.²²

Most studies have reported factors associated with the prevalence of HPV infection in cervical tissues due to HPV strains with genotype 16 and 18. Meanwhile, little is known about sociodemographic and gynecological-obstetric cofactors associated with infection by other HR-HPV genotypes. Therefore, the objective of this study was to assess the prevalence of HR-HPV infection not only due to HPV-16 and -18, as well as of cervical lesions and risk factors associated, within the baseline cohort study on HPV persistence in the Mexican population.

Material and Methods

Study Design and Population

This is a cross-sectional study nested in the baseline of a 5-year dynamic cohort study of HPV persistence, conducted between 2015 and 2021 in women who attended the Women’s Health Care Center (CAPASAM) of the Health Services of the State of Morelos, Mexico, consecutively after having abnormal results in cytology studies issued by a referring health center. This study meets the STROBE guidelines for cross-sectional studies.²³ Recruitment and eligibility criteria were described in the study protocol, published in 2018.^24,25 All women attending the CAPASAM were eligible for recruitment if they: (a) are 25 years old or older according to target population of the Mexican cervical cancer control program or younger women with cervical abnormalities; (b) have resided for 5 years or more in Morelos; (c) agree to participate in the study, sign an informed consent form, complete a questionnaire, and donate cervix exudate sample; (d) have a negative diagnosis for chronic inflammatory or autoimmune diseases at the beginning of the study and during follow-up, and a negative pregnancy status; and (e) received no previous anti-HPV vaccination, nor immunosuppressive treatment within 6 months before.

Assuming a power of 80% and a confidence level of 95%, a sample size of 73 was calculated, of the minimum population necessary to estimate the prevalence of HPV infection in the same population, taking as reference the estimated prevalence of HPV infection at the cervix level in population studies in Mexico of 11%–13%,^5,6 with an alternate prevalence reported in dysplasia clinics in Mexico of 25%.⁽⁸⁾ Thus, the inclusion of 373 women in the study was determined as sufficient. Calculations were done with Stata v. 16.0 (Stata-Corp, College Station, TX, USA).

Data Collection

At baseline (sampling 1), 373 women completed a structured questionnaire that included questions on lifestyle, sociodemographic, and reproductive factors. All participants agreed to participate in the study by signing an informed consent form. Information from each questionnaire was entered into a database. All data related to the patients were confidential and handled with a numerical code.

The following sociodemographic variables were included: Age (in years), marital status (single, married, concubinage, divorced or separated, widowed), indigenous language speaker (yes/no), educational level (literacy, elementary school, secondary school, high school, college, technical school), occupation (employee/unemployed).

The following variables describing a participants’ gynecological history were included: Age at menarche (age when a participant had her first menstrual period), sexual behavior (age at first intercourse [AFI]), number of lifetime sexual partners [LNSP], use of hormonal contraception (years of duration, current situation), and use of contraceptives (oral contraceptives [OC], injectable contraceptives [IC], condom, infrequent [<5%] contraceptive methods).

The following variables assessing a participant’s obstetric history were included: Parity, number of full-term pregnancies [FTPs]: nulliparous (no birth), primiparous (1 birth), multiparous (≥2 births), history of sexually transmitted diseases [STIs], previous HPV infection (yes/no), previous local treatment of cervical lesions, and familial history of cervical cancer—including consanguinity.

Finally, to explore lifestyle-related behaviors, smoking habit was included as a variable (years, number of cigarettes per day, whether the habit is old or current, previous years of smoking).

Procedures

Cervical swab samples were obtained from each participant who signed the informed consent form and completed the questionnaire, using a cervix brush (Rovers Medical Devices, Oss, Netherlands). The brush was immediately rinsed in a vial with PreservCyt solution (Cytyc Corporation, Marlborough, MA, USA), and the vial was placed in a ThinPrep Processor (Cytyc Corporation). DNA was extracted from cervical exudate following a previously described protocol.^24,26 A colposcopic diagnosis was issued by a gynecologist at the consultation time, and pathological analysis was performed by the CAPASAM Pathology Unit.

HPV Genotyping and Pathological Examination

HPV DNA detection and virus load quantification were performed using the Seegene Anyplex™ II HPV 28 system (Seegene, Seoul, Korea), based on a real-time multiplex PCR assay that allows simultaneous amplification, detection, and differentiation of target nucleic acids of 19 high-risk HPV types (16, 18, 26, 31, 33, 35, 35, 39, 45, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low-risk HPV types (6, 11, 40, 42, 43, 44, 54, 61, 70).²⁴ This system allowed us to confirm the HPV diagnosis and estimate viral loads by genotype in each patient. We determined the prevalence of HR-HPV either as single or multiple infections. Cytological diagnosis was graded according to the Bethesda classification system for Pap smear reports.²⁶

Statistical Analysis

A bivariate analysis was performed to determine proportion differences in sociodemographic variables, sexual behavior, obstetric-gynecologic history, and frequency of symptoms between the groups with and without HR-HPV infection (overall HR-HPV prevalence and of each identified HR-HPV types, both in single and multiple infections), using a chi square test for categorical variables. While age and the number of sexual partners were first recorded as continuous, numeric variables, they were analyzed as categorical variables. History of hormonal contraception, smoking habit, symptoms, reason for follow-up, results of last Papanicolaou smear, and diagnosis of premalignant lesions in the population under study were both collected and analyzed as categorical variables. A test for trend across ordered groups (np chi square test) was used to verify whether the number of negative and positive HR-HPV cases followed a linear frequency trend in each group for the variables analyzed. Variables with frequency of less than 5 values were evaluated with Fischer´s exact test.

In the analysis, the subjects were classified as HR-HPV-positive when tested positive for any of the 19 HR-HPV types, including HPV-16 and -18. Positive subjects were then subclassified either as HPV-16- or HPV-18-positive when they were positive to either of both subtypes regardless of any other HR-HPV type, or as non-16/18-HR-HPV-positive when they were positive for other HR-HPV types (26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) but not for HPV-16 nor HPV-18.

To determine the association between HR-HPV infection—either overall or segregated by genotype—with risk factors found to be differential distribution in the respective bivariate analysis, and the association of positivity to cervix premalignant lesion with risk factors found to be differential distribution in the respective bivariate analysis, a logistic regression analysis was performed, and 95% confidence intervals (CI) were calculated for odds ratios (OR), adjusting by variables with differential distribution in the bivariate analysis with the exception of exposition variable itself. The number of lifetime sexual partners, sexual behavior, oral contraceptive use, age, history of STIs, and current smoking were the variables used to fit the final model. All statistical analyses were performed with STATA v.16.0 (StataCorp, College Station, TX).

Results

Characteristics of Participants

Variables on sociodemographic information, lifestyle, sexual and reproductive behavior, and clinical data of the population analyzed in the baseline of the cohort study are shown in Table 1. The mean age of the participants at baseline was 36.6 ± 11.1 years (range, 15–68 years); the age group with the highest number of subjects (16.3%) was 40–44 years. In the studio, 68.6% of subjects were married or cohabitating, 55.2% were housewives and unemployed, and 45% had completed secondary education.

Table 1.

Baseline Characteristics of Study Population.

