Prevalence and clinical correlates of human papillomavirus infection: A study of 7,601 women attending a tertiary referral center in Greece

INTRODUCTION

Cervical cancer remains one of the most common malignancies among women globally, particularly in low- and middle-income countries, with an estimated 600,000 new cases and over 340,000 deaths annually.^[1] Human papillomavirus (HPV) infection constitutes a critical etiological factor in the pathogenesis of cervical cancer and other anogenital neoplasms.^[2] Despite the global implementation of preventive strategies, such as HPV vaccination and cytological screening programs, significant challenges remain in optimizing early detection, risk stratification, and long-term clinical management.^[3] Evidently, the heterogeneity in HPV type distribution, infection patterns across age groups, and the corresponding cytological findings necessitate tailored approaches to public health interventions. While HPV vaccination programs have substantially reduced the incidence of high-risk infections and precancerous lesions in many countries,^[4] ongoing disparities in vaccine uptake and HPV type coverage persist and challenge the effectiveness of current public health strategies.

In the Greek landscape, the national HPV vaccination program was introduced in 2008^[5] and, nowadays, includes the administration of the 9-valent vaccine. However, even though HPV vaccination is officially included in the Greek National Immunization Program and offered free of charge, coverage among adolescent girls remains suboptimal,^[5] particularly in rural regions and underserved populations. Cervical cancer screening is primarily opportunistic, rather than organized, leading to inconsistencies in participation and follow-up. These gaps highlight the importance of local epidemiological data to guide national prevention strategies.

Previous studies have already provided valuable data on HPV genotype distribution among the Greek population,^[6,7] but to-date, large-scale, epidemiological studies on HPV type prevalence and the associated cytological abnormalities across clinically meaningful and representative age cohorts remain limited. Addressing this gap, the present study undertakes a retrospective cross-sectional analysis of 7,601 cases involving concurrent Pap smear and HPV DNA testing, collected over 6 years (2019–2024) at Attikon University Hospital. Therefore, the primary objective of this study is to analyze the prevalence, genotype distribution, and infection burden of HPV, as well as their association with cytological findings, among this specific cohort.

MATERIALS AND METHODS

This retrospective study analyzed ~9,000 medical records collected between 2019–2024 at the Diagnostic Cytology Laboratory, Medical School of the National and Kapodistrian University of Athens (NKUA), located at Attikon University General Hospital, Greece. The dataset was obtained from the Diagnostic Cytology Laboratory of the hospital. All clinical encounters were chronologically sorted, and only the earliest entry per individual was retained for our study (n = 8,061). While excluding follow-up data may under-represent disease progression, the study objective was to assess the distribution of HPV genotypes and cytological outcomes at the point of first contact. Before analysis, the research team de-identified the dataset by removing patient names, dates of examination, sample codes, dates of birth, names of the clinicians collecting the samples, names of the cytologists examining the samples, and all diagnostic dates. All data were anonymized in accordance with institutional data protection protocols to safeguard patient privacy. The final dataset included patient age, HPV genotyping results, and corresponding cytological diagnoses. Due to the retrospective nature of the study, data on various confounding factors, such as HPV vaccination status, detailed sexual behavior history, smoking status, and socioeconomic status, were not consistently or reliably available in the medical records and could not be retrieved for statistical analysis. The detailed process of patient record identification, screening, and exclusion, including the number and specific reasons for all exclusions, has been outlined in Figure 1. The exclusion criteria specifically targeted diagnoses unrelated to cervical intraepithelial lesions (e.g., “inflammation,” “cervical atrophy,” or inadequate samples) to ensure that the analytical focus remained strictly on cytology outcomes relevant to the Bethesda classification system (atypical squamous cells of undetermined significance [ASC-US], low-grade squamous intraepithelial lesion [LSIL], high-grade squamous intraepithelial lesion [HSIL]) and to minimize confounding from benign or non-HPV-related morphological changes. The final analytical sample consisted of n = 7,601 women after applying the predefined inclusion and exclusion criteria.

