Journal of Medical Virology

Studies on the Prevalence of Oncogenic HPV Types Among Lithuanian Women With Cervical Pathology Vaida Simanaviciene,1* Zivile Gudleviciene,2 Violeta Popendikyte,3 Dovile Dekaminaviciute,1 Ausra Stumbryte,2 Vilija Rubinaite,1 and Aurelija Zvirbliene1 1

Institute of Biotechnology, Vilnius University, Vilnius, Lithuania Institute of Oncology, Vilnius University, Vilnius, Lithuania 3 Thermo Fischer Scientific Baltics, Vilnius, Lithuania 2

Human papillomavirus (HPV) is the main cause of cervical cancer. Therefore, the detection of oncogenic HPV types is important in predicting the risk of cervical cancer. The aim of the current study was to estimate the prevalence of 16 carcinogenic and potentially carcinogenic HPV types in the study group of Lithuanian women with various grades of cervical pathology in comparison to healthy women. A total of 824 cervical specimens were investigated for HPV DNA: 547 specimens of women with abnormal cytology and 277 specimens of healthy women. Cytological diagnosis was confirmed by histology. For the detection of HPV infection, HPV DNA was amplified by PCR using three different primer systems. HPV DNA was detected in 67.6% of specimens collected from women with abnormal cytology and 24.2% of specimens collected from healthy women. The frequency of HPV-positive specimens correlated with the severity of cervical pathology: it ranged from 50.0% in the subgroup of atypical squamous cells to 80.6% in cervical cancer. In cases confirmed by histology the frequency of HPV-positive specimens ranged from 68.6% in the subgroup of cervical intraepithelial neoplasia grade 1 to 89.2% in cervical intraepithelial neoplasia grade 3 or carcinoma in situ. HPV DNA-positive samples were further investigated for the presence of 16 HPV types by multiplex PCR. The most common HPV type was HPV 16 (detected in 42.3% of HPV-positive specimens) followed by HPV 31 (10.1%), HPV 33 (8.2%), and HPV 56 (5.7%). In contrast, the frequency of HPV 18 was lower as compared to other countries. J. Med. Virol. # 2014 Wiley Periodicals, Inc.

KEY WORDS:

human papillomavirus; cervical pathology; HPV genotypes

C 2014 WILEY PERIODICALS, INC. 

INTRODUCTION Infection with certain types of human papillomavirus (HPV) is a causative factor in the development of many benign and cancer diseases of skin and mucosa. More than 120 HPV genotypes have been identified, 40 of which are known to infect anogenital tract [Villiers et al., 2004; Bernard et al., 2010]. HPV genotypes that are linked to the development of cervical neoplasia are classified as high-risk (HR) genotypes. The International Agency for Research on Cancer (IARC) has classified 12 HPV types as group 1 carcinogens: these are HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, belonging to four species in a single evolutionary branch of the Alphapapillomavirus genus. The same evolutionary branch includes HPV 68, an IARC group 2A carcinogen, and 11 possibly carcinogenic HPV types (IARC group 2B), such as HPV 66, 73, 82 [Schiffman et al., 2011]. HR HPV types have been detected in 99% of biopsy samples of cervical carcinoma that is the third most common cancer in women worldwide [Ferlay et al., 2010]. Therefore, the detection of HR HPV infection is important in predicting the risk of cervical cancer. Studies on the prevalence of HR HPV types have been performed in many countries. HPV 16 has been reported to be the most common HR HPV genotype across all five continents and HPV 18 is the second Grant sponsor: Research Council of Lithuania; Grant number: AUT-16/2010.; Grant sponsor: Agency for Science, Innovations and Technology; Grant number: 31V-116. The work was performed at the Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.  Correspondence to: Vaida Simanaviciene, Institute of Biotechnology, Vilnius University, V.A. Graiciuno Str.8, LT-02241 Vilnius, Lithuania. E-mail: [email protected] Accepted 13 August 2014 DOI 10.1002/jmv.24073 Published online in Wiley Online Library (wileyonlinelibrary.com).

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most frequent HR HPV genotype [Bosch et al., 2008]. The use of improved PCR-based genotyping systems has revealed that more than two HPV genotypes are present in a large part of HPV-infected women [Trottier et al., 2006; Trottier and Franco, 2006; Smith et al., 2007; Kovacs et al., 2008; Sandri et al., 2009; Wentzensen et al., 2009]. Worldwide, the five most common HR HPV types in HPV-positive women are HPV 16, 18, 31, 58, and 52 [Sanjose et al., 2007]. However, a significant geographic variation in the prevalence of HR HPV genotypes has been reported in different countries. The most important geographic difference is high prevalence of HPV 58 in Eastern Asia, which greatly contributes to the significant worldwide prevalence of this HR HPV genotype [Li et al., 2011]. Geographic variations in the prevalence of HR HPV genotypes are also observed among samples from women with normal cytology, with noticeable increase in prevalence of HPV 31 in Europe and HPV 52 in Northern America [Bruni et al., 2010]. Therefore, it is necessary to estimate the prevalence of HR HPV genotypes in primary cervical screening in different geographic regions for developing a better strategy of protection against HR HPV infection. This information is essential for planning prevention measures by prophylactic HPV vaccines and for large-scaled screening programs based on HPV testing. Currently available prophylactic HPV vaccines protect against the two most prevalent HR HPV types (HPV 16 and HPV 18) that cause 70% of cervical cancers, 80% of anal cancers, 60% of vaginal cancers, and 40% of vulvar cancers [De Vuyst et al., 2009]. However, high prevalence and oncogenic potential of other HR HPV types suggest that future generations of HPV vaccines should also include the other most common HR HPV genotypes. The aim of the present study was to investigate the prevalence of HPV infection and the distribution of 12 carcinogenic and four potentially carcinogenic HPV genotypes in the groups of Lithuanian women with known cytological and histological diagnosis. MATERIALS AND METHODS Materials