Characteristics	n (% of Total Population) Total n = 373	HR-HPV Positive n	HR-HPV Prevalence %
Sociodemographic and lifestyle characteristics
Age (years)
15–29	110 (29.5)	84	76.3
30–49	217 (58.2)	144	66.3
≥50	46 (12.3)	33	71.7
Married/living as married
No	117 (31.4)	92	78.6
Yes	256 (68.6)	169	66.02
Socioeconomic level
Low	274 (73.5)	191	69.7
Medium	99 (26.5)	70	70.7
Occupation
Housewife/Unemployed	206 (55.2)	137	66.5
Housekeeping	52 (14.0)	35	67.3
Dependent employee	89 (23.9)	68	76.4
Student/Professional	26 (6.9)	21	80.7
Educational level
Unlettered/incomplete basic education	56 (15)	39	69.6
Elementary school	68 (18.2)	49	72
Secondary or technical education	168 (45)	113	67.2
High school	60 (16.1)	43	71.6
College or higher	21 (5.7)	17	80.9
Occupation of partner
Student/Professional	22 (5.9)	15	68.1
Employee	140 (37.5)	97	69.2
Bricklayer	62 (16.6)	37	59.6
Driver	30 (8.1)	26	86.6
Farmer	28 (7.5)	15	53.5
No partner/Unemployed	91 (24.4)	71	78
Lifestyle characteristics
History of smoking
No	285 (76.4)	192	67.3
Yes	88 (23.6)	69	78.4
Smoking behavior
Never smoked	319 (85.5)	217	68.03
Smoked at least once	54 (14.5)	44	81.4
Among smokers, no. of cigarettes/week
No smoker	319(85.5)	—	—
1–5	23 (6.1)	19	82.6
6–20	19 (5.2)	15	78.9
>20	12 (3.2)	10	83.3
History of alcoholism
No	336 (90.1)	232	69
Yes	37 (9.9)	29	78.3
Current alcoholism
No	315 (84.4)	213	67.6
Yes	58 (15.6)	48	82.7
Sexual characteristics
Age at menarche (years)
Early menarche, ≤11	148 (39.7)	108	72.9
12–14	204 (54.7)	137	67.1
Late menarche, ≥15	21 (5.6)	16	76.1
Age at first sexual contact
≤15	94 (25.2)	64	68
16–17	113 (30.3)	82	72.5
≥18	166 (44.5)	115	69.2
Number of lifetime sexual partners
1	138 (37)	77	55.8
2–3	169 (45.3)	130	76.9
≥4	66 (17.7)	54	81.8
Contraceptive use (among sexually active)
Use of oral contraceptives
Never	365 (97.9)	254	69.5
Ever	8 (2.1)	7	87.5
Use of injectable contraceptives
Never	352 (94.4)	251	71.3
Ever	21 (5.6)	10	47.6
Use of condoms
Never	346 (92.8)	241	69.6
Ever	27 (7.2)	20	74
Use of other contraceptive method
Never	56 (15)	37	66
Ever	317 (85)	224	70.6
No. of full-term pregnancies
Nulliparous (no birth)	24 (6.4)	20	83.3
1–2	136 (36.5)	93	68.3
≥3	213 (57.1)	148	69.4
Previous sexually transmitted diseases
No	338 (90.6)	232	68.6
Yes	35 (9.4)	29	82.8
Herpes simplex virus type 2
Negative	266 (71.3)	188	70.6
Positive	105 (28.2)	72	68.5
Missing data	2 (.5)	—	—
Clinic variables
Family history of cancer
No	252 (67.6)	167	66.2
Yes	121 (32.4)	94	77.6
History of HPV positive test
No	345 (92.5)	236	68.4
Yes	28 (7.5)	25	89.2
Histopathologic diagnosis
Negative for SICL or malignancy	208 (55.77)	131	62.9
Low-grade SICL, CIN1, ordinary dysplasia	126 (33.79)	98	77.7
High-grade SICL, CIN2, moderate dysplasia	30 (8.04)	23	76.6
High-grade SICL, CIN3, severe dysplasia, squamous cell carcinoma in situ	8 (2.14)	8	100
Invasive squamous cell carcinoma	1 (.27)	1	100
History of gynecological treatment
No	360 (96.5)	249	69.1
Cryotherapy	6 (1.6)	5	83.3
Electrosurgery	7 (1.9)	7	100
HR-HPV load
Negative	112 (30.0)	—	—
10 000–49 000	208 (55.8)	208	56.2
50 000–99 000	47 (12.6)	47	12.7
≥100 000	3 (.81)	3	.81
Missing data	3(.81)	—	—
Dyspareunia
No	286 (76.7)	200	69.9
Yes	87 (23.3)	61	70.1
Dysuria
No	311 (83.4)	220	70.7
Yes	62 (16.6)	41	66.1
Pruritus
No	241 (64.6)	171	70.9
Yes	132 (35.4)	90	68.1
Vaginal dryness
No	301 (80.7)	209	69.4
Yes	72 (19.3)	52	72.2
Leukorrhea
No	191 (51.3)	128	67.02
Yes	182 (48.7)	133	73.08
Body mass index at enrollment (kg/m²)
Underweight (<18.5)	11 (2.9)	7	63.6
Normal (18.5–24.9)	108 (28.9)	78	72.2
Overweight (25–29.9)	127 (34.1)	91	71.6
Obesity (≥30)	127 (34.01)	85	66.9

n (% of total population, col).

HR-HPV positive n (col): number of cases with at least 1 high-risk HPV genotype detected, including cases with co-infection by 2 or more high-risk HPV genotypes.

HR-HPV prevalence (row), is defined as the percentage of subjects with at least 1 high-risk HPV genotype detected by specific stratum for each evaluated characteristic.

Use of other contraceptive method: coitus interruptus, intrauterine device, bilateral tubal occlusion, vasectomy, subdermal implant, or not using any.

Herpes simplex virus type 2 test was performed in a StepOne Rea-Time PCR system, using the SYBR select master mix.

Of these women, 76.4% reported not having used tobacco at any time in their lives. Smoking participants (23.5%) reported having smoked 1–5 cigarettes per week. The mean age at menarche was 12.7 ± 1.5 years; the average age at first intercourse was 17.6 ± 3.1 years; the mean number of lifetime sexual partners was 2.4 ± 1.8; and the mean number of pregnancies to term was 3.1 ± 2.04. Only 13.6% of participants had used hormonal contraceptives at any time. Interestingly, 9.3% of participants reported having had a sexually transmitted infection at some point in their lives, but a confirmatory test revealed that 28.2% of them were positive for herpes simplex virus type 2 (HSV-2); the prevalence of HSV-2 infection was 68.5% in HR-HPV positive women. About 56% of HR-HPV infected women had a moderate viral load, 10,000–49,000 total viral total copies. The highest proportion of women were overweight and obese, according to their body mass index (Table 1).

Overall HR-HPV Prevalence

The overall prevalence of HR-HPV infection, defined as the percentage of women with abnormal results in cervix cytology tests from the CAPASAM dysplasia clinic and with at least 1 high-risk HPV genotype detected, was 69.97%. The prevalence of single HR-HPV infection with any genotype was 37.5%, while the prevalence of coinfection by HR-HPV with any genotype was 32.9%. The prevalence of HPV-16, HPV-18, or non-16/18 HR-HPV single infection was 5.9%, 2.9%, and 27.6%, respectively. The prevalence of coinfection with HPV-16, HPV-18, or non-16/18 HR-HPV was 5.6%, 2.9%, and 24.4%, respectively (Figure 1).

Figure 1.

Distribution of high-risk HPV genotypes in single and multiple infection.

HR-HPV Prevalence by Age

The prevalence of HR-HPV with any genotype by age group shows a bimodal distribution with an increased prevalence for the women aged 15–29 years, while the number of positive cases decreased in middle-aged subjects and increased again in women older than 55 years (Figure 2). The age group with the highest prevalence of HR-HPV infection was 25–29 years (81.4%). Women aged 30–49 years had a higher prevalence of single HR-HPV infections (71.4%), whilst the highest prevalence of HR-HPV multiple infection was found in the group aged 15–29 years (41.8%) (Figure 2).

Figure 2.

Age-specific prevalence of any HR-HPV genotype. HR-HPV prevalence is defined as the percentage of women with at least one high-risk HPV genotype detected (including coinfections with other HR-HPV genotypes).

Distribution of HR-HPV Genotypes

The most prevalent HR-HPV genotypes in our study population, both as single infections and as coinfections, were HPV-53 (13.4%), HPV-16 (11.8%), HPV-58 (10.9%), HPV-31 (10.9%), and HPV-66 (10.7%). The HR-HPV genotypes with the highest prevalence in single infections were HPV-16 (5.8%), HPV-58 (4.5%), HPV-31 (3.2%), HPV-66 (3.2%), and HPV-18 (2.9%). The genotypes with the highest prevalence in coinfections were HPV-53 (11.5%), HPV-31 (7.7%), HPV-66 (7.5%), HPV-58 (6.4%), HPV-16 (5.6%), and HPV-59 (5.6%) (Figure 3). HPV-26 and HPV-69 were only found in single infections, while HPV-73 and HPV-82 were only found in coinfection. HR-HPV genotypes were found to infect the subjects in 90 possible combinations. The genotypes with the highest number of combinations were HPV-53, HPV-31, HPV-58, HPV-16, and HPV-66 (Figure 4).