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Figure 1: Flowchart illustrating the selection process of study participants. The chart details the initial identification of medical records, the exclusion stages with corresponding numbers and reasons, and the final analytical sample size (n = 7,601). This figure was constructed using SmartArt Graphics functionality in Microsoft Word for Microsoft 365 (Microsoft Corporation, Redmond, WA, USA).

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RESULTS

Description of the study cohort and overall HPV prevalence

The mean age in our sample was 42.2 years (SD = 13.2), with an age range of 14-94 years. Participants were categorized into five age groups [Figure 2] using a data-driven approach to ensure adequate sample sizes within each group, while also reflecting meaningful life stages relevant to HPV exposure and cervical cancer screening practices.^[9]

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Figure 2: Number of study participants categorized by age group. Participants were grouped into five age categories: ≤24 (n = 656), 25–34 (n = 1764), 35–44 (n = 2073), 45–54 (n = 1681), and ≥55 years (n = 1427). A total of 7,601 women were included in the analysis.

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Cytological findings were predominantly normal (NILM), observed in 6,254 individuals (82.3%). Abnormal findings were LSIL in 15.9% (n = 1,210), ASC-US in 1.3% (n = 101), and HSIL in 0.5% (n = 36).

Most women (69.0%) had no detectable HPV infection. In contrast, 18.4% harbored a single HPV type, and 12.6% were infected with multiple types [Table 1].

Table 1: Frequencies of HPV infection group.

HPV infection group	Counts	% of Total	Cumulative %
None	5249	69	69
Single	1395	18.4	87.4
Multiple	957	12.6	100.0

The Cumulative % represents the sequential sum of the percentage of participants belonging to the current HPV infection group or any preceding infection group (None/Single/Multiple)

In terms of risk classification, 10.8% of participants (n = 822) were infected with low-risk HPV types, and 20.1% (n = 1,530) tested positive for high-risk HPV (hrHPV) types [Table 2].

Table 2: Frequencies of HPV risk group.

HPV risk group	Count	% of Total	Cumulative %
None	5249	69.1	69.1
Low-risk HPV genotypes	822	10.8	79.9
High-risk HPV genotypes	1530	20.1	100.0

Low-risk HPV genotypes 822 10.8 79.9 High-risk HPV genotypes 1530 20.1 100.0 The “HPV Risk Group” categorization (Low-Risk HPV Genotypes and High-Risk HPV Genotypes) is based solely on the oncogenic potential of the detected HPV genotype (s) and is independent of the concurrent cytological diagnosis (Normal, ASC-US, LSIL, HSIL). ASC-US: Atypical squamous cells of undetermined significance, LSILs: Low-grade squamous intraepithelial lesions, HSILs: High-grade squamous intraepithelial lesions, HPV: Human papillomavirus

The five most frequently detected HPV genotypes were HPV-42 (4.3%), HPV-51 (3.8%), HPV-16 (3.6%), HPV-53 (3.6%), and HPV-54 (3.5%). The full distribution of HPV genotypes detected in the study population is visualized in Figure 3.

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Figure 3: Number of positive detections for each human papillomavirus (HPV) genotype among the study participants. The bar chart shows the frequency of different HPV genotypes detected in the collected samples. HPV genotypes are shown on the X-axis, and the number of positive detections is on the Y-axis.

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Distribution of single vs. multiple HPV infections across age groups

Among HPV-positive women (n = 2,352), the distribution of single vs. multiple HPV infections varied significantly by age group (P <0.001). The proportion of multiple infections decreased steadily with age, from 52.9% in the ≤24 age group to 33.0% among women aged ≥55. Conversely, the prevalence of single-type infections increased from 47.1% in the youngest group to 67.0% in the oldest. The numerical distribution of infection types across age groups has been shown in Figure 4.