Clinical Specimens and Study Groups Cervical specimens were collected at the Institute of Oncology of Vilnius University (Vilnius, Lithuania) by a cytobrush. In total, 824 women (aged from 18 to 80 years) with known histological and/or cytological diagnosis were included into the study. The study was approved by the Vilnius Regional Committee of Biomedical Research (Lithuania, permission no. 158200-6-062-16). All cervical specimens were divided into two groups: the group of specimens with normal cytology (n ¼ 277, control group) and the group of specimens with various grades of cervical pathology (n ¼ 547, study group). The age of women in both groups ranged from 18 to 81 years (mean age in both groups 41.37 years: SD  12.03; mean age in the control group 40.37 years; mean age in the study group 42.37 years; 95% CI). The distribution of women according their age in both the study group and the control group was similar. In the study group, 4.7% of women were 56 years. In the control group, the distribution of women was similar: 5.4%, 33.2%, 31.0%, 22.2 and 8.2% in the indicated age groups, respectively. The group of specimens with cytological pathology (the study group) included 174 cases of atypical squamous cells, 67 cases of low-grade squamous intraepithelial lesions, 244 cases of high-grade squamous intraepithelial lesions and 62 cases of cancer, which consisted of 60 specimens with squamous cell carcinoma, one specimen with atypical glandular cells and one specimen of adenocarcinoma in situ. After cytological diagnosis 443 women were followed for biopsy and histological investigation. The group of specimens with histological pathology included 35 cases of cervical intraepithelial neoplasia grade 1, 43 cases of cervical intraepithelial neoplasia grade 2, 157 cases of cervical intraepithelial neoplasia grade 3 or carcinoma in situ, 75 cases of cervical cancer, which consisted of 70 specimens of squamous cell carcinoma and 5 adenocarcinoma. After histological investigation of specimens with cytological pathology, 133 cases with not confirmed pathology were identified (this subgroup was defined as “Normal histology”).

TM

Maxima Hot Start Green PCR Master Mix, 2 mM dNTP/dUTP, 25 mM MgCl2, UDG (Uracil-DNA Glycosylase), agarose, BSA, O’GeneRulerTM 50 bp DNA Ladder, ready-to-use, “GeneJetTM Genomic DNA purification Kit,” were purchased from Thermo Fisher Scientific Baltics UAB (Vilnius, Lithuania), Betaine was obtained from Sigma–Aldrich (Taufkirchen, Germany), all oligonucleotides were obtained from Metabion (Steinkirchen, Germany). The cervical cell lines used in the study: CaSki (60–600 copies of HPV 16 per genome) and HeLa (10–50 copies of HPV 18 per genome) were obtained from American Type Culture Collection (Manassas, VA). J. Med. Virol. DOI 10.1002/jmv

Collection of Cervical Specimens and DNA Extraction Cervical specimens were collected from the cervix using cervical brush and put into the special medium for transportation of clinical samples (22% ethanol, 3% methanol, 18% isopropanol, 100 mM sodium acetate, 150 mM sodium chloride, 5 mM EDTA; 0.005% saponine, 0.2% zinc chloride). DNA was extracted from all samples using DNA extraction GeneJetTM Genomic DNA Purification Kit (Thermo Scientific Fermentas Vilnius, Lithuania) according to the manufacturer’s instruction. Purified DNA solutions were

HPV Types in Cervical Pathology

stored at 20˚C until use. Before HPV PCR, all DNA samples were subjected to the end-point PCR using human b-globin gene primers (b-gloF: 50 -AAC TGT TGC TTT ATA GGA TTTT; b-gloR: 50 AGG AGC TTA TTG ATA ACT CAG A) as an internal control to avoid false-negative test results caused by possible contaminations. Human b-globin end-point PCR was performed in 25 ml reaction volume with the following composition: 12.5 ml of MaximaTM Hot Start Green PCR Master Mix, 3 mM MgCl2, 1 M betaine, 5 mg BSA, 0.5 mM mix of two b-globin primers, 3 ml DNA extracted from cervical specimens and nuclease-free water up to 25 ml. The PCR products were analyzed in 1.5% agarose gel. Only DNA samples that generated human b-globin gene amplicons (650 bp in size) were used to determine HPV DNA and HPV genotypes as described below. HPV DNA Detection and Genotyping HPV DNA was amplified by previously described general primer systems GP5þ/GP6þ and PGMY09/11 within the L1 open-reading frame [Husman et al., 1995; Gravitt et al., 2000]. In addition to these two PCR systems, HPV DNA was amplified by new in-house developed PCR system using a set of 19 primers specific for 16 carcinogenic or potentially carcinogenic HPV genotypes (HR_HPV PCR system). In all PCR systems, PCR mix without any DNA sample was used as a negative control and PCR mix containing 25 ng DNA purified from HeLa and CaSki cells was used as a positive control. As reported previously, the PGMY09/11 mix of 18 primers within the L1 open-reading frame of HPV genome is able to detect the following HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 40, 45, 51, 52, 56, 59, 39, 42, 53, 54, 55, 58, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, IS39, CP8304, CP6108, MM4, MM7, and MM8 with the sensitivity of 10 HPV genomes per reaction [Gravitt et al., 2000]. PCR products were analyzed in 1.5% agarose gel. In HPV DNA-positive samples, amplification product of 450 bp was observed. As reported previously, GP5þ/GP6þ primer system originally selected from the HPV L1 region is able to detect at least 27 HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 40, 45, 51, 52, 56, and 59. HPV genotypes, including HPV types 39, 42, 53, 54, 55, 58, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, IS39, CP8304, CP6108, MM4, MM7, and MM8 [Husman et al., 1995]. PCR products were analyzed in 2.0% agarose gel. In HPV DNA-positive samples, amplicon of 150 bp was observed. The in-house developed HR HPV PCR consisted of a set of 19 primers designed according to the bioinformatics analysis of HPV L1 gene sequences. The primers are specific for 16 carcinogenic or potentially carcinogenic HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 66, 73, and 82. The HR HPV PCR was performed in reaction volume of 25 ml with the following composition: 22 ml of HR