Figure 3.

Distribution of prevalence of high-risk HPV genotypes for single and multiple infection.

Figure 4.

Heat map: Patterns of high-risk HPV infection. Heat map of the patterns of multiple HR-HPV infections. Horizontal and vertical axes represent the 28 high-risk genotypes that were evaluated in each patient. Blue cells indicate a higher number of combinations, and yellow cells indicate a lower number of combinations for each infection pattern. The color scale progresses from yellow to blue as the number of patterns per genotype increases. As shown, genotype 53 has the highest number of combinations.

Factors Related to Sociodemographic, Lifestyle, and Gynecological-Obstetric Information Associated with HR-HPV Prevalence

A bivariate analysis of sociodemographic characteristics, gynecological-obstetric history, and lifestyle-related data showed a significantly different distribution in the number of sexual partners, number of pregnancies, smoking history, current smoking habit, and number of cigarettes per week in patients with HR-HPV multiple infection with respect to non-infected subjects. On the other hand, patients with multiple infection by HPV-16 showed significant differences with patients infected by non-16/18 HR-HPV in the number of sexual partners and history of smoking. For the most prevalent genotypes in multiple infections in our study, the number of sexual partners, age at first intercourse, number of pregnancies, and smoking history showed a significantly different distribution in HR-HPV-positive and negative groups (Table 2).

Table 2.

Clinical and Sex-Behavioral Characteristics in Genotype-Specific Prevalence of Single and Multiple HR-HPV Infections.

	Single HR-HPV Infection			Multiple HR-HPV Infection			Single HPV 16 Infection			Multiple HPV 16 Infection			Single HPV18 Infection			Multiple HPV 18 Infection			Single HR- HPV non-16/18			Multiple HR-HPV non 16/18
Characteristics	HR-HPV n (%)	HR-HPV Prevalence %	P Value	HR-HPV n (%)	HR-HPV Prevalence %	P Value	HPV 16 Positive n (%)	HPV 16 Positive Prevalence %	P Value	HPV 16 Positive n (%)	HPV 16 Positive Prevalence %	P Value	HPV 18 Positive n (%)	HPV 18 Positive Prevalence %	P Value	HPV 18 Positive n (%)	HPV 18 Positive Prevalence %	P Value	Non-16/18 HR-HPV Positiven	Non-16/18 HR-HPV Prevalence %	P Value	Non-16/18 HR-HPV Positive n	Non-16/18 HR-HPV Prevalence %	P value
Age (years)			.491			.081			.819			1			.647			.264			.293			.017
15–29	38 (27.1)	34.5		46 (37.4)	41.8		8 (36.3)	7.2		6 (28.5)	5.45		2 (18.1)	1.8		2 (18.1)	1.8		27 (26.2)	24.5		38 (31.7)	34.5
30–49	84 (60)	38.7		62 (50.4)	28.5		12 (54.5)	5.5		13 (61.9)	5.9		7 (63.6)	3.2		6 (54.5)	2.7		61 (59.2)	28.11		43 (47.2)	19.8
≥50	18 (12.8)	39.1		15 (12.2)	32.6		2 (9)	4.3		2 (9.5)	4.3		2 (18.1)	4.3		3 (27.2)	6.5		15 (14.5)	32.6		10 (10.9)	21.7
Herpes simplex virus type 2			.302			.436			.978			.128			.620			.134			.286			.548
Negative	104 (74.8)	39.1		85 (69.1)	31.9		15 (71.4)	5.6		12 (57.1)	4.5		8 (72.7)	3.01		10 (90.9)	3.7		78 (75.7)	29.3		63 (69.2)	23.6
Positive	35 (25.1)	33.3		38 (30.8)	36.1		6 (28.5)	5.7		9 (42.8)	8.5		3 (27.2)	2.8		1 (9.09)	0.9		25 (24.2)	23.8		28 (30.7)	26.6
Previous sexually transmitted diseases			.294			.582			.631			.394			.074			.276			.354			.310
No	124 (88.5)	36.6		110 (89.4)	32.5		20 (90.9)	5.9		20 (95.2)	5.9		8 (72.7)	2.3		9 (81.8)	2.6		91 (88.3)	26.9		80 (87.9)	23.6
Yes	16 (11.4)	45.7		13 (10.5)	37.1		2 (9)	5.7		1 (4.7)	2.8		3 (27.2)	8.5		2 (18.1)	5.7		12 (11.6)	34.2		11 (12.09)	31.4
Age at menarche (years)			.313			.541			.769			.414			.421			.580			.288			.897
≤11	59 (42.1)	39.8		52 (42.2)	35.1		9 (40.9)	6.08		11 (52.3)	7.4		3 (27.2)	2.03		3 (27.2)	2.03		44 (42.7)	29.7		35 (38.4)	23.6
12–14	72 (51.4)	35.2		64 (52)	31.3		11 (50)	5.3		9 (42.8)	4.4		7 (63.6)	3.4		8 (72.7)	3.9		52 (50.4)	25.4		50 (54.9)	24.5
≥15	9 (6.4)	42.8		7 (5.6)	33.3		2 (9)	9.5		1 (4.7)	4.7		1 (9.09)	4.7		0 (0)	0		7 (6.8)	33.3		6 (6.5)	28.5
Age at first intercourse			.989			.404			.361			.789			.627			.926			.825			.487
≤15	34 (24.2)	36.1		30 (24.3)	30		3 (13.6)	3.1		5 (23.8)	5.3		4 (36.3)	4.2		2 (18.1)	2.1		28 (27.1)	29.7		23 (25.2)	24.4
16–17	43 (30.7)	38		41 (33.3)	41		9 (40.9)	7.9		6 (28.5)	5.3		2 (18.1)	1.7		4 (36.3)	3.5		31 (30.1)	27.4		30 (32.9)	26.5
≥18	63 (45)	37.9		52 (42.2)	52		10 (45.4)	6.02		10 (47.6)	6.02		5 (45.5)	3.01		5 (45.5)	3.01		44 (42.7)	26.5		38 (41.7)	22.8
Number of lifetime sexual partners			.878			.000			.235			.032			.095			.853			.613			.001
1	47 (33.5)	34.06		30 (24.3)	21.7		5 (22.7)	3.6		3 (14.2)	2.1		1 (9)	0.7		5 (45.4)	3.6		39 (37.8)	28.2		23 (25.2)	16.6
2–3	72 (51.4)	42.6		60 (48.7)	35.5		13 (59)	7.6		11 (52.3)	6.5		8 (72.7)	4.7		5 (45.4)	2.9		48 (46.6)	28.4		43 (47.2)	25.4
≥4	21 (15)	31.8		33 (26.8)	50		4 (18.1)	6		7 (33.3)	10.6		2 (18.1)	3		1 (9)	1.5		16 (15.5)	24.2		25 (27.4)	37.8
No. of full-term pregnancies			.116			.006			.761			.336			.479			.776			.201			.030
Nulliparous (no birth)	6 (4.2)	25		14 (11.3)	58.3		0 (0)	0		2 (9.5)	8.3		2 (18.1)	8.3		1 (9)	4.1		4 (3.8)	16.6		12 (13.1)	50
1–2	48 (34.2)	35.2		48 (39)	35.2		10 (45.4)	7.3		9 (42.8)	6.6		0 (0)	0		4 (36.3)	2.9		36 (34.9)	26.4		32 (35.1)	23.5
≥3	86 (61.4)	40.3		61 (49.5)	28.6		12 (54.5)	5.6		10 (47.6)	4.6		9 (81.8)	4.2		6 (54.5)	2.8		63 (61.1)	29.4		47 (51.6)	22
Contraceptive use (among sexually active)
Use of oral contraceptives			.998			.448			.475			.016			.619			.619			.867			.968
Never	137 (97.8)	37.5		119 (96.7)	32.6		22 (100)	6		19 (90.4)	5.2		11 (100)	3		11 (100)	3		101 (98)	27.6		89 (97.8)	24.3
At least once	3 (2.1)	37.5		4 (3.2)	50		0 (0)	0		2 (9.5)	25		0 (0)	0		0	0		2 (1.9)	25		2 (2.2)	25
Use of injectable contraceptives			.383			.232			.820			.250			.412			.412			.689			.557
Never	134 (95.7)	38.07		119 (96.7)	33.8		21 (95.4)	5.9		21 (100)	5.9		11 (100)	3		11 (100)	3		98 (95.1)	27.8		87 (95.6)	24.7
At least once	6 (4.2)	28.5		4 (3.2)	19		1 (4.5)	4.7		0 (0)	0		0 (0)	0		0 (0)	0		5 (4.8)	23.8		4 (4.4)	19
Use of condoms			.196			.031			.177			.653			.348			.810			.839			.012
Never	133 (95)	38.4		109 (88.6)	31.5		22 (100)	6.3		20 (95.2)	25.7		11 (100)	3.1		10 (90.9)	2.8		96 (93.2)	27.7		79 (86.8)	22.8
At least once	7 (5)	25.9		14 (11.3)	51.8		0 (0)	0		1 (4.7)	3.7		0 (0)	0		1 (9)	3.7		7 (6.8)	25.9		12 (13.1)	44.4
Use of other contraceptive method			.134			.277			.157			.924			.158			.577			.636			.144
Never	16 (11.4)	28.5		22 (17.8)	39.2		1 (4.5)	1.7		3 (14.2)	5.3		0 (0)	0		1 (9)	1.7		14 (13.5)	25		18 (19.7)	32.1
At least once	124 (88.5)	39.1		101 (82.1)	31.8		21 (95.4)	6.6		18 (85.7)	5.6		11 (100)	3.4		10 (90.9)	3.1		89 (86.4)	28		73 (80.2)	23
History of smoking			.311			.001			.538			.033			.771			.668			.531			.033
No	111 (79.2)	38.9		81 (65.8)	28.4		18 (81.8)	6.3		12 (57.1)	4.2		8 (72.7)	2.8		9 (81.8)	3.1		81 (78.6)	28.4		62 (68.1)	21.7
Yes	29 (20.7)	32.9		42 (34.1)	47.7		4 (18.1)	4.5		9 (42.8)	10.2		3 (27.2)	3.4		2 (18.1)	2.2		22 (21.3)	25		29 (31.8)	32.9
Smoking behavior			.700			.004			.460			.012			.607			.221			.721			.532
Never	121 (86.4)	37.9		96 (78)	30.09		20 (90.9)	6.2		14 (66.6)	4.3		10 (90.9)	3.1		8 (72.7)	2.5		87 (84.4)	27.2		76 (83.5)	23.8
At least once	19 (13.5)	35.1		27 (21.9)	50		2 (9)	3.7		7 (33.3)	12.9		1 (9)	1.8		3 (27.2)	5.5		16 (15.5)	29.6		15 (16.4)	27.7
Among smokers, No. of cigarettes/week			.732			.009			.594			.049			.851			.583			.874			.312
1–5	9 (6.4)	39.1		11 (8.9)	47.8		1 (4.5)	4.3		2 (9.5)	8.7		1 (9)	4.3		1 (9)	4.3		7 (6.8)	30.4		7 (7.6)	30.4
6–20	5 (3.5)	26.3		10 (8.1)	52.6		1 (4.5)	5.2		3 (14.2)	15.7		0 (0)	0		1 (9)	5.2		4 (3.8)	21		6 (6.5)	31.5
>20	5 (3.5)	41.6		6 (4.8)	50		0 (0)	0		2 (9.5)	16.6		0 (0)	0		0	0		5 (4.8)	41.6		3 (3.3)	25

