Introduction

Human papilloma virus (HPV) is the most common sexually transmitted infection (STI) among sexually active young women, being acquired shortly after the first sexual intercourse, with the highest prevalence seen in women aged 25 years or younger [1, 2].

Based on epidemiological and biological data HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 should be considered carcinogenic, or high-risk types (IARC Group 1); HPV26, 53, 66, 67, 68, 70, 73, and 82 probably/possibly carcinogenic or probable/possible high-risk types (IARC Groups 2A and 2B) and HPV 6 and 11 non-carcinogenic, or low-risk types (IARC Group 3) [3].

Persistent infection with HPV, especially high-risk types, leads to the development of cervical cancer [4]. HPV-16 and HPV-18 are the most common carcinogenic types, responsible for almost 70% of all cervical cancers. Five additional high-risk types, 31, 33, 45, 52, and 58, are responsible for another 15% of all cervical and 11% of all HPV-associated cancers. HPV-6 and HPV-11 are the two primary “low-risk” types that cause anogenital warts [5].

Cervical cancer is the fourth most common cancer among women, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020, with nearly 90% of these occurring in low- and middle-income countries [6]. In Latin America and the Caribbean, approximately 60,000 new cases of cervical cancer and more than 31,000 deaths due to the disease occurred in 2020. In Paraguay, the incidence is much higher than that observed in other countries in the region, with incidence and mortality rates of 34.1 and 19.1 per 100,000 women, respectively [7].

Cervical cancer is a disease which reflects inequities among different populations depending on the availability of a national vaccination program and population-based cervical cancer screening, and access to quality treatment. In 2020, the World Health Organization (WHO) called for action to eliminate cervical cancer as a public health problem. With three key strategies and clear 2030 targets: 90% of girls fully vaccinated with HPV vaccine by 15 years of age, 70% of women screened using a high-performance test by 35 years of age and again by 45 years, and 90% of women identified with cervical disease treated [8].

The primary prevention approach focuses on preventing disease before it develops. WHO recommends including the HPV vaccine in the national immunization schedule. Currently, three HPV vaccines have been internationally approved: a bivalent (Cervarix), a quadrivalent (Gardasil), and a nonavelent (Gardasil-9) vaccine, consisting of type-specific HPV L1 virus-like particles (VLPs) that induce type-restricted protection. All three vaccines prevented HPV-16 and HPV-18 infection, with the quadrivalent Gardasil also protecting against HPV-6 and HPV-11, and the nonavalent Gardasil-9 targeting an additional five HPV types (HPV-31, 33, 45, 52, and 58) [9]. These vaccines may also have some cross-protection against other less common oncogenic types, such as HPV-31, 33, and 45 [10].

Secondary prevention, based on cervical screening program, is widely implemented. The adoption of Pap smear and HPV testing resulted in a significant increase in the diagnosis of cervical dysplasia and a significant decrease in cervical cancer, mainly in more developed countries. New screening strategies, such as HPV self-sampling, and the use of artificial intelligence in colposcopy assessment, must be disseminated in a near future [11].

Some markers, including p16 ink4a, p16, E-cadherin, Ki67, pRb, and p53, have been found to be useful in identifying intra-epithelial lesions that are more likely to develop into invasive lesions. Others, such as CEA, SCC-Ag, CD44, were developed to detect invasive forms. These biomarkers represent a promising diagnostic, prognostic and dynamic tool and can also be easily performed in the management of cervical cancer [12].

And tertiary prevention in cervical cancer includes its treatment with surgery (radical hysterectomy), radiotherapy, and systemic treatments (chemotherapy, bevacizumab, and immunotherapy) [11]. Minimally invasive surgery has been studied and associated with advantages in the treatment of various gynecological cancers [13].

In Paraguay, the quadrivalent HPV vaccine was introduced into the national immunization program in 2013, prioritizing girls ages 9–10 years old with 0-1-6-month immunization schedule using school-based vaccination [14].

The detection of HPV infection can precede advanced stages of the disease, such as cervical intraepithelial neoplasia grade 3 (CIN 3), for a period of five to ten years; therefore, identifying prevalent HPV genotypes can provide earlier clues about the impact of the vaccine program [9, 15].

In Paraguay, primary screening aims to detect precursor lesions of cervical cancer in women over 30 years of age [14], making screening data inadequate for assessing the early impact of the vaccine. Consequently, establishing a national HPV surveillance program that includes a series of cross-sectional studies to assess the prevalence of HPV in cervical samples from young women would aid in identifying the impact of the vaccination program [9].

This study aimed to estimate the type-specific HPV frequency in unvaccinated women aged 18–25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination program. Here, we provide the first data about circulating genotypes in the unvaccinated population, generating a baseline to compare future changes in overall and type-specific HPV prevalence after HPV vaccination.