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HPV PCR master mix (optimal buffer, Hot Start Taq DNA Polymerase, dNTP, PCR enhancers, mix of 19 primers, UDG, two tracking dyes and density reagents for direct loading of PCR products on a gel) and 3 ml DNA extracted from cervical specimens. The PCR was run according to the StepDown PCR protocol: 50˚C 2 min (UDG); 95˚C 7 min; 5 cycles: 95˚C 30 sec; 60˚C 30 sec; 72˚C 60 sec; 5 cycles: 95˚C 30 sec; 55˚C 30 sec; 72˚C 60 sec; 25 cycles: 95˚C 30 sec; 50˚C 30 sec; 72˚C 60 sec. PCR products were analyzed in 1.5% agarose gel. In HPV DNA-positive samples, amplicon of 400–430 bp was observed. The sensitivity of the HR HPV PCR is 10–20 copies of HPV DNA. All HPV DNA-positive samples identified by either PCR system (PGMY09/11, GP5þ/GP6þ, or HR HPV) were further genotyped using an in-house developed multiplex PCR system (PCR MM 1–4) using four sets of specific primers for 16 carcinogenic or potentially carcinogenic HPV types, including all 12 IARC group 1 carcinogens: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58; 59 one IARC group 2A carcinogen: HPV68; and three IARC group 2B carcinogens: HPV66, 73, 82. Each primer set of the PCR MM 1–4 system consisted of four pairs of primers designed for four different HPV genotypes [Popendikyte et al., 2008]. The amplicons obtained using each of these primer sets differed in size at least by 80 bp thus allowing their identification in agarose gels. The HPV MM-1 primer set was used to detect HPV 16 (481 bp), HPV 18 (346 bp), HPV 39 (253 bp), HPV 58 (152 bp); the MM-2 primer set was used to detect HPV 52 (541 bp), HPV 33 (422 bp), HPV 56 (354 bp), and HPV 31 (287 bp); the MM-3 primer set was used to detect HPV 35 (448 bp), HPV 68 (357 bp), HPV 59 (239 bp), and HPV 45 (175 bp); the MM-4 primer set was used to detect HPV 82 (623 bp), HPV 73 (424 bp), HPV 66 (301 bp), and HPV 51 (247 bp). To develop positive controls for HPV genotyping, specific amplicons of different HPV genotypes were cloned into phage l DNA. DNA samples from different phage l clones were extracted and verified by sequencing. According to obtained sequencing data, a collection of phage l DNA with the highly specific sequences for studied HPV genotypes was generated. The mixes of four phage l DNA samples containing amplicons (50 pg DNA per reaction) of the corresponding HPV genotypes were used as positive controls in the multiplex PCR system MM (1–4). The PCR products were analyzed in 2% agarose gels. HPV genotypes were identified according to the size of obtained amplicons as indicated above. The sensitivity of the multiplex PCR system MM1–4 is 100– 200 copies of target HPV DNA. Statistical Analysis All data were analysed using Microsoft Excel and Statistica eight programs. To estimate the association of cervical pathology with HPV infection and certain HPV genotypes, odds ratios (OR) and 95% confidence J. Med. Virol. DOI 10.1002/jmv

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interval (CI) were calculated. Differences between the groups were compared using the x2 test. The P-value of 0.05 was considered statistically significant. RESULTS The Prevalence of HPV Infection in the Groups of Women With Known Histological and/or Cytological Diagnosis A total of 824 cervical specimens with confirmed histological and/or cytological diagnosis were analysed for HPV DNA. All specimens were distributed into two groups: 547 specimens of women with various grades of cervical pathology (study group) and 277 specimens of healthy women (control group). All specimens were analyzed for HPV DNA by three PCR-based systems: PGMY09/11, GP5þ/GP6þ and the newly developed primer set for 16 carcinogenic or potentially carcinogenic HPV genotypes (HR_HPV PCR system). In total, 437 specimens were positive for HPV DNA by either PCR test. The prevalence of HPV DNA in the study group was 67.6% and in the control group 24.2% (Table I). The association of cervical pathology with HPV infection was statistically significant (OR ¼ 6.6, 95% CI 4.7–9.1, P < 0.0001). The numbers of HPV DNA-positive specimens detected by GP5þ/GP6þ, PGMY09/11 and HR_HPV primer systems in the study group were 297/547 (54.3%), 279/547 (51.0%) and 299/547 (54.7%) and in the control group 36/277 (13.0%), 51/277 (18.4%) and 29/277 (10.5%), respectively (Fig. 1). TABLE I. The Prevalence of HPV Infection and HPV Types in the Study Group (Women With Cervical Pathology) and the Control Group (Healthy Women With Normal Cytology) Study group Control group HPV status HPV DNA HPV DNAþ Total HPV 16 HPV 18 HPV 39 HPV 58 HPV 31 HPV 33 HPV 52 HPV 56 HPV 59 HPV 68 HPV 35 HPV 45 HPV 66 HPV 51 HPV 73 HPV 82 Unspecified HPV types

n

%a

n

177 370 547 173 15 11 14 36 33 16 22 6 5 13 9 12 21 4 5 71

32.4 67.6 100.0 46.8 4.1 3.0 3.8 9.7 8.9 4.3 5.9 1.6 1.4 3.5 2.4 3.2 5.7 1.1 1.4 19.2

210 67 277 12 2 5 3 8 3 7 3 3 1 2 1 4 5 0 0 23

%a

OR, 95% CI

75.8 6.6 (4.7–9.1) 24.2 100.0 17.9 4.03 (2.08–7.76) 3.0 7.5 4.5 11.9 4.5 10.4 0.39 (0.15–0.98) 4.5 4.5 1.5 3.0 1.5 5.9 7.5 0.0 0.0 34.3 0.45 (26–0.8)

a Percentages of the prevalence of HPV types are calculated among the total number of HPV DNA-positive specimens.  P < 0.05.  P < 0.001.

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. The prevalence of HPV DNA-positive cervical specimens detected by different PCR-based systems in the study group (women with cervical pathology) and in the control group (healthy women with normal cervical cytology). “Total” indicates the prevalence of HPV DNA-positive specimens identified with all three PCR-based systems.