Use of other contraceptive methods: coitus interruptus, intrauterine device, bilateral tubal occlusion, vasectomy, subdermal implant, or not using any.

Herpes simplex virus type 2 test was performed in a StepOne Real-Time PCR system, using the SYBR select master mix.

Previous sexually transmitted diseases were self-reported by the patient.

HR-HPV positive n (%, col): number of cases with at least 1 high-risk HPV genotype detected, including cases with co-infection by 2 or more high-risk HPV genotypes.

HR-HPV prevalence (%, row), is defined as the percentage of subjects with at least 1 high-risk HPV genotype detected by specific stratum for each evaluated characteristic.

Values in bold type indicate significant P-values (P < .05) chi square test np_trend to determine differences in proportions between groups with and without HR-HPV infection for categorical variables.

Variables with cell <5 values were evaluated with Fischer´s exact test. Values in bold type indicate significant P-values (P < .05).

Bivariate analyses segregated by viral genotype showed a differential distribution in hormonal contraceptive use and current smoking habit in patients with single HPV-66 infections. Meanwhile, significant differences were observed in marital status among subjects with multiple infections by the genotypes HPV-16, non-16/18 HR-HPV, HR-HPV-58, HPV-51, and HPV-39. The number of sexual partners was significantly different in women with multiple infections by the genotypes HPV-16, non-16/18 HR-HPV, HPV-58, HPV-31, and HPV-53. A differential distribution of smoking history was found in patients coinfected with the genotypes HPV-16, non-16/18 HR-HPV, HPV-58, and HPV-53 with respect to negative subjects. Finally, the number of pregnancies was significantly different in patients coinfected with non-16/18 HR-HPV, HPV-58, and HPV-39 with respect to negative cases (Table S1).

The association of HR-HPV single and multiple infections with risk factors was assessed for each genotype by logistic regression, adjusting for variables that showed a differential distribution in previous bivariate analyses. Marital status was found to be positively associated with HPV-16, non-16/18 HR-HPV, HPV-58, HPV-51, and HPV-39 multiple infections. The number of lifetime sexual partners was associated with HPV-16, non-16/18 HR-HPV, HPV-58, HPV-31, and HPV-53 multiple infections. Smoking history was associated with HPV-16, non-16/18 HR-HPV, HPV-58, and HPV-53 multiple infections (Tables 3 and S2).

Table 3.

Association of HR-HPV Infection With Sexual Health and Behavior-Related Variables.

	HR-HPV (by any Genotype)						HR-HPV single Infection						HR-HPV Multiple Infection
Reproductive Characteristics	Unadjusted OR (95%CI)	P-value	Adjusted OR (95%CI)^a	P-value	Adjusted OR by Age (95%CI)^b	P-value	Unadjusted OR (95%CI)	P-value	Adjusted OR (95%CI)^a	P-value	Adjusted OR by Age (95%CI)^b	P-value	Unadjusted OR (95%CI)	P-value	Adjusted OR (95%CI)^a	P-value	Adjusted OR by Age (95%CI)^b	P-value
Married/living as married
No	1.8 (1.1-3.1)	.014	1.4 (.8-2.5)	.177	1.3 (.7-2.4)	.245	.8 (.5-1.2)	.367	.8)0.5-1.3)	.404	.8 (.4-1.3)	.394	2.3 (1.4-3.6)	0	1.6 (1.03-2.7)	.036	1.6 (.9-2.7)	.053
Yes	Reference
Number of lifetime sexual partners
1	Reference
2–3	2.6 (1.6–4.3)	.000	2.3 (1.3–3.8)	.001	2.4 (1.4–4)	.001	1.4 (.9–2.2)	.127	1.5 (.9–2.4)	.076	1.5 (.9–2.5)	.073	1.9 (1.1–3.3)	.009	1.6 (.9–2.8)	.055	1.7 (1–2.9)	.047
≥4	3.5 (1.7–7.2)	.000	2.7 (1.2–5.7)	.008	2.6 (1.2–5.5)	.012	.9 (.4–1.6)	.751	1 (.5–1.9)	.961	1.01 (.5–1.9)	.97	3.5 (1.9–6.7)	0	2.5 (1.3–5.05)	.055	2.5 (1.3–5)	.006
Use hormonal contraceptives
Never	Reference
At least once			1.08 (.5–2.1)	.819	.9 (.4–1.8)	.79	1.08 (.5–1.9)	.789	1.2 (.6–2.3)	.502	1.2 (.6–2.3)	.52	.8 (.4–1.5)	.561	.7 (.3–1.5)	.416	.7 (.3–1.4)	.376
No. of full-term pregnancies
Nulliparous (no birth)	Reference
1–2	.43 (.13–1.3)	.147	.4 (.1–1.6)	.252	.5 (.1–1.7)	.311	1.6 (.6–4.3)	.329	1.5 (.5–4.2)	0.4	1.5 (.5–4.3)	.388	.3 (.1–.9)	.037	.4 (.1–1.2)	.133	.4 (.1–1.2)	.151
≥3	.45 (.14–1.3)	.166	.5 (.1–1.7)	.327	.8 (.2–2.8)	.752	2.03 (.7–5.3)	.149	1.9 (.7–5.1)	.201	1.9 (.6–5.7)	.199	.2 (.1–.6)	.005	.3 (.1–.9)	.048	.4 (.1–1.1)	.100
Previous sexually transmitted diseases
No	Reference
Yes	2.2 (.89–5.4)	.087	1.5 (.5–4.02)	.389	1.4 (.5–3.8)	.473	1.4 (.7–2.9)	.296	1.5 (.7–3.1)	.243	1.5 (.7–3.2)	.244	1.2 (.5–2.5)	.582	1 (.4–2.1)	.98	.9 (.4–2.1)	.970
History of smoking
No	Reference
Yes	1.7 (.9–3.09)	.050	1.2 (.6–2.5)	.536	1.1 (.5–2.4)	.681	.7 (.4–1.2)	.311	.7 (.3–1.4)	.357	.7 (.3–1.4)	.346	2.2 (1.4–3.7)	.001	1.7 (.8–3.3)	.101	1.6 (.8–3.2)	.113
Smoking behavior
Never smoked	Reference
At least once	2.06 (1.00–4.27)	.050	1.4 (.5–3.6)	.416	1.4 (.5–3.6)	.441	.8 (.4–1.6)	.700	1.2 (.5–2.7)	.569	1.2 (.5–2.7)	.565	2.3 (1.2–4.1)	.005	1.1 (.5–2.5)	.684	1.1 (.5–2.6)	.654
Among smokers, no. of cigarettes/week
1–5	Reference
6–20	.78 (.16–3.6)	.764	.9 (.1–4.5)	.904	.9 (.1–4.8)	.98	.5 (.1–2)	.383	.4 (.1–1.8)	.282	.4 (.1–1.8)	.281	1.2 (.3–4)	.757	1.6 (.4–6.1)	.424	1.6 (.4–6.1)	.4121
>20	1.05 (.16–6.7)	.957	1.1 (.1–8.1)	.854	1.4 (.2–10.2)	0.6	1.1 (.2–4.5)	.884	1.05 (.2–4.5)	.938	1.08 (.2–4.6)	.915	1.09 (.2–4.4)	.903	1.3 (.3–5.8)	.709	1.3 (.3–6.1)	.658