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Figure 4: Percentage of participants within each age category by human papillomavirus (HPV) infection group. The bar chart compares the proportion of women with single-type HPV infections vs. multiple-type HPV infections across five age groups (≤24, 25–34, 35–44, 45–54, and ≥55 years). Colors correspond to age categories as shown in the legend. Numerical counts for each age category: ≤24 (single/multiple: n = 153/172), 25–34 (single/multiple: n = 432/335), 35–44 (single/multiple: n = 357/200), 45–54 (single/multiple: n = 266/158), and ≥55 (single/multiple: n = 187/92).

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A Chi-square test of independence confirmed that the association between age group and HPV infection burden (single vs. multiple infections) was statistically significant (χ² (4) = 37.2, P <0.001), with a small effect size (Cramér’s V = 0.126), indicating an age-related shift in the pattern of HPV coinfection.

DISCUSSION

In the present study, we analyzed HPV genotyping and cytological results from a sample of 7,601 women across a wide age range. Our findings reinforce established epidemiological patterns and support the view that age should be a central consideration in cervical cancer screening strategies.

The age distribution in our sample reflects the standard segmentation used in screening programs, with the lowest representation observed for the age group of women under 24. This is consistent with international guidelines, such as those issued by the World Health Organization (WHO),^[11] which recommend initiating screening at age 21. To date, evidence on how HPV vaccination influences the screening behavior of younger women is contradictory. On the one hand, HPV vaccination among younger women may contribute to a sense of security, potentially reducing the likelihood of consistent annual screening.^[12] On the other hand, receiving the HPV vaccine usually requires contact with healthcare providers, which offers an opportunity to raise awareness and educate about the importance of consistent annual cervical screening. Doctors and medical staff often do, and should, emphasize the insufficiency of existing vaccines against all oncogenic HPV types, reaffirming the need for yearly routine Pap or HPV DNA testing among the vaccinated women, too. This way, vaccination may signal an overall behavioral shift toward healthy and preventive behaviors, particularly among individuals with regular and easy access to health care,^[13] thus making their screening routine more consistent. Regardless, the underrepresentation of younger women in our sample highlights the importance of individual risk assessment and personal medical history in tailoring screening protocols, both to reduce the risk of overdiagnosis in low-risk individuals^[14] and to ensure timely diagnosis and intervention for those at higher risk.

The majority of participants in our study (82.3%) presented with normal cytology results, a finding that aligns with large-scale epidemiological studies.^[15] Among all participants, LSIL was the most frequently observed abnormality (15.9%), followed by ASC-US (1.3%) and HSIL (0.5%). These patterns reflect the natural history of HPV infection and the known pathophysiological progression from low-grade lesions to high-grade dysplasia and, eventually, carcinogenesis.^[16] The substantial difference in the number of women with low- vs. high-grade dysplasia further confirms the well-documented potential for spontaneous regression of precancerous cervical lesions.^[17]

Among the women studied, 69.1% tested negative for HPV, a prevalence rate that closely mirrors those reported in previous population-based studies conducted in Greece using PCR methods, with overall HPV prevalence rates ranging from 22-33%.^[18,19] Of all women assessed in our study, 18.4% were infected with a single HPV type, while 12.6% harbored multiple genotypes. Low-risk HPV genotypes were detected in 10.8% of participants, while hrHPV types were found in 20.1%. These results are higher than those reported in a large U.S. post-hoc study,^[20] which found a 10.1% prevalence of hrHPV among over 40,000 women. However, direct comparisons should be made cautiously, as our study had a smaller sample size and included women aged <25 years, an age group excluded from the U.S. study. The inclusion of younger women, among whom hrHPV prevalence is known to be higher, likely contributed to the increased overall prevalence in our sample. Several other large-scale studies report findings that align more closely with ours. A population-based study in Germany found an overall hrHPV prevalence of 23% in women aged 20-30 years.^[21] A Greek study reported a hrHPV prevalence of approximately 25.7% among women aged 16-65, further supporting the representativeness of our findings in a regional context.^[22] A large meta-analysis conducted in subSaharan Africa, encompassing 27 studies across 19 countries, reported a pooled hrHPV prevalence of 34%^[23] with significant variability in hrHPV rates, ranging from 10.7% to 97.2%, depending on several local epidemiological factors and health system differences. This heterogeneity in hrHPV prevalence globally, driven by differences in population structure, sampling strategies, and access to health services, necessitates the interpretation of prevalence rates within their specific epidemiological and healthcare systems.

