Genetic diversity of human papillomavirus (HPV) as specified by the detection method, gender, and year of sampling: a retrospective cross-sectional study

doi:10.21203/rs.3.rs-1653831/v1

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Research Article

Genetic diversity of human papillomavirus (HPV) as specified by the detection method, gender, and year of sampling: a retrospective cross-sectional study

https://doi.org/10.21203/rs.3.rs-1653831/v1

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published 10 Jan, 2023

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Purpose.

This study assesses HPV prevalence and genotype distribution in Lebanon, and to identify differentials in HPV infection, infection with multiple genotypes, and with high-risk genotypes, by sex, age, and year of data collection.

Methods.

Study participants comprised 1,042 female and 160 male participants between 2006–2018. HPV genotyping was done by PCR and hybridization (2006–2013) or real-time PCR (2013 onwards). Diversity of HPV genotypes across gender, age groups, and years of data collection was tested by applying Shannon Diversity Index.

Results.

The overall HPV prevalence was 44.8% among study participants, and 3-fold higher in women than men. Single HPV infection was seen in two-third of HPV-positive participants. Women were less likely to be infected with multiple HPV strains, but more likely to be infected with high-risk or mixed risk HPV genotypes. HPV16 (11.0%, 9.8%) and HPV53 (8.5%, 4.9%) were the most prevalent high-risk HPV genotypes in women and men, respectively, while HPV18 prevalence was 4.9% in men and 3.1% in women, while HPV59 prevalence was 6.6% in men and 2.1% in women. Samples collected post-2011 from women showed twice higher odds of HPV infection than those collected earlier and were 3-fold more likely to be infected with multiple HPV strains, and twice more likely to be infected with high-risk genotypes compared to those tested earlier. Women scored higher on Shannon index indicating high diversity in HPV types and frequency, with trend of increased diversity over time. While the odds of HPV infection remained associated with sex and temporal trend in multivariable analysis, odds of having high-risk genotypes was mainly associated with infection with multiple HPV strains.

Conclusion.

Our study showed high diversity in HPV genotypes and an increasing trend of infection with multiple and high-risk genotypes in recent years. Findings underscore the need for effective screening/surveillance and HPV vaccination programs.

Cervical cancer

Genetic diversity

Genotypes

Human papillomavirus

Cervical cancer is the fourth most prevalent cancer among women [1], with 570,000 women developed cervical cancer in 2018, and more than 300,000 related deaths were reported globally [2, 3], of which 80% occurred in low-income countries [1]. Cervical cancer is the 6th most frequent cancer among Lebanese women aged 15–44 years [4, 5], and the 12th leading cause of deaths among all women [5, 6]. Human papillomavirus (HPV) is a well-established risk factor for cervical cancer, of which around 200 genotypes were identified, with pathogenic potential limited to few genotypes [7, 8].

The double-stranded HPV DNA virus belongs to Papillomaviridae family, which targets skin and epithelial cells and mucous membrane [9, 10]. Its circular genome consists of 8 open reading frames encoding early (E1-E7) region needed for replication, late region (L1-L2) encoding the structural proteins, and noncoding long control region [10] divided into 5^’, central, and 3^’ segments [11, 12]. HPV infection is linked with several pathologies, including anogenital cancers [13, 14], head-and-neck cancers [13, 15], particularly oropharyngeal cancer [16], and infection of the penis, reported in 16–69% of otherwise healthy men [13, 17].

HPV genotypes were classified based on their carcinogenic profile into low-risk (LR) and high-risk (HR) [8, 18]. Of these, 14 are highly carcinogenic linked to cervical cancer [19], including HPV-16 and HPV-18 identified as the major cervical cancer-causing HPV genotypes [20]. By comparison, LR HPV types 6, 11, 40, 42, and 54 are associated with cellular proliferation, including condyloma acuminata [8, 19], along with recently identified HPV genotypes with ill-defined pathogenic capacity. Accordingly, HPV genotypes were clustered based on genetic distancing between viral genomes. As described for HBV [20, 21], HPV genotype diversification results from co-divergence between viruses and their hosts, with most HPV infections being asymptomatic, others causing latent infections, and few are carcinogenic.