HR-HPV n (%): number of cases and HR-HPV prevalence are defined as the absolute number and corresponding percentage of women with at least 1 high-risk HPV genotype detected by specific stratum for each evaluated characteristic.

^aOdds ratio for all variables, adjusted by variables with differential distribution in the bivariate analysis with the exception of exposition variable itself.

^bOdds ratio for all variables adjusted by age and variables with differential distribution in the bivariate analysis with the exception of exposition variable itself.

Distribution and Association of HR-HPV Genotypes and Risk Factors per Histopathological Status

According to histopathological diagnosis of the cervix, the prevalence of cases positive for HR-HPV was 66.35% in women with negative results for squamous intraepithelial cervical lesion or malignancy. In the group with intraepithelial cervical neoplasia 1 (CIN1), the prevalence of HR-HPV infection was 73.81%, and the most frequently detected HR-HPV genotypes were HPV-16 (12.7%), HPV-66 (12.7%), and HPV-31 (11.9%). The prevalence of HR-HPV infection in patients with CIN2 was 73.3%, being HPV-16 (20%), HPV-18 (16.6%), and HPV-58 (16.6%) the most frequently HR-HPV genotypes. In patients with CIN3, the prevalence of HR-HPV infection was 87.5%, with HPV-35 (25%), HPV-52 (25%), and HPV-58 (25%) as the most prevalent viral genotypes (Table 4).

Table 4.

Prevalence of High-Risk HPV Infection by Histopathologic Diagnosis.

Histopathologic Diagnosis	HR-HPV Infection	Negative for SICL or Malignancy, n = 208			Low-grade SICL, CIN1, n = 126			High-grade SICL, CIN2, n = 30			High-grade SICL, CIN3, Squamous Cell Carcinoma in Situ, n = 8
HPV-HR Genotypes	HR-HPV Infection	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value
16	Total	22 (10.5)	50	.413	16 (12.7)	36.3	0.7	6 (20)	13.6	.147	—	—	—
	Single	12 (5.7)	54.5	.906	8 (6.3)	36.3	.792	3 (10)	13	.279	—	—	—
	Multiple	10 (4.8)	47.6	.44	8 (6.3)	38.1	.667	3 (10)	14.2	.233	—	—	—
18	Total	7 (3.3)	31.8	.02	10 (7.9)	45.4	.233	5 (16.6)	22.7	.009	—	—	—
	Single	4 (1.9)	36.3	.157	5 (3.9)	45.5	.407	2 (6.6)	18.1	.219	—	—	—
	Multiple	3 (1.4)	27.2	.052	5 (3.9)	45.5	.407	3 (10)	27.2	.050	—	—	—
26	Total	—	—	—	1 (.79)	100	.338	—	—	—	—	—	—
	Single	—	—	—	1 (.79)	100	.338	—	—	—	—	—	—
	Multiple	—	—	—	—	—	—	—	—	—	—	—	—
31	Total	23 (11.06)	56.1	.964	15 (11.9)	36.5	.688	2 (6.6)	4.8	.334	1 (12.5)	2.4	.610
	Single	7 (3.3)	58.3	.856	5 (3.9)	41.6	.558	—	—	—	—	—	—
	Multiple	16 (7.6)	55.1	.947	10 (7.9)	34.4	.934	2 (6.6)	6.9	.581	1 (12.5)	3.4	.480
33	Total	8 (3.8)	53.3	.847	6 (4.7)	40	.604	—	—	—	1 (12.5)	6.6	.282
	Single	4 (1.9)	66.6	.456	1 (.79)	16.6	.341	—	—	—	1 (12.5)	16.6	.012
	Multiple	4 (1.9)	44.4	.359	5 (3.9)	55.5	.163	—	—	—	—	—	—
35	Total	9 (4.3)	52.9	.811	4 (3.1)	23.5	.263	1 (3.3)	5.8	.595	3 (37.5)	17.6	.004
	Single	2 (.96)	50	.597	1 (.79)	14.2	.585	—	—	—	—	—	—
	Multiple	7 (3.3)	46.6	.847	3 (2.3)	20	.194	1 (3.3)	6.6	.656	3 (37.5)	20	.003
39	Total	15 (7.2)	62.5	.493	9 (7.1)	37.5	.691	—	—	—	—	—	—
	Single	6 (2.8)	75	.269	2 (1.5)	25	.456	—	—	—	—	—	—
	Multiple	9 (4.3)	56.2	.968	7 (5.5)	33.7	.389	—	—	—	—	—
45	Total	4 (1.9)	50	.505	4 (3.1)	33.7	.266	—	—	—	—	—	—
	Single	1 (.4)	33.3	.414	2 (1.5)	66.6	.265	—	—	—	—	—	—
	Multiple	3 (1.4)	60	.608	2 (1.5)	40	.549	—	—	—	—	—	—
51	Total	12 (5.7)	54.5	.906	8 (6.3)	36.3	.792	1 (3.3)	4.5	.456	1 (12.5)	4.5	.388
	Single	6 (2.8)	66.6	.506	2 (1.5)	22.2	.364	1 (3.3)	11.1	.534	—	—	—
	Multiple	6 (2.8)	46.1	.478	6 (4.7)	46.1	.338	—	—	—	1 (12.5)	7.6	.249
52	Total	10 (4.8)	45.4	.316	9 (7.1)	40.9	.467	1 (3.3)	4.5	.456	2 (25)	9.09	.075
	Single	4 (1.9)	80	.266	1 (.79)	20	.451	—	—	—	—	—	—
	Multiple	6 (2.8)	35.2	.082	8 (6.3)	47.06	.237	1 (3.3)	5.8	.595	2 (25)	11.7	.047
53	Total	38 (18.2)	76	.002	10 (7.9)	20	.027	2 (6.6)	4	.202	—	—	—
	Single	4 (1.9)	57.1	.625	2 (1.5)	28.5	.560	1 (3.3)	14.2	.447	—	—	—
	Multiple	34 (16.3)	79	.001	8 (6.3)	18.6	.025	1 (3.3)	2.3	.114	—	—	—
56	Total	15 (7.2)	57.6	.838	9 (7.1)	34.6	.926	2 (6.6)	7.9	.651	—	—	—
	Single	4 (1.9)	40	.243	4 (3.1)	40	.453	2 (6.6)	20	.188	—	—	—
	Multiple	11 (5.2)	68.7	.286	5 (3.9)	31.2	.827	—	—	—	—	—	—
58	Total	20 (9.6)	48.7	.341	13 (10.3)	31.7	.766	5 (16.6)	12.2	.301	2 (25)	4.8	.216
	Single	6 (2.8)	35.2	.082	6 (4.7)	35.2	.893	3 (10)	17.6	.148	1 (12.5)	5.8	.314
	Multiple	14 (6.7)	58.3	.794	7 (5.5)	29.1	.622	2 (6.6)	8.3	.594	1 (12.5)	4.1	.416
59	Total	10 (4.8)	40	.101	13 (10.3)	52	.046	2 (6.6)	8	.675	—	—	—
	Single	3 (1.4)	66.6	.586	1 (.79)	33.3	.735	—	—	—	—	—	—
	Multiple	7 (3.3)	33.3	.033	12 (9.5)	57.1	.02	2 (6.6)	9.5	.518	—	—	—
66	Total	20 (9.6)	50	.438	16 (12.7)	40	.379	2 (6.6)	5	.352	2 (25)	5	.208
	Single	7 (3.3)	58.3	.856	4 (3.1)	33.3	.620	1 (3.3)	8.3	.640	—	—	—
	Multiple	13 (6.2)	46.4	.302	12 (9.5)	42.8	.292	1 (3.3)	3.5	.318	2 (25)	7.1	.115
68	Total	8 (3.8)	61.5	.67	5 (3.9)	38.4	.717	—	—	—	—	—	—
	Single	1 (.48)	33.3	.442	1 (.79)	100	.338	—	—	—	—	—	—
	Multiple	7 (3.3)	53.8	.785	4 (3.1)	40	.453	—	—	—	—	—	—
69	Total	1 (.48)	33.3	.414	1 (.79)	33.3	.735	—	—	—	—	—	—
	Single	1 (.48)	33.3	.414	1 (.79)	33.3	.735	—	—	—	—	—	—
	Multiple	—	—	—	—	—	—	—	—	—	—	—	—
73	Total	2 (.96)	33.3	.241	3 (2.3)	50	.328	—	—	—	—	—	—
	Single	0 (0)	—	—	—	—	—	—	—	—	—	—	—
	Multiple	2 (.96)	33.3	.241	3 (2.3)	50	.328	—	—	—	—	—	—
82	Total	1 (.48)	20	.122	4 (3.1)	80	.046	—	—	—	—	—	—
	Single	—	—	—	—	—	—	—	—	—	—	—	—
	Multiple	1 (.48)	20	.122	4 (3.1)	80	.046	—	—	—	—	—	—