To analyse the correlation between HPV infection and cytological alterations, all specimens with known cytological diagnosis were subdivided into four subgroups: 174 cases of atypical squamous cells, 67 cases of low-grade squamous intraepithelial lesions, 244 cases of high-grade squamous intraepithelial lesions and 62 cases of cancer. The prevalence of HPV correlated with the severity of cervical pathology (P < 0.0001). In atypical squamous cells, 50.0% of specimens were found to be HPV DNA-positive. In this group, HPV DNA was detected in 40.2% of specimens with GP5þ/GP6þ primer system, in 33.3% of specimens with PGMY09/11 primer system, and in 32.8% of specimens with HR_HPV primer system. In low-grade squamous intraepithelial lesions, 55.2% of specimens were found to be HPV DNA-positive. In this group, HPV DNA was detected in 38.8% of specimens with GP5þ/GP6þ primer system, in 52.2% of specimens with PGMY09/11 primer system and in 43.3% of specimens with HR_HPV primer system. In the subgroup of high-grade squamous intraepithelial lesions, 80.3% of specimens were found to be HPV DNA-positive. In this group, HPV DNA was detected in 65.9% of specimens with GP5þ/GP6þ primers, in 61.5% of specimens with PGMY09/11 primers and in 68.9% of specimens with HR_HPV primers. In cancer, 80.6% of specimens were found to be HPV DNA-positive. In this group, HPV DNA was detected in 64.5% of specimens with GP5þ/GP6þ primers, in 58.1% of specimens with PGMY09/11 primers and in 72.6% of specimens with HR_HPV primers (Table II). The difference in HPV prevalence between the following subgroups: atypical squamous cells and high-grade squamous intraepithelial lesions (P < 0.0001), the atypical squamous cells and cancer (P < 0.0001), the low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions (P < 0.0001), the low-grade squamous intraepithelial lesions and cancer (P < 0.001) was statistically significant. Also, statistically significant differences

HPV Types in Cervical Pathology

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TABLE II. The Prevalence of HPV DNA Among Specimens With Known Cytological and Histological Diagnosis Cytological diagnosis ASC HPV status HPV HPVþ Total HPV 16 HPV 18 HPV 39 HPV 58 HPV 31 HPV 33 HPV 52 HPV 56 HPV 59 HPV 68 HPV 35 HPV 45 HPV 66 HPV 51 HPV 73 HPV 82

n

%a

LSIL n

%a

Histological diagnosis

HSIL n

%a

Ca n

%a

CIN I n

%a

CIN II n

%a

87 50.0 30 44.8 48 19.7 12 19.4 11 31.4 6 14.0 87 50.0 37 55.2 196 80.3 50 80.6 24 68.6 37 86.0 174 100.0 67 100.0 244 100.0 62 100.0 35 100.0 43 100.0 24 27.6 11 29.7 108 55.1 30 60.0 9 37.5 18 48.6 3 3.4 2 5.4 4 2.0 6 12.0 3 12.5 1 2.7 2 2.3 2 5.4 6 3.1 1 2.0 0 0.0 1 2.7 2 2.3 1 2.7 10 5.1 1 2.0 0 0.0 5 13.5 7 8.0 6 16.2 20 10.2 3 6.0 4 16.7 3 8.1 8 9.2 1 2.7 24 12.2 0 0.0 2 8.3 2 5.4 5 5.7 3 8.1 8 4.1 0 0.0 1 4.2 1 2.7 8 9.2 4 10.8 7 3.6 3 6.0 2 8.3 5 13.5 0 0.0 2 5.4 3 1.5 1 2.0 0 0.0 2 5.4 3 3.4 0 0.0 1 0.5 1 2.0 0 0.0 1 2.7 5 5.7 1 2.7 6 3.1 1 2.0 2 8.3 1 2.7 4 4.6 1 2.7 1 0.5 3 6.0 0 0.0 1 2.7 5 5.7 4 10.8 3 1.5 0 0.0 3 12.5 1 2.7 8 9.2 5 13.5 7 3.6 1 2.0 3 12.5 4 10.8 1 1.1 1 2.7 2 1.0 0 0.0 2 8.3 1 2.7 1 1.1 1 2.7 2 1.0 1 2.0 0 0.0 0 0.0

CIN III/CIS

CC

n

%a

17 140 157 81 2 3 5 16 18 7 8 1 0 3 1 3 3 0 2

10.8 89.2 100.0 57.9 1.4 2.1 3.6 11.4 12.9 5.0 5.7 0.7 0.0 2.1 0.7 2.1 2.1 0.0 1.4

n

%a

13 17.3 62 82.7 75 100.0 38 61.3 7 11.3 2 3.2 1 1.6 3 4.8 2 3.2 0 0.0 3 4.8 1 1.6 1 1.6 1 1.6 3 4.8 0 0.0 1 1.6 0 0.0 1 1.6

Normal histology n

%a

78 55 133 13 1 2 3 4 4 3 3 1 1 3 1 2 5 1 2

58.6 41.4 100.0 23.6 1.8 3.6 5.5 7.3 7.3 5.5 5.5 1.8 1.8 5.5 1.8 3.6 9.1 1.8 3.6

ASC, atypical squamous cells; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; Ca, cervical cancer confirmed by cytology; CIN I, cervical intraepithelial neoplasia grade 1; CIN II, cervical intraepithelial neoplasia grade 2; CIN III/CIS, cervical intraepithelial neoplasia grade 3 or carcinoma in situ; CC, cervical cancer confirmed by histology; Normal Histology, not confirmed pathology. a Percentages of the prevalence of HPV types are calculated among the total number of HPV DNA-positive specimens.