Materials and methods

Results

The average age of study participants was 22.5±2.1 years. Of these, 70.2% had university as education level. The mean age of first sexual intercourse was 16.8±2.1 years, and 33.2% reported a previous history of STI. Additionally, 59.1% of the participants reported having fewer than two sexual partners in the last year, 81.7% had no history of smoking, and 72.6% reported alcohol consumption (Table 1). A total of 208 cervical samples were analyzed, all of which yielded a positive result for the human CFTR gene, proving the DNA integrity and good sample quality. HPV DNA was detected in 114 specimens, resulting in an overall prevalence of 54.8% (95% CI 48.0–61.6).

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Table 1. Participant characteristics and behavioral factors associated with HPV infection in young women from Paraguay.

https://doi.org/10.1371/journal.pone.0283542.t001

The results were further analyzed for cross-sectional associations between HPV infection and sociodemographic, obstetric, and behavioral characteristics. We observed significant associations between HPV infection and women who reported not living with their partner (OR 3.25; 95% CI 1.53–6.88; p = 0.01), women with more than one sexual partner within the past year (OR 2.16; 95% CI 1.22–3.83; p = 0.008), inconsistent condom usage (OR 2.20; 95% CI 1.15–4.22; p = 0.02), and prior non-HPV genital tract infections (OR 2.09; 95% CI 1.14–3.81; p = 0.01). The other variables were not significantly associated with HPV infection.

Among the positive samples, multiple infections with two to seven different HPV types represented 43.0% (95% CI 33.9–52.1) (Table 2) of the total identified infections.

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Table 2. Frequency of single and multiple HPV infections (n = 114).

https://doi.org/10.1371/journal.pone.0283542.t002

At least one HR-HPV genotype was identified in 42.3% (95% CI 36.6–49.0) of the women included in the study. High frequencies of HR-HPV were prevalent at all ages analyzed: 18–19 [50.0%; (95% CI 30.0–70.0)]; 20–21 [40.0%; (95% CI 23.8–56.2)], 22–23 [56.5%; (95% CI 44.1–68.8)], and 24–25 [63.6%; (95% CI 52.0–75.2)] (Fig 1).

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Fig 1. Frequency of HR-HPV, pHR-HPV, and LR-HPV in young women from Paraguay (N = 208).

HR-HPV: High risk HPV types; pHR-HPV: possible high risk; and LR-HPV: low risk HPV types. HR types, including Group 1 IARC, carcinogenic to humans (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) and Group 2A IARC, probable carcinogenic to humans (HPV68). pHR types, Group 2B IARC, possible carcinogenic to humans (HPV26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85, and 97). LR types, non-carcinogenic to humans (HPV6, 11, 40, 42, 54, 61, 71, 72, 81, 83, and 84).

https://doi.org/10.1371/journal.pone.0283542.g001

A total of 29 different viral types were identified, present in both single and multiple infections among 208 studied women (Fig 2). The most commonly detected HPV types were HPV-58 [14.9% (95% CI 8.4–21.5)], followed by HPV-16, HPV-51, and HPV-66 at similar frequencies [12.3%; (95% CI 6.3–18.3)].

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Fig 2. Distribution of viral types among HPV positive genital samples in women with single and multiple infections (N = 208).

HR-HPV: High risk HPV types; pHR-HPV: possible high risk; and LR-HPV: low risk HPV types. HR types, including Group 1 IARC, carcinogenic to humans (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) and Group 2A IARC, probable carcinogenic to humans (HPV68). pHR types, Group 2B IARC, possible carcinogenic to humans (HPV26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85, and 97). LR types, non-carcinogenic to humans (HPV6, 11, 40, 42, 54, 61, 71, 72, 81, 83, and 84).

https://doi.org/10.1371/journal.pone.0283542.g002

The prevalence of HPV types preventable with currently available vaccines, was 8.2% for the bivalent vaccine (16/18) (95% CI 4.5–11.9), 13.0% for the quadrivalent vaccine (6/11/16/18) (95% CI 8.4–17.6), and 38.0% for the nonavalent vaccine (6/11/16/18/31/33/45/52/58) (95% CI 31.4–44.6%). Infection prevalence of HPV types against which vaccine cross-protection has been reported, such as 31/33/45, was 10.6% (95% CI 6.4–14.8) (Table 3).

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Table 3. Frequencies of vaccine-preventable HPV types in young women (N = 208).

https://doi.org/10.1371/journal.pone.0283542.t003

Discussion

The WHO global strategy for the elimination of cervical cancer as a public health problem proposes a multidisciplinary approach that includes community education, social mobilization, vaccination, detection, treatment, and palliative care.