The most frequently identified HPV genotypes in our study were HPV-42 (4.3%), HPV-51 (3.8%), HPV-16 (3.6%), HPV-53 (3.6%), and HPV-54 (3.5%). Both the high prevalence of HPV-16 and its strong oncogenic potential align with its established role as the leading cause of cervical cancer globally.^[24] However, the absence of HPV-18 among the five most common types, alongside the notable prevalence of HPV-42 and other less frequently documented types,^[25] emphasizes the importance of regionally tailored vaccination strategies. While current vaccines cover a significant number of oncogenic HPV types, shifts in type distribution remain a possibility and should not be underestimated. Such shifts refer to changes in the prevalence of HPV genotypes over time, potentially driven by vaccine-induced selective pressure. As vaccine-targeted types decline, non-vaccine types may become more common by potentially gaining a competitive advantage, which could impact the overall effectiveness of current vaccines unless they are periodically updated to reflect the circulating genotypes.^[26,27]

Another important finding from our study is that the presence of multiple infections is more frequently observed in younger individuals; a predominance that can be attributed to a combination of behavioral, biological, and immunological factors. At first, lifestyle behaviors, such as the use of oral contraceptives and tobacco products, which are more prevalent in younger populations, have been associated with increased HPV acquisition and reduced viral clearance, thereby facilitating the persistence of multiple infections.^[28] One proposed mechanism for this association is the adverse impact of both smoking and oral contraceptive use on serum folate and vitamin B12 levels, which are key nutrients in immune function and DNA methylation. Their deficiency has been linked to impaired immune surveillance and increased risk of viral persistence and progression.^[26] Moreover, sexual behavior, which varies by age group, constitutes another primary factor. Younger women are more likely to contract HPV, either as single or multiple-type infections, due to a higher frequency of partner change^[29] and higher levels of sexual activity, potentially exposing themselves to a broader spectrum of HPV genotypes within a short timeframe.^[30] This behavioral pattern is more likely to result in concurrent infections before immunological clearance can occur. Finally, increased susceptibility to HPV infections has also been attributed to physiological characteristics, like cervical ectopy, a condition commonly observed in adolescents and younger women of reproductive age, in which the columnar epithelium is present on the ectocervix, therefore possibly exposing women to a higher risk of multiple HPV types acquisition.^[31] However, studies have yielded conflicting results,^[32] as some research supports a link between ectopy and heightened vulnerability to HPV infection due to increased cellular turnover and exposure of columnar epithelium, but other studies have not found a consistent or statistically significant correlation, suggesting that ectopy alone may not be a definitive risk factor. Therefore, further research is needed to clarify the role of cervical ectopy in HPV susceptibility, particularly in relation to co-infection dynamics and age-specific biological vulnerability.

In addition, our study also revealed significant differences in the pathogenesis of cytological abnormalities (ASC-US, LSIL, and HSIL) in relation to various HPV genotypes. Most HPV genotypes (e.g., HPV 16, 18, 31, 33, 42, and 73) did not show a significant association with ASC-US, aligning with existing literature that suggests this diagnosis often reflects transient infections or non-HPV-related inflammatory processes.^[33] The only statistically significant and clinically concerning association was observed with HPV 51, a genotype known to be involved in both early lesions and invasive cancer.^[34] Notably, current vaccines do not directly target HPV 51,^[35] highlighting a crucial gap in vaccine coverage. This finding is epidemiologically significant, as it underscores the sustained risk from non-9vHPV types and raises questions about potential genotype replacement. The significant oncogenic contribution of HPV 51 in this cohort strongly advocates for ongoing, targeted surveillance of nonvaccine-covered hrHPV genotypes and supports the future development or implementation of broader-spectrum vaccines, tailored to regional epidemiological profiles. HPV genotypes 16, 18, 31, 33, 42, and 51 demonstrated strong and statistically significant associations with LSIL, reinforcing the notion that not only high-risk but also certain low-risk types (e.g., HPV-42) can contribute to early dysplastic changes. These genotypes can integrate into the host genome, leading to the expression of oncogenes (E6/E7) and suppression of tumor suppressor genes such as p53 and Rb.^[36] The strongest findings emerged for HSIL, with HPV types 16, 18, and 33 showing the highest statistical associations, confirming their well-established high oncogenic potential. HPV 16, in particular, consistently appears as the most prevalent and carcinogenic genotype globally.^[24]