HR-HPV positive n (%, col): number of cases with at least 1 high-risk HPV genotype detected, including cases with co-infection by 2 or more high-risk HPV genotypes.

HR-HPV prevalence (%, row), is defined as the percentage of women with at least 1 high-risk HPV genotype detected by cervical histopathological diagnosis.

Values in bold type indicate significant P-values (P < .05), chi square test np_trend to determine differences in proportions between groups with cervical histopathologic diagnosis and HR-HPV genotype.

Variables with cell <5 values were evaluated with Fischer´s exact test. Values in bold type indicate significant P-values (P < .05).

A bivariate analysis of histopathology diagnosis and coinfection by each genotype showed significant differences in HPV-53 and HPV59 positivity in patients with CIN1; significant differences were also observed in patients with CIN2 and with infection by HPV-18, and in patients with CIN3 with respect to infection by HPV-33, HPV35, and HPV-52. No significant differences in histopathology diagnosis were observed in patients with single infections (Table 4).

In a bivariate analysis of risk factors with the prevalence of premalignant lesions, statistically significant differences were found in age in the CIN1 group, as well as in age, number of pregnancies, and current smoking habit in the CIN2 group (Table 5). However, the logistic regression analysis only showed association with current smoking habit in the latter group (Table 6).

Table 5.

Sociodemographic, Sexual, and Reproductive Characteristics of Patients and Cervical Cancer Stage.

	Negative for SICL or Malignancy, n = 209			Low-grade SICL, CIN1, Condyloma Ordinary Dysplasia, n = 126			High-grade SICL, CIN2, Moderate Dysplasia, n = 30			High-grade SICL, CIN3, Severe Dysplasia, Squamous Cell Carcinoma in situ, n = 8
Characteristics	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value	n (%)	Prevalence	P Value
Age (years)			.147			.002			.004			.678
15–29	55 (26.4)	50		50 (39.6)	45.4		2 (6.6)	1.8		2 (25)	1.8
30–49	125 (60.1)	57.6		65 (51.5)	29.9		21 (70)	9.6		6 (75)	2.7
≥50	28 (13.4)	60.8		11 (8.7)	23.9		7 (23.3)	15.2		0 (0)	0
Herpes simplex virus type 2			.353			.424			.727			.289
Negative	146 (69.8)	54.6		93 (74.4)	34.8		20 (68.9)	7.4		7 (87.5)	2.6
Positive	63 (30.1)	60		32 (25.6)	30.4		9 (31.03)	8.5		1 (12.5)	.95
Previous sexually transmitted diseases			.375			.758			.449			.451
No	186 (89.4)	55.03		115 (91.2)	34.02		28 (93.3)	8.2		8 (100)	2.3
Yes	22 (10.5)	62.8		11 (8.7)	31.4		2 (6.6)	5.7		0 (0)	0
Age at menarche (years)			.610			.741			.951			.156
≤11	73 (35.1)	49.3		56 (44.4)	37.8		13 (43.3)	8.7		6 (75)	4.05
12–14	120 (57.6)	58.8		65 (51.5)	31.8		16 (53.3)	7.8		2 (25)	.98
≥15	15 (7.2)	71.4		5 (3.9)	23.8		1 (3.3)	4.7		0 (0)	0
Age at first intercourse			.588			.376			.542			.303
≤15	48 (23.08)	51.06		33 (26.1)	35.1		9 (30)	9.5		4 (50)	4.2
16–17	63 (30.2)	55.7		41 (32.5)	36.2		7 (23.3)	6.1		2 (25)	1.7
≥18	97 (46.6)	58.4		52 (41.2)	31.3		14 (46.6)	8.4		2 (25)	1.2
Number of lifetime sexual partners			.787			.840			.956			.186
1	79 (37.9)	57.2		45 (35.7)	32.6		12 (40)	8.7		1 (12.5)	.72
2–3	92 (44.2)	54.4		59 (46.8)	34.9		12 (40)	7.1		6 (75)	3.5
≥4	37 (17.7)	56.06		22 (17.4)	33.3		6 (20)	9.09		1 (12.5)	1.5
No. of full-term pregnancies			.211			.978			.029			.834
Nulliparous (no birth)	16 (7.6)	66.6		7 (5.5)	29.1		1 (3.3)	4.1		0 (0)	0
1–2	78 (37.5)	57.3		48 (38.1)	35.2		5 (16.6)	3.6		4 (50)	2.9
≥3	114 (54.8)	53.5		71 (56.3)	33.3		24 (80)	11.2		4 (50)	1.8
Contraceptive use (among sexually active)
Use of oral contraceptives			.068			.185			.508			.839
Never	201 (96.6)	55.07		125 (99.2)	34.2		30 (100)	8.2		8 (100)	2.1
At least once	7 (3.3)	87.5		1 (.79)	12.5		0 (0)	0		0 (0)	0
Use of injectable contraceptives			.896			.366			.163			.626
Never	196 (94.2)	55.6		117 (92.8)	33.2		30 (100)	8.5		8 (100)	2.2
At least once	12 (5.7)	57.1		9 (7.1)	42.8		0 (0)	0		0 (0)	0
Use of condom			.409			.959			.161			.455
Never	195 (93.7)	56.3		117 (92.8)	33.8		26 (86.6)	7.5		7 (87.5)	2.02
At least once	13 (6.2)	48.1		9 (7.1)	33.3		4 (13.3)	14.8		1 (12.5)	3.7
Use of other contraceptive method			.822			.980			.521			.657
Never	32 (15.3)	57.1		19 (15.08)	33.9		4 (13.3)	7.1		1 (12.5)	1.7
At least once	176 (84.6)	55.5		107 (84.8)	33.7		26 (86.6)	8.2		7 (87.5)	2.2
History of smoking			.986			.337			.079			.599
No	159 (76.4)	55.7		100 (79.3)	35.09		19 (63.3)	6.6		6 (75)	2.1
Yes	49 (23.5)	55.6		26 (20.6)	29.5		11 (36.6)	12.5		2 (25)	2.2
Smoking behavior			.742			.700			.048			.283
Never smoked	179 (86)	56.1		109 (86.5)	34.1		22 (73.3)	6.9		8 (100)	2.5
At least once	29 (13.9)	53.7		17 (13.4)	31.4		8 (26.6)	14.8		0 (0)	0
Among smokers, no. of cigarettes/week			.964			.421			.079			—
1-5	13 (6.2)	56.5		7 (5.5)	30.4		3 (10)	13.04		0 (0)	0
6-20	11 (5.2)	57.8		4 (3.1)	21.05		4 (13.3)	21.05		0 (0)	0
>20	5 (2.4)	41.6		6 (4.7)	50		1 (3.3)	8.3		0 (0)	0
Number of HPV-HR types			.343			.741			.354			.124
Negative	70 (33.6)	61.9		33 (26.1)	29.2		9 (30)	7.9		1 (12.5)	.88
1	73 (35.1)	52.1		49 (38.8)	35		14 (46.6)	10		3 (37.5)	2.1
2	41 (19.7)	53.2		29 (23)	37.6		6 (20)	7.7		1 (12.5)	1.3
3	19 (9.1)	57.5		11 (8.7)	33.3		0 (0)	0		3 (37.5)	9.09
≥4	5 (2.4)	50		4 (3.1)	40		1 (3.3)	10		0 (0)	0