HPV vaccination in combination with timely detection and appropriate treatment of cervical cancer precursor lesions is considered to have the potential to achieve the elimination of this disease, defined by the WHO as fewer than 4 new cases per 100,000 women-years [8].

With the introduction of the HPV vaccine, changes in the transmission dynamics of the virus are expected, making necessary to monitor changes in the prevalence of HPV and the patterns of HPV-associated diseases. Population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low- and middle-income countries, so cross-sectional surveys in sentinel populations could be a suitable alternative [8].

Paraguay, like many other countries worldwide, has incorporated the HPV vaccine into its national immunization program, requiring a virological surveillance to measure its impact. It can provide an early measure of vaccine effectiveness by quantifying the magnitude of reduction in HPV type-specific prevalence, including possible evidence of herd and cross protection and provide timely assessment of vaccination strategy. The principle of HPV prevalence surveillance is to recruit samples in age groups at risk of HPV exposure and conduct repeat-cross sectional analyses [16].

In this study, we analyzed a cohort of unvaccinated sexually active women between 18–25 years of age and found an overall HPV prevalence of 54.8%, comparable to others cross-sectional studies in South American as Colombia with a prevalence of 60.3% in a same age group [17], and Argentina with a prevalence of 56.3% in 15 to 17-year-old female adolescents [18], showing the high of HPV infections in young women in the region. Our observed HPV prevalence rate is also like the 55% infection rate observed at enrollment in a longitudinal study of young women [19]. However, this rate was higher than that observed in studies in unvaccinated young women from United States (47.5% for 21–24 years) [20] and Europe, which has ranged from 25% to 33% among women aged <25 years [21–23]. Thus, the overall HPV prevalence is low in developed countries [24].

Previous studies have established sexual contact as the main route of HPV transmission. Thus, the higher HPV prevalence in women ≤25 years of age could be explained by recent first sexual contact, a condition that increases the chance of exposure to the virus [5, 24, 25].

Although the demographic data and all risk factors for acquiring HPV showed that the participants were moderate risk e.g., only 24.0% used condom, 33% reported STI, 40.9% had reported 2 or more partners, the results reported high risk HPV types. At least one type of HR-HPV was found in 42.3% of young women, with HR-HPV prevalence ranging from 18.7%–45.7% among women under 25 years old worldwide [26–33]. A similar rate (42.2%) has been observed in regional surveys conducted in Colombia and Argentina [17, 18]. However, an elevated HR-HPV infection rate in young women does not necessarily lead to the development of precancerous lesions and cancer. The decreasing frequency of infection with age has been demonstrated in national, regional, and global studies, indicating the self-limited nature and control of the infection by the action of the host’s immune system in most cases [24, 25, 34, 35].

As expected, sexual behavior is associated with an increased risk of HPV infection, as well as the number of sexual partners, new sexual partners, non-use of condoms, and a history of other sexual infections. A high number of sexual partners is the most important risk factor for HPV positivity, and newly acquired sexual partners further increase this risk [5, 25, 36]. Condom use may help reduce the risk of cervical and vulvovaginal HPV infection [37]. Other STI like Chlamydia trachomatis have been associated with cell inflammatory processes in the cervix, facilitating HPV entry and reducing its elimination [25, 38].

We identified multiple infections in 43.0% of all cases, as previously reported in younger groups of women [39, 40]. A few studies have associated multiple HPV infections with an increased risk of cervical intraepithelial neoplasia [41, 42]. However, other studies found no significant difference in the development of cervical intraepithelial neoplasia and cancer in women with single or multiple infections [43, 44]. The total risk of precancer in women infected with several HPV types was increased compared to women infected with a single HPV type [45]. It is not clear whether the risk from multiple infections is greater than the sum of the risks posed by individual HPV types. However, despite the presence of multiple viral types in a sample, only one HR-HPV leads to potential malignant transformation [46].

HPV types differ in their prevalence worldwide in diverse populations based on age range, cytologic condition, laboratory method used, and the interaction between HPV types and host immunogenetic factors. We identified 29 distinct HPV types, with HPV-58 being the most detected in 14.9% samples, followed by HPV-16, 51, 52, 31, and 33 among the HR-HPV types. The five most frequent types in women with normal cytology worldwide are HPV-16, 18, 31, 58, and 52, and in South America are HPV-16, 58, 18, 45, and 31 [24]. HPV-58, 51, and 52 are relatively abundant in Costa Rica, followed by HPV-31 and 18 [43]. The most common high-risk viral types in Chile are HPV-16, 56, 31, 58, 59, 18, and 52 [28] in Brazil are HPV-52, 16, 58, 51, 31, and 59 [47]. In Colombia, infections with HPV-16 are the most frequent, followed by HPV-58 [39]. Likewise, HPV-16, 58, and 31 have been reported as the most predominant types in Paraguayan women with normal cytology [34].