New categorization based on complete genome analysis, divided HPV types into lineages (1–10%) and sublineages (0.5-1%) linked with differential infection prognosis [22, 23]. Disruption of HPV proteins is key for emergence of new HPV types related to papillomaviruses phylogenetic assembly [24], while sublineages possess biological characteristics consistent with persistent infection and cervical cancer [23]. Despite the increased HPV infection prevalence among women and men in Lebanon [5, 25], and the heightened risk of progression to cervical cancer, comprehensive data on the prevalence and distribution of HPV genotypes in Lebanon are sparse. Here we assess HPV prevalence and genotype diversity, and examine associations with HPV infection, infection with multiple genotypes, and with high-risk genotypes in asymptomatic men and women between 2006–2018.

Study design and data collection

Between 2006–2018 HPV testing information were collected for 1,202 participants, of whom 1,042 (86.7%) were females seen at OB/GYN and Urology outpatient clinics. Patients’ socio-demographic information were retrieved through review of medical charts. HPV genotyping was warranted in case of recent abnormal Papanicolaou test, after immunization with HPV vaccine (Cervarix Gardasil-4, Gardasil-9), known HPV carrier, self-declared multiple sexual partners, or partner with multiple sexual partners, before starting new relationship, and after breaking high-risk relationship. HPV DNA testing was also indicated in non-protected sex, vulvar warts, extra conjugal affair, or concern about potential sexually transmitted infections (STI). Inclusion criteria for females were ≥ 21 years of age, presence of vaginal portion of uterine cervix, sexually active (vaginal, anal), willing to undergo primary HPV high-risk (PCR) screening individually or as co-test, testing concern of potentially having STI. Exclusion criteria were young age (< 20 years), pregnancy, history of cervical lesions, and current/recent history of radiotherapy, chemotherapy, or surgical treatment. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Research & Ethics Committee of St. Marc Medical Center (Number, SMMC-2019-0047), granted on October 17, 2019.

HPV Genotyping

Cervical and vaginal scrapes were collected from females using Ayre spatula, and genital scraps (urethral epithelium, penile epidermis) were collected from males using nylon flocked swabs and placed in sterile saline. Between 2006–2010, total genomic DNA was extracted from isolated scrapes by the mini-spin column method using DNeasy Blood and Tissue kits, per manufacturer’s instructions (Qiagen, Courtaboeuf Cedex, France). From 2011 onwards, genomic DNA extraction was done using MagPurix automated extractor (Zinexts Life Science Corp., Taiwan). Between 2006 and 2013, HPV genotyping was done by PCR followed by hybridization, using AID HPV TYPING PCR kit (AID AUTOIMMUN DIAGNOSTIKA GMBH, Strasberg, Germany). This involved PCR, line probe assay, and manual reading using AID Scanner (AID AUTOIMMUN DIAGNOSTIKA). Control bands were present on all strips for validation of DNA extraction, amplification, and hybridization. From 2013 onwards, real time (RT)-PCR was used for HPV genotyping, using automated DNA extraction (PREP-NA kits; DNA Technology, Moscow, Russia), followed by RT-PCR using cycling conditions provided by the manufacturer (DNA Technology).

Statistical analysis

Analyses were performed in Stata/SE 15.1 (College Station, TX). Frequency distributions were used to describe participants’ baseline characteristics stratified by sex. Bivariable analyses applying Chi² test and univariable logistic regression were conducted to associations with HPV positivity. Additional bivariable analyses were conducted on subsamples, including only HPV-positive patients, to examine differences in the type and risk classification of HPV genotypes. Odds ratios (ORs) and relative risk ratios (RRRs) with corresponding 95% confidence intervals (CIs) were reported for dichotomous and polytomous outcomes, respectively. Frequency distributions were used to describe HPV genotype distribution (frequency of specific genotype relative to all identified genotypes), stratified by sex.

Study participants testing positive for multiple genotypes, contributed separately to quantitative estimation of each identified genotype. HPV genotype diversity by sex, age group, and year of data collection was assessed by Shannon Diversity Index [26] to resulting distributions, and using data on the global HPV genotype diversity [27]. Univariable and multivariable regression analyses were performed to identify associations with HPV infection. Factors with P-value ≤ 0.1 in univariable analysis were included in the multivariable model, which was conducted to assess the contribution of sex, age, and temporal trend to the observed variability in HPV infection. The strength of association with HPV infection was determined using adjusted ORs (AORs), 95% CIs, and P-value ≤ 0.05. Similar analyses applying multinomial logistic regression were performed to investigate the association of sex, age, temporal trend, and typology of HPV genotype (single/multiple) with high-risk HPV genotypes in HPV-positive patients.