Herpes simplex virus type 2 test was performed in a StepOne Real-Time PCR system, using the SYBR select master mix.

Previous sexually transmitted diseases were self-reported by the patient.

Use of other contraceptive methods: coitus interruptus, intrauterine device, bilateral tubal occlusion, vasectomy, subdermal implant, or not using any.

Prevalence (%, row), is defined as the percentage of women detected by cervical histopathological diagnosis by specific stratum for each characteristic evaluated.

Values in bold type indicate significant P-values (P < .05), chi square test np_trend to determine differences between groups with cervical histopathologic diagnosis for categorical variables.

Variables with cell <5 values were evaluated with Fischer´s exact test. Values in bold type indicate significant P-values (P < .05).

Table 6.

Association Between Cervical Cancer Stage and Risk Factors.

	Negative for SICL or Malignancy, n = 209				Low-grade SICL, CIN1, Condyloma Ordinary Dysplasia, n = 126				High-grade SICL, CIN2, Moderate dysplasia,n = 30				High-grade SICL, CIN3, Severe Dysplasia, Squamous Cell Carcinoma in situ, n = 8
Characteristics	Unadjusted OR (95% CI)	P-value	Adjusted OR by age^a (95% CI)	P-value	Unadjusted OR (95% CI)	P-value	Adjusted OR by age^a (95% CI)	P-value	Unadjusted OR (95% CI)	P-value	Adjusted OR by age^a (95% CI)	P-value	Unadjusted OR (95% CI)	P-value	Adjusted OR by age^a (95% CI)	P-value
Married/living as married
No	1.1 (.7–1.8)	.466	1.1 (.7-1.8)	.509	.9 (.5–1.4)	.719	.8 (.5-1.4)	.579	1.7 (.8–3.7)	.145	1.9 (.8–4.1)	.107	2.2 (.5–9)	.263	2.6 (.6–11)	.177
Yes	Reference
Age at first intercourse
≤15	1.3 (.8–2.2)	.251	1.2 (.7–2.1)	.327	1.1 (.6–2.02)	.533	1.07 (.6–1.8)	.804	1.1 (.4–2.7)	.756	1.3 (.5–3.3)	.492	3.6 (.6–20.2)	.14	4 (.7–22.7)	.118
16–17	1.1 (.6–1.8)	.657	1.07 (.6–1.7)	.764	1.2 (.7–2.06)	.389	1.1 (.6–1.9)	.59	.7 (.2–1.8)	.488	.8 (.3–2.1)	.683	1.4 (.2–10.6)	.698	1.5 (.2–11.1)	.675
≥18	Reference
Number of lifetime sexual partners
1	Reference
2–3	1.1 (.7–1.7)	.622	1.1 (.7–1.7)	.619	1.1 (.6–1.7)	.672	1.1 (.6–1.8)	.661	.8 (.3–1.8)	.605	.8 (.3–1.8)	.613	5 (.5–42.3)	.136	4.8 (.5–40.7)	.147
≥4	1.04 (.5–1.8)	.873	.9 (.5–1.8)	.989	1.03 (.5–1.9)	.918	.9 (.4–1.7)	.794	1 (.3–2.9)	.926	1.2 (.4–3.7)	.629	2.1 (.1–34.2)	0.6	2.1 (.1–34.9)	.597
No. Of full-term pregnancies
Nulliparous (no birth)	Reference
1–2	1.4 (.5–3.7)	.385	1.7 (.6–4.3)	.247	1.3 (.5–3.4)	.561	1.6 (.6–4.3)	.309	.8 (.09–7.8)	.907	.6 (.06–5.7)	.662	—	—	1.6 (.3–7.7)	.533
≥3	1.7 (.7–4.2)	.224	2.8 (1.06–7.5)	.037	1.2 (.4–3)	.681	2.5 (.9–7.1)	.073	2.9 (.3–22.6)	.305	1.1 (.1–10.4)	.886	1.5 (.3–6.4)	.521	—	—
Smoking behavior
Never smoked	Reference
At least once	1.1 (.6–1.9)	.742	1.02 (.5–1.8)	.935	.8 (.4–1.6)	.699	.7 (.3–1.4)	.358	2.3 (.9–5.5)	.054	3.5 (1.4–9)	.007	—	—	—	—
Among smokers, No. of cigarettes/week
1–5	Reference
6–20	.9 (.2–3.2)	.929	.9 (.2–3.3)	.959	.6 (.1–2.5)	.493	.6 (.1–2.6)	.538	1.7 (.3–9.1)	.492	1.7 (.2–9.7)	.55	—	—	—	—
>20	1.8 (.4–7.4)	.406	1.9 (.4–8.1)	.354	2.2 (.5–9.6)	.26	2.7 (.6–12)	.175	.6 (.05–6.5)	.68	.4 (.03–4.9)	.499	—	—	—	—

HR-HPV n (%): Number of cases and HR-HPV prevalence are defined as the absolute number and the corresponding percentage of women with at least 1 high–risk HPV genotype detected by specific stratum for each characteristic evaluated.

Values in bold type indicate significant P-values (P < .05) for OR.

Statistically significant P-values ≤.05 of OR are marked in bold type.

^aOdds ratio for all variables, adjusted by age.

Discussion

Cervical cancer was the fourth most important neoplastic disease in Mexico in females of all ages in 2020, according to GLOBOCAN.²⁷ Persistence of HR-HPV infection is a prerequisite for the modification of the squamous epithelium and progression to a preneoplastic lesion, which may result in cancer.¹ As the quadrivalent HPV vaccine (Gardasil, Merck Sharp & Dohme) only prevents infection by high-risk genotypes 16 and 18, observational studies evaluating the prevalence of other high-risk genotypes could help us to design new vaccines targeting most prevalent genotypes by region, increasing the coverage against non-16/18 genotypes. This study provided additional information on the relative distribution of HR-HPV genotypes other than HPV-16 and -18, and the risk factors associated in a Mexican population.