It is established that persistent infections by HPV-16 have a higher probability of causing precancerous lesions than infection with other viral types [48, 49]. Women with normal cytology and persistent HPV-16 infection have an approximately 30% chance of developing high-grade lesions in a 12-month period [50]. HPV-58 is also included in high-risk group and has been frequently reported in cervical cancer and high-grade lesions in several regions of the world [51], such as Asia [52, 53], Mexico [54, 55], Costa Rica [43], and Brazil [56]. Given the wide circulation of HPV-58 in the population studied and the evidence of its oncogenic potential, particular attention should be given to HPV-58 infection.

We found that 8.2% of the women were infected with HPV-16/18 that are included in the bivalent vaccine, close to that reported in similar populations from Colombia (12.9%) [17] and the United States (10.0%) [57]. Infections with types included in the quadrivalent vaccine (HPV-6/11/16/18) were detected in 13.0% of women, comparable to a nationally representative United States survey of females conducted from 2003 to 2006 with a prevalence of 18.5% in the 20–24-year-old group [58], but less than that reported in Argentine adolescents (22.5%) [18]. HPV 31, 33, and 45 were detected in 10.6% of women. These infections could also be prevented, as the bivalent and quadrivalent vaccines provide cross-protection against these genotypes [10].

HPV-58, not targeted by the vaccine currently in use in Paraguay’s vaccination program was the most prevalent type in this studied group. Consequently, the incidence and risk of non-vaccine HPV types must be considered to assess the impact of the quadrivalent vaccine used in the country in the national immunization program.

Have been reported women immunized with quadrivalent vaccine at young age who still developed cervical intraepithelial neoplasia with evidence that these lesions are commonly associated with HPV types not targeted by this vaccine, but some of the HPV types associated with these lesions in vaccinated women are targeted by the Gardasil-9 [59].

As we know, the nonavalent vaccine contains five additional oncogenic types (HPV-31, 33, 45, 52, and 58) and according to the current data, this vaccine could prevent 38.0% of HPV infections identified in this study. Therefore, the high prevalence of HPV types targeted by nonavalent HPV vaccines could support its introduction in the national immunization program to achieve greater coverage, considering that the preventable proportion of cervical cancers is estimated to increase to 90% [60].

The present study had some limitations as the small sample size of population studied, for this reason the results cannot be extrapolated to the total young women from Paraguay. The recruitment strategy by social networks and flyers at local health centers and higher education institutes may have induced a selection bias. Although this was not a population-based study, the results are particularly important, providing information regarded HPV prevalence in groups that are not often included in epidemiological surveys or cervical cancer prevention programs. We know that the population-based cohort studies are the most appropriate to assess the impact of HPV vaccination; however, the feasibility of conducting these studies is limited in low resources countries, thus cross-sectional surveys in sentinel populations could be a suitable alternative.

For hence this study even its important limitations, such as the small sample size and the convenience recruitment strategy, the results are particularly important, providing information regarding HPV prevalence in groups that are no often included in national cervical cancer prevention programs. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the short-term estimation of the impact of the national HPV immunization program.

Even though several studies provide evidence that genital HPV infection is very common in young sexually active women and in most cases is a transient and self-limited infection, the evaluation of overall HPV prevalence in this population has been proposed as a short-term endpoint for determining the impact of HPV vaccination in surveillance programs.

This research could provide key information to healthcare policy makers about the importance of increasing HPV vaccination coverage in national immunization programs. The results should be combined with other studies in cohorts of vaccinated women to investigate the reduction of types 16 and 18, as well as their possible replacement by other genotypes, thus evaluating the impact of the implementation of HPV vaccination in Paraguay and compare the results with other countries.

In addition, these data can improve the acceptance of the HPV vaccine in the Paraguayan population, highlighting the high prevalence of HPV, especially the high-risk types, observed in unvaccinated young women. Increased public awareness of the benefits of HPV vaccination for cervical cancer prevention could increase public support for immunization.

In conclusion, this is the first report on the prevalence of HPV and its genotype distribution, including HPV types targeted by current vaccines, in unvaccinated young sexually active women from Paraguay. These data provide the baseline against which to compare future HPV prevalence of vaccine and non-vaccine-types, allowing the estimation of the impact of the national HPV immunization program. Surveillance and monitoring of changes in viral transmission that lead to modification of HPV type-specific prevalence and the patters of HPV-associated disease are crucial to progress toward the global elimination of cervical cancer.