were detected between the control group (healthy women) and all subgroups of specimens with cytological pathology (P < 0.0001). The prevalence of HPV DNA among specimens with known cytological diagnosis and the efficiency of different PCR-based systems are shown in Figure 2. To analyse the correlation between HPV infection and histological alterations, the group of specimens with confirmed histological diagnosis were subdivided into five subgroups: 35 cases of cervical intraepithelial neoplasia grade 1, 43 cases of cervical intraepithelial neoplasia grade 2, 157 cases of cervical intraepithelial neoplasia grade 3 or carcinoma in situ, 75 cases of cervical cancer, and 133 cases of not

confirmed pathology (the subgroup of “Normal histology”). The prevalence of HPV correlated with the severity of cervical pathology (P < 0.0001). As shown in Figure 3, the highest prevalence of HPV DNA was determined in the subgroups of cervical intraepithelial neoplasia grade 3 or carcinoma in situ 89.2%. In the subgroups of cervical intraepithelial neoplasia grade 3 or carcinoma in situ, 73.9% of specimens were found to be HPV DNA-positive with GP5þ/ GP6þ primer system, 66.9% with PGMY09/11 and 80.3% with HR_HPV primer system. In the subgroup of cervical intraepithelial neoplasia grade 2, 86.0% of specimens were found to be HPV DNA-positive (79.1% with GP5þ/GP6þ PCR system, 76.7% with

The prevalence of HPV DNA-positive specimens among specimens with known cytological diagnosis analysed by different PCR-based systems (“Total” indicates the prevalence of HPV DNA-positive specimens identified with all three PCR-based systems); (ASC, atypical squamous cells; LSIL, lowgrade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; Ca, cervical cancer confirmed by cytology).

Fig. 3. The prevalence of HPV DNA-positive specimens among specimens with known histological diagnosis analysed by different PCR-based systems (“Total” indicates the prevalence of HPV DNA-positive specimens identified with all three PCR-based systems); (CIN I, cervical intraepithelial neoplasia grade 1; CIN II, cervical intraepithelial neoplasia grade 2; CIN III/CIS, cervical intraepithelial neoplasia grade 3 or carcinoma in situ; CC, cervical cancer confirmed by histology; Normal histology, not confirmed pathology).

Fig. 2.

J. Med. Virol. DOI 10.1002/jmv

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PGMY09/11 and 69.8% with HR_HPV primer system). In the subgroup of cervical cancer (cervical cancer), 82.7% of specimens were HPV DNA-positive as detected by all three PCR-based systems (GP5þ/ GP6þ 68.0%, PGMY09/11–56.0% and HR_HPV 73.3%). Similar rate of HPV DNA positivity in the advanced-stage disease is reported in other studies [Jarrell et al., 1992; Agoff et al., 2003; Smith et al., 2007]. The lowest prevalence of HPV was determined in the subgroups of cervical intraepithelial neoplasia grade 1 (68.6%), and “Normal histology” (41.4%) (Table II). Statistically significant differences were detected between all subgroups of specimens with histological pathology and both control (healthy women) and ”Normal histology” subgroups (P < 0.01), as well as between the cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 3 or carcinoma in situ subgroups (P < 0.01). When the specimens of the control group (healthy women with normal cytology) were compared with cervical cancer specimens, a strong association of cervical cancer and HPV infection was determined (OR ¼ 14.95, 95% 7.7–28.9, P < 0.0001). The prevalence of HPV DNA among specimens with known histological diagnosis and results obtained with different PCR-based systems are shown in Figure 3. The Prevalence of HPV Genotypes Among HPV DNA-Positive Specimens HPV DNA-positive specimens (n ¼ 437) were analyzed further for HPV genotypes using an in-house multiplex PCR system MM 1–4 designed for the detection of 16 HPV genotypes [Popendikyte et al., 2008]. The multiplex PCR system consists of four sets of specific primers and detects 12 IARC group 1 carcinogens (HPV 16, 18, 31, 33, 35, 39, 45,

51, 52, 56, 58, and 59); one IARC group 2A carcinogen (HPV 68) and three IARC group 2B carcinogens (HPV 66, 73, 82). Of the analysed 437 HPV-positive specimens, 94 (21.5%) specimens were negative for any of the tested HPV types: 71 (19.2%) specimens of women with abnormal cytology and 23 (34.3%) specimens with normal cytology (the control group, healthy women). This suggests that these specimens were infected with low-risk HPV types not detectable with the multiplex PCR system. HPV 16 was the most common HR HPV genotype, detected in 185 out of 437 (42.3%) HPV-positive specimens (P < 0.0001), followed by HPV 31 (10.1%), 33 (8.2%) and HPV 56 (5.7%). Only 3.9% of HPV-positive specimens were infected with HPV 18. In both groups (e.g., women with various grades of cervical pathology and healthy women), HPV 16 was the most prevalent HPV genotype (detected in 46.8% and 17.9% of HPVpositive specimens, respectively) (Table I). Moreover, HPV 16 infection was strongly associated with cervical pathology (OR ¼ 4.03, 95% CI 2.0–7.7, P < 0.0001). The prevalence of all tested 16 HPV types among HPVpositive specimens is shown in Figure 4. The prevalence of HPV types was also analyzed in specimens subdivided according to the cytological diagnosis. The frequency of HPV 16 among HPVpositive specimens correlated with the severity of cervical pathology: it increased from 27.6% in atypical squamous cells to 29.7% in low-grade squamous intraepithelial lesions, 55.1% in high-grade squamous intraepithelial lesions and 60.0% in cancer. Statistically significant difference was detected between the high-grade squamous intraepithelial lesions and atypical squamous cells subgroups (P < 0.0001), the high-grade squamous intraepithelial lesions and lowgrade squamous intraepithelial lesions subgroups (P < 0.01), the cancer and atypical squamous cells

Fig. 4. The distribution of 16 analysed HPV genotypes in a single (lined area) and in a multiple (spotted area) HPV infection among all HPV DNA-positive specimens. The study group represents women with cervical pathology and the control group represents healthy women with normal cytology.