We also found that age appeared to be associated with lesion severity. A marked reduction in ASC-US prevalence was observed with increasing age, particularly in women ≥55 years, supporting previous findings that associate age with a decline in transient, low-grade HPV infections due to immune clearance. Moreover, increasing age clearly appeared to be protective against LSIL development. Women aged ≥35 in the study cohort had significantly lower odds compared to those aged ≤24, consistent with the literature that underscores the transient nature of HPV-related lesions and the decline in new infections after the initial years of sexual activity.^[37] However, in contrast to these milder lesions (ASC-US and LSIL), age was not associated with HSIL occurrence. This suggests that HSIL represents a consequence of chronic, persistent infection with a specific hrHPV genotype. The absence of significant associations with multiple HPV infections or any specific age group highlights that lesion severity is primarily genotype-dependent, rather than influenced by infection burden or age, reinforcing that all patients, regardless of age, may be at risk. Thus, early detection, consistent surveillance, and organized screening programs are critical to preventing the progression of cervical lesions to invasive cancer.

Furthermore, our findings reveal a clear positive association between HPV viral load and the risk of developing cytological abnormalities. Women who tested positive for multiple HPV types were more likely to present with ASC-US or LSIL compared to those infected with a single type. This suggests a possible infection burden-response relationship, wherein the cumulative presence of multiple HPV types may increase the risk of low-grade cytological changes.^[38] In the case of HSIL, both our data and existing literature point to a different pattern: women infected with a single HPV type showed slightly higher rates of high-grade dysplasia compared to those with multiple-type infections. This finding aligns with the prevailing understanding that the presence of at least one high-risk oncogenic type, such as HPV-16 or HPV-18, is a stronger predictor of carcinogenic potential^[39] compared to co-infections involving several low-risk genotypes, which are more likely to be cleared by the immune system.^[40]

Overall, our results underscore the importance of targeted patient monitoring and HPV genotyping in cervical cancer screening programs. Identifying specific HPV types or the presence of multiple infections may help predict the likelihood of both low- and high-grade cervical dysplasia. In addition, further investigation into age-related patterns in HPV genotype prevalence is warranted to inform public healthcare policies. The possibility of carcinogenesis varies considerably by host immune factors, behavioral patterns, and screening access. Therefore, future research should also consider the role of immunosenescence,^[41] sexual behavior trends across age groups, and social determinants of health to optimize risk-based screening algorithms and enhance preventive strategies.

SUMMARY

This retrospective study successfully met its objective of defining the prevalence, genotype distribution, and clinical correlates of HPV infection burden within this specific Greek tertiary care cohort. Our findings confirmed a high overall HPV prevalence of 31% and established a clear burden– response relationship, as multiple infections were significantly associated with the highest Odds for low-grade lesions, particularly among younger women. While the highest risks for HSIL were concentrated among the established oncogenic types (HPV-16, -18, and -33), our analysis also demonstrated significant associations of the low-risk HPV-42 with LSIL and of the non-vaccine-covered HPV-51 genotype with both ASC-US and LSIL. These comprehensive results reinforce the clinical necessity of genotype and infection burden profiling for accurate risk stratification and underscore the critical need for continued age-stratified screening and localized genotype surveillance to inform future prophylactic strategies in Greece.