Sensitivity analysis was performed to assess the robustness of study results to changes in HPV testing post-2011. Data was restricted to study participants undergoing testing post-2011 before re-conducting the bivariable analyses examining differences in type and risk classification of HPV genotypes, and regression analyses examining associations with HPV infection and with high-risk HPV genotypes in HPV positive study participants.

HPV prevalence among study participants. Data were available for 160 men and 1,042 women (Table 1). Among men, over two-thirds were 30–49 years, 43.1% of whom in 30–39 years age group, compared to women who were slightly older (32.3% aged 30–39 years, 24.7% aged 40–49 years, and 21.8% aged 60 + years). HPV prevalence was 44.8%, with the odds of infection being 3-fold higher in women than in men [P < 0.001; OR(95%CI), 3.2(2.2–4.8)]. No significant differences in HPV positivity were noted among men across age groups, or years of data collection. However, samples collected from women post-2011 showed twice higher odds for HPV infection than those collected earlier [P < 0.001; OR(95%CI), 2.0(1.4–2.8)]; no difference noted across age groups. Using year of data collection as linear term suggested an increase in HPV of odds infection by 6%/year among women [P = 0.003; OR(95%CI), 1.06(1.02–1.10)].

Status of HPV infection. Most HPV-positive men (61.1%) and women (66.2%) were infected by single HPV strain (Table 2). Women were less likely than men to be infected with multiple HPV strains [OR(95%CI), 0.8(0.4–1.6)], but more likely to be infected with high-risk [RRR(95%CI), 1.6(0.7–3.5)] or mixed risk [RRR(95%CI), 1.4(0.6–3.4)] genotypes. Although this did not reach statistical significance due to low number of HPV-positive men, this suggested high-risk behavior among men. Subgroup analysis focused on women showed significant reduction in the infection probability with multiple HPV strains, and of high-risk or mixed-risk genotypes with increasing age; older women had a lower risk for HPV infection than younger cohorts. Compared to pre-2011 testing, women testing post-2011 were 3-fold more likely to be infected with multiple HPV strains [OR(95%CI), 3.2(1.7–5.9)] and twice more likely to be infected with high-risk genotypes [RRR(95%CI), 1.9(1.1–3.3)].

Distribution of HPV genotypes. HPV-16 (11.0% and 9.8%) and HPV-53 (8.5% and 4.9%) were the most prevalent high-risk HPV genotypes in women and men, respectively (Fig. 1). By comparison, the prevalence of HPV-18 was 4.9% in men and 3.1% in women, while HPV-59 prevalence was 6.6% in men and 2.1% in women. Assuming equal distribution of HPV genotypes [27], the largest Shannon Diversity Index value was 5.28 [26]. Women scored higher than men on this index (56.3%, 47.8%) indicating high diversity in the types and frequency of HPV genotypes (Table 3). Higher diversity was observed in younger (19–29 years) than older (60 + years) participants (57.4%, 51.1%), and trend of increased diversity over time was noted, as samples from recent years had over 55% diversity compared to samples from earlier years.

Adjusted associations with HPV infection and high-risk genotypes. Strong association of HPV infection was noted for sex, where women had 3-fold higher adjusted odds of infection than men [AOR (95% CI), 3.2 (2.2–4.7)], and for years of data collection, as samples collected post-2011 had twice higher adjusted odds of infection than those collected in earlier years [AOR(95%CI), 1.8(1.3–2.4)] (Table 4). Older age, recent years of data collection, and infection with multiple HPV genotypes were associated with high-risk genotypes in univariable analyses (Table 5). Of these, infection with multiple HPV genotypes was the only factor that remained associated with high-risk genotypes in the multivariable model [P < 0.001; ARRR(95%CI), 7.6(3.8–15.0)], indicating that it was predictor of high-risk genotypes.