J. Med. Virol. DOI 10.1002/jmv

HPV Types in Cervical Pathology

subgroups (P < 0.001) and the cancer and low-grade squamous intraepithelial lesions subgroups (P < 0.01). HPV 16 was detected significantly more often in high-grade squamous intraepithelial lesions (OR ¼ 5.6, 95% CI 2.84–11.16, P < 0.0001) and in cancer (OR ¼ 6.9, 95% CI 2.96–15.97, P < 0.0001) than in specimens with normal cytology. The prevalence of other HPV genotypes was very similar in all subgroups divided according to the cytological diagnosis and the total prevalence of other HPV types (excluding HPV 16) ranged from 35.6% in atypical squamous cells to 42.6% in high-grade squamous intraepithelial lesions (Fig. 5A, Table II). Similarly, the prevalence of HPV types was also analyzed in specimens subdivided according to the histological diagnosis. In these subgroups the frequency of HPV 16 among the HPV-positive specimens correlated with the severity of cervical pathology (P < 0.0001): HPV 16 was detected in 37.5% cases in cervical intraepithelial neoplasia grade 1, 48.6% in cervical intraepithelial neoplasia grade 2, 57.9% cervical intraepithelial neoplasia grade 3 or carcinoma in situ, 61.3% in cervical cancer and 23.6% in the subgroup with not confirmed pathology (defined as “Normal histology”). Statistically significant differ-

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ence in HPV 16 prevalence was determined between the “Normal histology” subgroup and cervical intraepithelial neoplasia grade 2, cervical intraepithelial neoplasia grade 3 or carcinoma in situ and cervical cancer subgroups (P < 0.05; P < 0.0001; P < 0.0001, respectively) and between the cervical intraepithelial neoplasia grade 1 and cervical cancer subgroups (P < 0.05). Also, statistically significant difference was detected between the control group (healthy women with normal cervical cytology,) and cervical intraepithelial neoplasia grade 2, cervical intraepithelial neoplasia grade 3 or carcinoma in situ and cervical cancer subgroups (P < 0.001). HPV 16 infection was strongly associated with cervical cancer and there was a statistically significant difference in the prevalence of HPV 16 in cervical cancer subgroup as compared to the control group (OR ¼ 7.3, 95% CI 3.24–16.26, P < 0.0001). HPV 18 was identified only in 3.9% of all HPV-positive women; however, in the subgroup of cervical cancer HPV 18 was the second most prevalent type after HPV 16 (11.3%, P < 0.05). The difference of the prevalence of other HPV types in the subgroups divided according to the histological diagnosis was not statistically significant (P > 0.05) (Fig. 5B, Table II).

Fig. 5. The distribution of 16 analysed HPV genotypes among specimens with known cytological (A) and histological (B) diagnosis (ASC, atypical squamous cells; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; Ca, cervical cancer confirmed by cytology; CIN I, cervical intraepithelial neoplasia grade 1; CIN II, cervical intraepithelial neoplasia grade 2; CIN III/CIS, cervical intraepithelial neoplasia grade 3 or carcinoma in situ; CC, cervical cancer confirmed by histology; Normal histology, not confirmed pathology).

J. Med. Virol. DOI 10.1002/jmv

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The Prevalence of Multiple HPV Genotypes Among HPV DNA-Positive Specimens Using a sensitive multiplex PCR system that allows detection of multiple HPV types in a single reaction, it was found that 94 specimens (21.5%) of all HPV DNA-positive specimens (n ¼ 437) had multiple HR HPV types: more than one HPV type was detected in 82 (22.2%) specimens of women with abnormal cytology and 12 (17.9%) specimens with normal cytology (control group of healthy women). The difference between the study group and the control group in regard to the frequency of multiple HPV genotypes was not statistically significant. The prevalence of multiple HPV types was also analyzed in specimens subdivided according to the cytological diagnosis. The highest prevalence of multiple HPV infection was detected in the subgroup of low-grade squamous intraepithelial lesions (14 cases, 37.8%) and the lowest in cancer subgroup (nine cases, 18%). Statistically significant difference was detected between the following subgroups: atypical squamous cells and high-grade squamous intraepithelial lesions (P < 0.01), the atypical squamous cells and cancer (P < 0.05), the low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions (P < 0.05), the low-grade squamous intraepithelial lesions and cancer (P < 0.05). Similar tendency of multiple HPV infection was observed in a group of specimens subdivided according to the histological diagnosis. The highest prevalence of multiple HPV infection was detected in the subgroup of cervical intraepithelial neoplasia grade 1 subgroup (11 cases, 45.8%) and the lowest in cervical cancer subgroup (11 cases, 17.7%). Statistically significant difference was observed between the subgroups of cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 3 or carcinoma in situ, cervical cancer, “Normal histology,” control (healthy women) subgroups (P < 0.05) (Table III).

When analysing the distribution of HPV types, all 16 HPV genotypes were identified in a multiple HPV infection. The prevalence of HPV types both in a single and in a multiple HPV infection among all HPV DNA-positive specimens is shown in Figure 4. HPV 16 was the most common HPV type both in the single and the multiple HPV infections. HPV 16 in combination with other HPV types was detected in 48 of 370 specimens (13.0%) in the study group and in 7 of 67 specimens (10.4%) in the control group. The distribution of HPV 16 type and its combinations with other HPV types among HPV-positive specimens in the study and the control groups is shown in Figure 6. DISCUSSION HPV infection plays a key role as the major risk factor of both cervical cancer and precancerous pathology [Longworth and Laimins, 2004; Hoory et al., 2008]. Epidemiological studies have shown that about 75–80% of sexually active women are infected with HPV at least once in their lives [Medeiros et al., 2005]. Cervical cancer is the third most common cancer in women worldwide [Ferlay et al., 2010]. Morbidity and mortality rates for cervical cancer have increased in Lithuania since 1992: cervical cancer incidence rate in 1999 reached 17.4 cases per 100,000 women when compared to 15.4/100,000 cases in 1993 [Aleknaviciene et al., 2002]. The incidence rate of cervical cancer is the highest in Romania and Lithuania among European countries [Bray et al., 2002]. Before the onset of the national cervical cancer screening program that was initiated in Lithuania in 2004 under the authority of National Health Insurance Fund, cervical cancer represented more than 5% of new malignancies among women. The European age-standardized mortality rate was 10.7/100,000 [Maver et al., 2013]. Due to the high morbidity and mortality rates for cervical cancer in