The overall prevalence of HR-HPV infection in women with precancerous lesions who were referred from first-level health centers was 69.8%, a value similar to that reported in other cross-sectional studies conducted in dysplasia clinics in some regions of Mexico (60%–90%);^28-32 these rates are lower than those reported in studies with regional representativeness (24.78%),⁷ but they are higher than the prevalence in the open population in Mexico.^5,6 The prevalence of HR-HPV infection age-specific in our population is distributed as a “U-shaped” curve. In contrast with other studies in Mexico and worldwide,^4,28,33,34 the first peak occurs at ages in the range 25–29 years, with a decline in the number of cases in middle-aged women (30–49 years); the second peak, observed after menopause, could be explained by the reactivation of a latent HR-HPV infection linked with a decline in the immune response.^35,36 Infection rates are strongly dependent on the number of sexual partners and a patient’s sexual behavior. Higher transmission rates result from the increase in the number of cases in young women, aged 20–25 years. This peak is followed by a decline in the number of cases due to a spontaneous elimination of infection resulting from a successful immune response and a lesser exposure to new sexual partners as the age of the patients increases.^11,37,38

The most prevalent HR-HPV genotypes in women attending the CAPASAM dysplasia clinic in 2015–2021 were HPV-53, HPV-16, HPV-58, HPV-31, HPV-66, and HPV-56. Our results showed that HPV-16 and HPV-18 were replaced by other HR-HPV types in the Mexican population. These results are comparable with other studies conducted in Mexico.^{4,28,31,39,40} The genotype distribution pattern showed that the most prevalent combinations were 31-53, 35-53, 53-58, and 33-35. Since these genotypes belong to the α-6 and α-9 species, this could suggest that phylogenetically related HR-HPV genotypes tend to cluster together, in agreement with reports by other authors.^41-43 Senapati et al. reported that patients coinfected with phylogenetically related genotypes had a 2.9-fold increased risk of invasive cervical carcinoma with respect to other genotypes; thus, phylogenetically-driven, specific interactions between genotypes in multiple infections could increase or decrease the risk of cervical cancer.⁴⁴

Variables related to sexual behavior, such as marital status and the number of lifetime sexual partners, along with history of smoking, were associated with HR-HPV multiple infection by any genotype, in agreement with findings of other authors.^45-47 However, no association was observed between these risk factors and single infection by any HR-HPV genotype.

When we evaluated the association of various risk factors with infections by HPV-16, HPV-18, and non-16/18 HR-HPV, no association was observed with HPV-18 positivity, neither as single nor multiple infection.

Single women had a higher risk of acquiring multiple infections by HPV-16 and oncogenic non-HPV-16/18 than married or cohabiting women. This result had already been reported by Kops et al⁴⁷ for multiple infections by the genotypes HPV-39, HPV-51, and HPV-58; in that study, the incidence of multiple infections was 1.29 times higher among single subjects compared with respect to married or cohabiting participants.

A positive association was found between having had more than 4 sexual partners and multiple infections by HPV-16 and non-16/18 HR-HPV, specifically HPV-31, HPV-53, and HPV-58; this is in line with the findings of other authors, who reported that unmarried subjects and those who had had a greater number of sexual partners over 5 years showed higher rates of multiple HPV infections.⁴⁷

The positive association between smoking history and multiple infections by HPV-16 and non-16/18 HR-HPV, specifically HPV-53 and HPV-58, has also been reported by other authors.^48,49 Several components of cigarette smoke and their metabolites have been detected in cervical mucus and may interact with HPV. Benzo[a]pyrene increased the number of HPV genome copies, modulated HPV DNA replication, and accelerated the HPV replication cycle.⁵⁰ Nicotine and nicotine-derived nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone impair the defense mechanisms in cervical epithelium.⁵¹ Xi et al. found that baseline viral load was higher among current smokers that in women who never smoked.⁵²

On the other hand, in our study, the number of pregnancies was a protective factor for positivity to multiple non-16/18 HR-HPV infections. Specifically, the risk of multiple HPV-39 and HPV-58 infections was lower in women who had at least 1 pregnancy. This could be explained by the fact that pregnant women are less likely to have new sexual partners, which decreases their exposure to HR-HPV infection. However, other authors have reported that having >7 full-term pregnancies increased the risk of invasive cervical carcinoma compared to women who had 1-2 full-term pregnancies. This has been attributed to the hormonal profile during pregnancy and eversion of the columnar epithelium over the ectocervix (ectopia) due to birth-related trauma, which favors the exposure of the squamocolumnar junction to HR-HPV infection.^17,53 In agreement to this, no differences were observed between risk factors for HPV-16 infection or non-16/18 HR-HPV multiple infections.

With regard to the prevalence of HR-HPV infection by histopathological diagnosis, we found that the prevalence of infection due to any HR-HPV genotype increased directly with the severity of cervical lesions, which is consistent with other studies in Mexico.^28,29,39,54 Interestingly, the lack of significant differences in the prevalence of single or multiple HR-HPV infections between patients with differing histopathological diagnoses in our study are in contrast with reports in other studies, that high-grade cervical lesions are linked with simple infections.^31,55

The distribution of HR-HPV genotypes showed that HPV-16 predominated in patients diagnosed with CIN1 and CIN2, whereas the most prevalent genotype in patients with CIN3 was HPV-35. The higher prevalence of single and multiple HPV-16 infections in women with CIN1 and CIN2 indicates a higher oncogenic capacity than that of other genotypes, as reported by Padilla et al; according to that author, HPV-16 infection led to a 10.3-fold increase in the risk of developing cervical cancer.³¹ Association with current smoking habit was found only in the CIN2 group. This association has also been observed by other authors, who reported that current smoking increases the risk of developing CIN2 by a factor of 1.89, as the rate of HR-HPV infection increased.⁵⁶

The main strengths of this study are the identification of risk factors associated with non-16/18 HPV infection, in addition to determining the prevalence of non-16/18 HPV infections in dysplasia clinics in central Mexico. The main limitation of our study is that the risk factors associated with premalignant lesions could be underestimated, since the sample size of women with premalignant lesions in the baseline study was small. Additionally, our population is not representative of other regions of the world, so these results are not generalizable to other populations.

In conclusion, the prevalence of single and multiple HR-HPV infections revealed a predominance of non-16/18 multiple HR-HPV infections. The risk factors for acquiring non-16/18 HR-HPV multiple infections are no different than those linked to HPV-16 multiple infections. Finally, HPV-16 was the most prevalent genotype in low- and high-grade cervical lesions. Our results describe the characteristics of a population in a dysplasia clinic, and these data could help us to determine the genotype circulation and identify women at a higher risk to develop cervical lesions.

Supplemental Material

Supplemental Material - Prevalence and Risk Factors for High-Risk Human Papillomavirus Infection and Cervical Disorders: Baseline Findings From an Human Papillomavirus Cohort Study

Supplemental Material for Prevalence and Risk Factors for High-Risk Human Papillomavirus Infection and Cervical Disorders: Baseline Findings From an Human Papillomavirus Cohort Study by Paula Saldaña-Rodríguez, Margarita Bahena-Román, Karina Delgado-Romero, Vicente Madrid-Marina, and Kirvis Torres-Poveda in Cancer Control

Supplemental Material

Supplemental Material - Prevalence and Risk Factors for High-Risk Human Papillomavirus Infection and Cervical Disorders: Baseline Findings From an Human Papillomavirus Cohort Study

Footnotes

Acknowledgments

The authors thank Juraj Lord for assistance in the preparation of the manuscript. This work was submitted in partial fulfillment of the requirements for the PhD degree of Paula Saldaña-Rodríguez from the Doctoral Program in Sciences of the School of Public Health of Mexico.

Author Contributions

KTP conceived the idea and had a full role in the conceptualization and design of the study, formal analysis, draft writing, and revision of the manuscript; PSR participated in the collection of clinical materials, analysis of the results, and manuscript draft. KDR participated in the selection of study population, gynecological specimen sampling, and patient care. MBR carried out HPV genotyping analyses. VMM participated in the analysis of the results and manuscript draft. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest Statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the Instituto Nacional de Salud Pública, Mexico, and grants from the Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico) to KTP (FONSEC SSA/IMSS/ISSSTE-2018 grant # A3-S-49099) and to VMM (grant CATEDRAS-2014-C01-#245520).

Ethical Statement

ORCID iD

Kirvis Torres-Poveda

Supplemental Material

Supplemental material for this article is available online.

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