Sensitivity analysis. Sensitivity analyses conducted using post-2011 data affirmed the robustness of results, specifically significant decrease in the probability of multiple HPV strains, and of high-risk or mixed-risk genotypes with increasing age (Table S1). Further analysis showed increased frequency of high-risk and mixed-risk genotypes per year post-2011 (Table S1). Whereas the odds of HPV infection remained strongly associated with sex in multivariable analysis, no evidence for variability in prevalence was found in 2011–2018 (Table S2). There was evidence for increased probability of having high-risk genotypes in participants infected with multiple HPV strains, and over year-by-year, starting from 2011 (Table S3).

Few studies reported on HPV genotype diversity in MENA region, and fewer examined HPV genotype distribution among men in Lebanon [28]. This study contributed to filling this gap through assessment of HPV prevalence and genotype diversity and association with HPV infection, infection with multiple and high-risk genotypes in asymptomatic subjects. Study showed high diversity in HPV genotypes and increasing trend of infection with multiple and high-risk genotypes in recent years, highlighting the need for sexual health, screening/surveillance, and targeted HPV vaccination.

Cervical and penile samples provided to accredited laboratories documented HPV prevalence of 44.8%, with 3-fold higher odds of infection in women. This high diversity is attributed to liberalization in sexual norms in Lebanon and other MENA countries [29, 30]. This was partially comparable to Iranian study, which documented 53% HPV positivity in 5,176 cases [31], with notable difference that HPV prevalence was higher among men, and among younger (20–40 years) women [31]. In contrast, a recent smaller Chinese study reported lower incidence of HPV among men, with no effect of age groups, or data collection years on its prevalence [32]. This varied prevalence of HPV among men and women are attributed to factors including high-risk group screening [33], method used [33, 34], and availability of screening facilities.

Earlier HPV genotyping relied on the universal MY09/11 primers [18, 35, 36], and detection by DNA Enzyme ImmunoAssays, RFLP, RT-PCR, or sequencing [18, 35–37]. We compared HPV incidence and genotype profile of specimens tested by PCR-hybridization (2006–2013) with those tested by RT-PCR (post-2011), as reliance on one method may underestimate the HPV prevalence in clinical samples [38, 39]. Significant increased likelihood of infection with multiple HPV strains and high-risk genotypes were noted, in agreement with recent studies which documented varied prevalence of HPV, and novel HPV genotypes based on detection method [37, 40]. This recommends selection of the most appropriate HPV detection based on high specificity and sensitivity, and cost for developing/low-income countries [34, 38, 39, 41].

Effective HPV screening depends on the genotyping method used [41, 42]. Women tested post-2011 were 3-fold more likely to be infected with multiple HPV strains, and twice more likely to be infected with high-risk genotypes, compared to women tested pre-2011. These differences are attributed to increased sensitivity, and broader detection capacity of RT-PCR compared to conventional (strip-based) hybridization [31, 41, 42]. This was in agreement with Chinese [43] and Turkish [38] studies, which documented superiority of RT-PCR in detecting variants not captured by hybridization assays, and in determining HPV viral loads, thus serving as quantitative marker for diagnosing and treating single and multiple HPV infections.

Women were less likely to be infected with multiple HPV strains than men, suggesting high-risk behavior among men. This was consistent with evidence implicating positive spouse as the main source of STI among women in the region. HPV vaccination among men remains limited. Our study suggests that curtailing HPV infections and sequelae entail designing sexual health and vaccination programs, specifically tailored for men.

We evaluated HPV genotypic diversity among men and women from the large and diverse collection of HPV-genotyped samples. While HPV-16 and HPV-53 were the most prevalent HR genotypes among women, higher prevalence of HPV-18 and HPV-59 was noted in men. As women, particularly younger subjects, scored higher than men on Shannon Diversity index [26], this confirms high diversity and frequency of HPV genotypes, in agreement with recent studies on gender-dependent distribution of HPV genotypes [31, 44]. We demonstrated gender-dependent distribution of select HPV genotype, rather than functional genotypic distribution, in contrast to Iranian study documenting high prevalence of HR genotypes in females, and high prevalence of LR genotypes in males [31], and Czech study that suggested higher prevalence of HR and LR HPV genotypes in males [44], These are attributed to anatomical factors (anal, genital, oral) [44, 45], method of detection [34, 38], and sample size [31, 44].