TABLE III. The Prevalence of Multiple HPV Infection Among Specimens With Known Cytological and Histological Diagnosis Cytological diagnosis ASC

LSIL

HPV status

n

%a

n

Single HPV type 2 HPV types 3 HPV types 4 HPV types 5 HPV types Multiple HPV types Total HPVþ

37 17 1 3 0 21

42.5 19.5 1.1 3.4 0.0 24.1

15 12 2 0 0 14

%a

HSIL

CIN I

CIN II CIN III/CIS

%a

n

%a

n

%a

n

32 10 1 1 0 12

47.8 14.9 1.5 1.5 0.0 17.9

8 10 1 0 0 11

33.3 41.7 4.2 0.0 0.0 45.8

18 9 2 0 1 12

48.6 24.3 5.4 0.0 2.7 32.4

94 28 1 0 0 29

87 100.0 37 100.0 196 100.0 50 100.0 67 100.0 24 100.0 37 100.0 140

n

%a

Control n

n

%a

Ca

Histological diagnosis

40.5 131 66.8 32 64.0 32.4 35 17.9 7 14.0 5.4 2 1.0 2 4.0 0.0 0 0.0 0 0.0 0.0 1 0.5 0 0.0 37.8 38 19.4 9 18.0

%a

CC

n

%a

67.1 40 64.5 20.0 9 14.5 0.7 2 3.2 0.0 0 0.0 0.0 0 0.0 20.7 11 17.7

27 8 2 0 0 10

49.1 14.5 3.6 0.0 0.0 18.2

100.0 62 100.0

55

100.0

n

%a

Normal histology

ASC, atypical squamous cells; LSIL, low-grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithelial lesions; Ca, cervical cancer confirmed by cytology; CIN I, cervical intraepithelial neoplasia grade 1; CIN II, cervical intraepithelial neoplasia grade 2; CIN III/CIS, cervical intraepithelial neoplasia grade 3 or carcinoma in situ; CC, cervical cancer confirmed by histology; Normal Histology, not confirmed pathology). a Percentages of the prevalence of HPV types are calculated among the total number of HPV DNA-positive specimens.

J. Med. Virol. DOI 10.1002/jmv

HPV Types in Cervical Pathology

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Fig. 6. The distribution of HPV 16 type and its combinations with other HPV types among HPV DNA-positive specimens in the study group (women with cervical pathology) (A) and in the control group (healthy women with normal cytology) (B).

Lithuania, studies on the prevalence of HR HPV genotypes are of high importance. These data may be useful for the assessment of cancer risk and development of vaccine-based preventive strategies. In the current study, the prevalence of HPV infection and 16 carcinogenic or potentially carcinogenic HPV genotypes in the group of Lithuanian women with cervical pathology in comparison to healthy women was investigated. In total, 824 cervical specimens were investigated for HPV DNA: 547 specimens of women with abnormal cytology and 277 specimens of healthy women. For the analysis of HPV DNA in cervical specimens, three PCR systems based on different primer sets were employed. In addition to the previously described PGMY09/11 and GP5þ/GP6þ primer systems [Husman et al., 1995; Gravitt et al., 2000] a new set of primers targeting L1 gene sequences of 16 HPV types was designed. The newly developed HR_HPV PCR system has improved specificity and sensitivity in regard to carcinogenic or potentially carcinogenic HPV genotypes as it does not detect low-risk HPV types. The HR_HPV PCR system has been proven efficient for HPV DNA detection in 21 cervical specimens found to be negative by any of the other two PCR systems. As expected, the prevalence of HPV infection in women with cervical pathology was significantly higher than that in the control group (67.6% and 24.2%, respectively, P < 0.0001). The frequency of HPV DNA-positive specimens correlated with the severity of histological and cytological alterations. The analysis of HPV genotypes revealed HPV 16 as the most common genotype detected in 42.3% of all HPVpositive specimens (P < 0.0001), followed by HPV 31 (10.1%), HPV 33 (8.2%) and HPV 56 (5.7%). HPV 18 was detected only in 3.9% of all studied HPV-positive women, however this HPV type was more common in a subgroup of cervical cancer (detected in 11.3% of HPV-positive specimens). Meanwhile, other HPV types were distributed similarly among all subgroups divided according to the cytological and histological

diagnosis. The current study revealed a relatively high number of HPV DNA-positive specimens with multiple HPV infection. The prevalence of multiple HPV types among HPV DNA-positive specimens ranged from 45.8% in cervical intraepithelial neoplasia grade 1 to 17.7% in cervical cancer subgroups (P < 0.05). In a previous study, similar frequency of multiple HPV types in cervical specimens collected from women with cervical pathology has been reported [Zuna et al., 2007]. However, in contrast to the previous study where the differences in the frequency of multiple HPV infections among the subgroups were not significant, the current study revealed a statistically significant differences between the subgroups with known cytological diagnosis such as atypical squamous cells and high-grade squamous intraepithelial lesions, atypical squamous cells and cancer, lowgrade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, low-grade squamous intraepithelial lesions and cancer. Furthermore, in the group of specimens with known histological diagnosis a statistically significant difference between the subgroups with cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 3 or carcinoma in situ, cervical cancer, “Normal histology,” control (healthy women) in regard to multiple HPV infection was observed. In the follow-up studies, it would be important to investigate the correlation between the number of different HR HPV types in a single specimen and the risk of developing high-grade lesions and invasive cancer. The prevalence of HPV DNA in cervical cancer specimens was 82.7% in the current study and it was similar to that reported for other countries. According to the large meta-analyses, the HPV DNA-positivity rate was 79–95% in cervical cancer, depending on the continent [Smith et al., 2007]. The HPV-positivity rate in the group of healthy Lithuanian women was relatively high (24.2%) and similar to that reported in two previous studies: 26.7% [Gudleviciene et al., 2005] and 23.6% [Gudleviciene et al., 2006], J. Med. Virol. DOI 10.1002/jmv