Significant increase in HPV positivity was noted in 40–49 year-old and elder (≥60 years) women. This was in disagreement with German [46] and Chinese [47, 48] studies, which suggested that peak HPV positivity was highest in younger women (20–22 years) linked with earlier sexual activity with (unprotected) risky behavior, and with Iranian study suggesting 30–32-year peak HPV infection in males and females [49]. It is noteworthy that the German study was limited to women up to 30 years of age [46], while the study of Wang was based on uneven age group distribution [48]. Later peak of HPV positivity seen in 55–59-year category attributed to reduced immunity at menopause [47, 50], was also reported.

There are some limitations to this study. The number of male participants was low, hindering conducting further stratified analyses. The study took place in Greater Beirut, where participants had easier access to HPV genotyping, thus limiting the generalizability of our findings. Furthermore, its retrospective nature limited assessment of the outcome of HPV infection. Future studies involving larger number of subjects and from different regions of the country, in particular HPV vaccinated individuals, are of paramount importance towards assessment of HPV infection scope, which is needed for effective national vaccination.

AOR, adjusted odds rate; CI, confidence intervals; HPV, human papillomavirus; HR, high risk; LCR, long control region; LR, low risk; MENA, Middle East-North Africa; OR, Odds ratios; RRR, relative risk ratios

Funding.

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests.

The authors have no relevant financial or non-financial interests to disclose.

Author contributions.

All authors contributed to the study conception and design. Material provision and preparation were provided by Ramzi R. Finan and Eddie Racoubian, data collection and analysis were performed by Hiam Chemaitelly, Eddie Racoubian and Gulzhanat Aimagambetova. The first draft of the manuscript was written by Hiam Chemaitelly, and Gulzhanat Aimagambetova and all authors commented on previous versions of the manuscript. Supervision of the project and extensive editing of the text was provided by Wassim Y Almawi. All authors read and approved the final manuscript.

Ethics approval.

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of St. Marc Medical Center (Number, SMMC-2019-0047), granted on October 17, 2019.

Consent to participate:

Informed consent was obtained from all individual participants included in the study.

Consent to publish:

N/A.

Acknowledgements:

This publication was made possible through infrastructure support provided by the Biostatistics, Epidemiology, and Biomathematics Research Core at the Weill Cornell Medicine-Qatar.

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Table 1 HPV prevalence and socio-demographic characteristics of study participants.

Socio-demographic characteristics	Total sample N (%)	HPV positive N (%)	OR (95% CI)	p-value	LR test p-value
Sex
Men	160 (13.3)	36 (22.5)	1.00		<0.001^*
Women	1,042 (86.7)	503 (48.3)	3.21 (2.18-4.75)	<0.001
MEN
Age (years)
19-29	19 (11.9)	5 (26.3)	1.00		0.305
30-39	69 (43.1)	13 (18.8)	0.65 (0.20-2.13)	0.477
40-49	34 (21.3)	5 (14.7)	0.48 (0.12-1.95)	0.306
50-59	19 (11.9)	6 (31.6)	1.29 (0.32-5.28)	0.721
60+	19 (11.9)	7 (36.8)	1.63 (0.41-6.51)	0.487
Years of data collection
2006-10	46 (28.8)	11 (23.9)	1.00		0.964
2011-15	50 (31.3)	11 (22.0)	0.90 (0.35-2.33)	0.824
2016-18	64 (40.0)	14 (21.9)	0.89 (0.36-2.19)	0.801
Year of data collection
2006-2018 (β; 95% CI)	160 (100.0)	36 (22.5)	0.97 (0.88-1.08)	0.605	0.605
WOMEN
Age (years)
19-29	128 (12.3)	72 (56.3)	1.00		0.251
30-39	336 (32.3)	164 (48.8)	0.74 (0.49-1.12)	0.153
40-49	257 (24.7)	116 (45.1)	0.64 (0.42-0.98)	0.040
50-59	94 (9.0)	48 (51.1)	0.81 (0.48-1.38)	0.444
60+	227 (21.8)	103 (45.4)	0.65 (0.42-1.00)	0.050
Years of data collection
2006-10	233 (22.4)	82 (35.2)	1.00		<0.001^*
2011-15	389 (37.3)	202 (51.9)	1.99 (1.42-2.78)	<0.001
2016-18	420 (40.3)	219 (52.1)	2.01 (1.44-2.79)	<0.001
Year of data collection
2006-2018 (β; 95% CI)	1,042 (100.0)	503 (48.3)	1.06 (1.02-1.10)	0.003	0.003^*

^*Evidence for an association assessed at p-value ≤0.1

Abbreviations: CI, confidence interval; HPV; human papilloma virus; OR, odds ratio.

Table 2 Type and risk classification of HPV genotypes in HPV positive study participants.

HPV genotypes	Single	Multiple	OR multiple gen (95% CI)	Low risk only	High risk only	Mix or unknown	RRR High risk gen (95% CI)	RRR mix/unknown gen (95% CI)
Sex
Men	22 (61.1)	14 (38.9)	1.00	15 (41.7)	12 (33.3)	9 (25.0)	1.00	1.00
Women	333 (66.2)	170 (33.8)	0.80 (0.40-1.61)	160 (31.8)	206 (41.0)	137 (27.2)	1.61 (0.73-3.53)	1.43 (0.61-3.36)
WOMEN^†
Age (years)
19-29	35 (48.6)	37 (51.4)	1.00	14 (18.2)	34 (44.2)	29 (37.7)	1.00	1.00
30-39	105 (64.0)	59 (36.0)	0.53 (0.30-0.93)^*	54 (30.5)	79 (44.6)	44 (24.9)	0.60 (0.30-1.23)	0.39 (0.19-0.83)^*
40-49	80 (69.0)	36 (31.0)	0.43 (0.23-0.78)^*	40 (33.1)	52 (43.0)	29 (24.0)	0.54 (0.25-1.13)	0.35 (0.16-0.78)^*
50-59	37 (77.1)	11 (22.9)	0.28 (0.12-0.64)^*	19 (35.2)	19 (35.2)	16 (29.6)	0.41 (0.17-1.00)^*	0.41 (0.16-1.02)^*
60+	76 (73.8)	27 (26.2)	0.34 (0.18-0.64)^*	48 (43.6)	34 (30.9)	28 (25.5)	0.29 (0.14-0.62)^*	0.28 (0.13-0.62)^*
Years of data collection
2006-10	69 (84.2)	13 (15.9)	1.00	40 (43.0)	29 (31.2)	24 (25.8)	1.00	1.00
2011-18	264 (62.7)	157 (37.3)	3.16 (1.69-5.90)^*	135 (30.3)	189 (42.4)	122 (27.4)	1.93 (1.14-3.27)^*	1.51 (0.86-2.64)
Year of data collection
2006-2018 (β; 95% CI)	333 (66.2)	170 (33.8)	1.36 (1.26-1.48)^*	175 (32.5)	218 (40.5)	146 (27.1)	1.25 (1.16-1.33)^*	1.14 (1.06-1.22)^*

^*Evidence for an association assessed at p-value ≤0.1.

^†Subgroup analysis was restricted to HPV positive women due to the smaller sample size of HPV positive men.

Abbreviations: CI, confidence interval; Gen, genotypes; HPV; human papilloma virus; OR, odds ratio; RRR; relative risk ratio.

Table 3 Diversity in identified HPV genotypes assessed using Shannon Diversity Index

Characteristic	Shannon Diversity Index (H)	Index relative to total possible diversity (%)
Gender
Males	2.53	47.77
Females	2.98	56.28
Age (years)
19-29	3.04	57.44
30-39	2.93	55.42
40-49	2.85	53.93
50-59	2.67	50.47
60+	2.70	51.12
Year of data collection
≤2008	1.89	35.82
2009	1.82	34.33
2010	1.60	30.29
2011	0.90	17.05
2012	1.25	23.68
2013	1.47	27.82
2014	2.45	46.41
2015	2.92	55.32
2016	2.90	54.76
2017	2.98	56.37
2018	2.95	55.73
Overall diversity	2.96	56.07

Table 4 Univariable and multivariable regression analyses in identifying associations with positive HPV test results