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respectively. Meanwhile, HPV incidence in other world regions is lower (Africa 22.1%, Central America and Mexico 20.4%, Northern America 11.3%, Europe 8.1% and Asia 8.0%) [De Sanjose´ et al., 2007]. Part of the differences in HPV prevalence might be caused by the methods used to collect cervical specimens, the assays used for HPV DNA detection as well as the quality and type of specimens (biopsies, surgical specimens, or fresh tissue) analysed [Gravitt et al., 2007]. Nevertheless, the current study shows a relatively high prevalence of HPV DNA in cervical specimens collected both from the study and the control groups of Lithuanian women. According to the meta-analyses of HPV genotype distribution in cervical cancer, HPV 16 is the most common, and HPV 18 is the second most common genotype in all continents [Smith et al., 2007]. Previous studies suggest that HPV 16 and HPV 18 are detected in approximately 70% of all cases of cervical cancer worldwide and these HPV genotypes play an important role in causing cell transformation [Baseman and Koutsky, 2005; Bosch et al., 2008]. These data are in line with the current study, indicating that HPV 16 and HPV 18 are the most prevalent HR HPV types in the subgroup of women with confirmed cervical cancer. In this subgroup, both HPV 16 and 18 were detected in 72.6% of HPV-positive cervical specimens, which corresponds to 46.2% of all studied specimens with confirmed cytological diagnosis. However, the frequency of HPV 18 (3.9% of HPV-positive specimens or 11.3% of all studied cervical cancer specimens) was lower in the current study as compared to other countries. According to the previous reports, HPV 18 is detected in 17.2% of all cervical cancer specimens in Europe, 22.1% in North America and 15.3% in Asia [Pagliusi and Aguado, 2004]. In all analysed specimens of this study without subdividing them according to the cytological and/or histological diagnosis, the frequency of other HR HPV types (HPV 31, 33 and 56) was higher than that of HPV 18. Thus, the current study demonstrated high prevalence of other HR HPV types besides of HPV 16 with a potential to cause precancerous pathology and cervical cancer in the groups of Lithuanian women. The obtained data confirm the geographic variability in the prevalence of HR HPV genotypes. As the HR HPV infection is the main risk factor of cervical carcinogenesis, the information on the frequency of HR HPV genotypes in different countries is essential for planning prevention measures by prophylactic HPV vaccines and for large-scaled screening programs based on HPV testing. Two HPV vaccines are currently on the market: Gardasil and Cervarix that protect from two oncogenic HPV types: HPV 16 and HPV 18. Gardasil also protects from two non-oncogenic HPV types: HPV 6 and HPV 11. These HPV vaccines have been shown to afford some degree of cross-protection from closely related non-vaccine types HPV 31, HPV 33 and HPV 45 [Lu et al., 2011; Romanowski, 2011]. However, other high-risk HPV J. Med. Virol. DOI 10.1002/jmv

Simanaviciene et al.

types are not affected by the currently available vaccines. The development of a new nine-valent HPV vaccine V503 against nine HPV types (HPV 16, 18, 31, 33, 45, 52, 58, 6 and 11) was recently reported (ClinicalTrials.gov NCT00543543, 2007). The current study that demonstrates a high prevalence of HPV 56 and HPV 51 among Lithuanian women suggests the importance of developing new HPV vaccines that could protect from multiple high-risk HPV types. CONCLUSIONS The current study presents new data on the prevalence of HPV infection and distribution of 12 carcinogenic and four potentially carcinogenic HPV genotypes in the groups of Lithuanian women with known cytological and histological diagnosis (n ¼ 824). Among studied Lithuanian women, HPV 16 was the most common HPV type identified in the groups of women both with normal (n ¼ 277) and abnormal cytohistology (n ¼ 547). High prevalence of other high-risk HPV types such as HPV 31, HPV 33, and HPV 56 was demonstrated. In contrast, the frequency of HPV 18 was lower as compared to other countries. The current study also revealed a relatively high number of HPV DNA-positive specimens with multiple HPV infection. The obtained data confirm the geographic variability in the prevalence of highrisk HPV genotypes and provide useful information for planning prevention measures of cervical cancer. REFERENCES Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA. 2003. p16(INK4a) expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of highrisk HPV types. Mod Pathol 16:665–673. Aleknaviciene B, Smailyte G, Elaawar B, Kurtinaitis J. 2002. Cervical cancer: Recent trends of incidence and mortality in Lithuania. Medicina 38:223–230. Baseman JG, Koutsky LA. 2005. The epidemiology of human papillomavirus infections. J Clin Virol 32:16–24. Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen HZ, de Villiers EM. 2010. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virol J 401:70–79. Bosch FX, Burchell AN, Schiffman M, Giuliano AR, Sanjose S, Bruni L, Tortolero-Luna G, Kjaer SK, Mu~ noz N. 2008. Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia. Vaccine 26:1–16. Bray F, Sankila R, Ferlay J, Parkin DM. 2002. Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer 38:99– 166. Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, Sanjose SD. 2010. Cervical human papillomavirus prevalence in 5 continents: Meta-analysis of 1 million women with normal cytological findings. J Infect Dis 202:1789–1799. De Sanjose´ S, Diaz M, Castellsague´ X, Clifford G, Bruni L, Mu~ noz N, Bosch FX. 2007. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: A meta-analysis. Lancet Infect Dis 7:453–459. De Vuyst H, Clifford GM, Nascimento MC, Madeleine MM, Franceschi S. 2009. Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: A meta-analysis. Int J Cancer 124:1626–1636. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. 2010. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127:2893–2917.

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J. Med. Virol. DOI 10.1002/jmv

Studies on the prevalence of oncogenic HPV types among Lithuanian women with cervical pathology.

Human papillomavirus (HPV) is the main cause of cervical cancer. Therefore, the detection of oncogenic HPV types is important in predicting the risk o...
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