Socio-demographic characteristics	Tested	Univariable analysis			Multivariable analysis
Socio-demographic characteristics	N	OR (95% CI)	p-value	Global P-value	AOR (95% CI)	P-value
Gender
Men	160	1.00			1.00
Women	1,042	3.21 (2.18-4.75)	<0.001^*	<0.001^*	3.23 (2.18-4.79)	<0.001^**
Age (years)
19-29	147	1.00			1.00
30-39	405	0.71 (0.48-1.03)	0.071^*		0.79 (0.53-1.17)	0.236
40-49	291	0.65 (0.43-0.96)	0.032^*		0.70 (0.46-1.07)	0.100
50-59	113	0.83 (0.51-1.36)	0.463		0.97 (0.58-1.62)	0.902
60+	246	0.74 (0.49-1.11)	0.141	0.265	0.78 (0.51-1.20)	0.263
Years of data collection
2006-10	279	1.00			--	--
2011-15	439	1.88 (1.38-2.57)	<0.001^*		--	--
2016-18	484	1.86 (1.37-2.52)	<0.001^*	<0.001^*	--	--
Year of data collection^†
2006-2018 (β; 95% CI)	1,202	1.05 (1.01-1.09)	0.005^*	0.005^*	1.04 (1.00-1.08)	0.031^**

AOR, Adjusted odds ratio; CI, confidence interval; HPV; human papilloma virus; OR, odds ratio.

^*Evidence for an association assessed at P-value ≤0.1

^**Strong evidence for an association assessed at P-value ≤0.05

^†Only the continuous form for the year of data collection was considered for the multivariable analysis to avoid collinearity. A sensitivity analysis considering the categorical form for the year of data collection in the multivariable model did not have an impact on regression analysis results.

Table 5 Regression analyses for identify associations with higher risk HPV genotypes in HPV positive subjects

		Univariable analysis					Multivariable analysis
HPV positive cases	Tested	High risk genotypes		Mix/unknown genotypes		Global P-value	High risk genotypes		Mix/unknown genotypes
HPV positive cases	N	RRR (95% CI)	P-value	RRR (95% CI)	P-value	Global P-value	ARRR (95% CI)	P-value	ARRR (95% CI)	P-value
Gender
Men	36	1.00		1.00			1.00		1.00
Women	503	1.61 (0.73-3.53)	0.236	1.43 (0.61-3.36)	0.416	0.472	1.79 (0.75-4.29)	0.191	2.46 (0.85-7.10)	0.095
Age (years)
19-29	77	1.00		1.00			1.00		1.00
30-39	177	0.60 (0.30-1.23)	0.163	0.39 (0.19-0.83)	0.015^*		0.80 (0.37-1.71)	0.565	0.43 (0.18-1.04)	0.060
40-49	121	0.54 (0.25-1.13)	0.101^*	0.35 (0.16-0.78)	0.010^*		0.89 (0.39-2.01)	0.778	0.43 (0.17-1.12)	0.085
50-59	54	0.41 (0.17-1.00)	0.051^*	0.41 (0.16-1.02)	0.056^*		0.67 (0.26-1.74)	0.415	0.64 (0.22-1.88)	0.422
60+	110	0.29 (0.14-0.62)	0.002^*	0.28 (0.13-0.62)	0.002^*	0.021^*	0.47 (0.21-1.07)	0.071	0.38 (0.15-0.98)	0.045^**
Years of data collection
2006-10	93	1.00		1.00			--	--	--	--
2011-15	213	0.97 (0.55-1.72)	0.918	1.06 (0.58-1.94)	0.845		--	--	--	--
2016-18	233	3.92 (2.18-7.06)	<0.001^*	2.42 (1.28-4.57)	0.006^*	<0.001^*	--	--	--	--
Year of data collection^†
2006-2018 (β; 95% CI)	539	1.25 (1.16-1.33)	<0.001^*	1.14 (1.06-1.22)	<0.001^*	<0.001^*	1.18 (1.09-1.27)	<0.001^**	0.96 (0.87-1.05)	0.329
Type of HPV genotype
Single	355	1.00		1.00			1.00		1.00
Multiple	184	8.0 (4.1-15.6)	<0.001^*	29.5 (14.7-59.5)	<0.001^*	<0.001^*	5.54 (2.75-11.14)	<0.001^**	33.3 (15.8-70.3)	<0.001^**

ARRR, Adjusted relative risk ratio; CI, confidence interval; HPV; human papilloma virus; RRR, relative risk ratio.

^*Evidence for an association assessed at P-value ≤0.1

^**Strong evidence for an association assessed at P-value ≤0.05

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Genetic diversity of human papillomavirus (HPV) as specified by the detection method, gender, and year of sampling: a retrospective cross-sectional study

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