The aim of the present population-based cohort study was to analyze the association between the prevalence of 32 types of human papilloma virus (HPV) in 615 female patients with abnormal cervical cytopathology findings. In total, 32 HPV types were screened by DNA array technology. HPV infection was detected in 470 women (76.42%), 419 of whom (89.15%) were infected with ≥1 high-risk (HR)-HPV type. HPV16, which is recognized as the main HR-HPV type responsible for the development of cervical cancer, was observed in 32.98% of HPV+ participants, followed by HPV42 (18.09%), HPV31 (17.66%), HPV51 (13.83%), HPV56 (10.00%), HPV53 (8.72%) and HPV66 (8.72%). The prevalence of HR-HPV types, which may be suppressed directly (in the case of HPV16 and 18), or possibly via cross-protection (in the case of HPV31) following vaccination, was considerably lower in participants ≤22 years of age (HPV16, 28.57%; HPV18, 2.04%; HPV31, 6.12%), compared with participants 23–29 years of age (HPV16, 45.71%; HPV18, 7.86%; HPV31, 22.86%), who were less likely to be vaccinated. Consequently, the present study hypothesizes that there may be a continuous shift in the prevalence of HPV types as a result of vaccination. Furthermore, the percentage of non-vaccine HR-HPV types was higher than expected, considering that eight HPV types formerly classified as ‘low-risk’ or ‘probably high-risk’ are in fact HR-HPV types. Therefore, it may be important to monitor non-vaccine HPV types in future studies, and an investigation concerning several HR-HPV types as risk factors for the development of cervical cancer is required.
Human papilloma viruses (HPVs) are small non-enveloped DNA viruses that belong to the papilloma virus family, and infect cutaneous and mucosal epithelia in humans (
HPV is usually acquired via sexual transmission, and may induce the development of cervical cancer within several years following a persistent infection (
Currently, there are two approved HPV vaccines in Germany (
With the increasing impact of vaccination against certain HPV types in young women (
The study cohort consisted of 615 Caucasian women aged between 16 and 93 years. The participants had undergone routine cytological evaluation in three Southern Bavaria pathology centers (Institutes of Pathology at Kaufbeuren, Ravensburg and Rosenheim, Germany) using the Second Munich Cytological Classification (
Cytological specimens were collected between December 2010 and September 2014 by Pap smear. The specimens were dehydrated, and subsequently stained with Harris' hematoxylin (Carl Roth GmbH, Karlsruhe, Germany), bleached with hydrated hydrochloric acid (Hernicht GmbH, Sulzberg, Germany), and stained orange-red with Papanicolaou's staining solution Orange G (EA 50; VWR International GmbH, Darmstadt, Germany), according to the manufacturer's protocol. The slides were reviewed by qualified pathologists at the Institutes of Pathology at Kaufbeuren, Ravensburg and Rosenheim using the Bethesda classification system (
In addition to the Pap smear, cervical conization specimens were obtained from a subgroup of 86 out of 615 participants. The cervical conization specimens were reviewed and classified as grades CIN1, CIN2 and CIN3, according to the WHO criteria (
DNA was isolated from the formalin-fixed, paraffin-embedded (FFPE) tissues using the QIAamp DNA FFPE Tissue kit (Qiagen GmbH, Hilden, Germany) and the QIAcube (Qiagen GmbH), according to the manufacturer's protocol. HPV testing was performed using a DNA-based liquid-crystal display (LCD)-Array kit (LCD Array HPV 3.5; Chipron GmbH, Berlin, Germany), which contained 32 specific capture probes for the identification of 32 types of HPV. In total, 20 µl polymerase chain reaction (PCR) AmpliTaq Gold® 360 Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used, which contained 1.2 µl pre-labeled primer mix, 0.2 µM dNTPs, 2 mM magnesium, 0.2 U Taq-Gold polymerase and 2 µl template DNA. Amplification was performed in a DNA Engine® Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the following PCR program: Initial denaturation of 10 min at 95°C followed by 42 cycles of 1 min at 94°C, 1.5 min at 45°C and 1.5 min at 72°C, with a final elongation of 3 min at 72°C. The present study adapted the classification of HR- and LR-HPV types according to previous studies concerning the carcinogenic properties of particular HPV types (
PCR results were evaluated using SlideReader Software v2.0 (Chipron GmbH). PCR, hybridization, labeling and staining were performed according the manufacturer's protocol (Chipron GmbH). HPV types that were detected by PCR but did not generate signals on the LCD array were sequenced and subjected to Basic Local Alignment Search Tool analysis (National Center for Biotechnology Information, Bethesda, MD, USA). One tissue sample with HPV87 was detected by sequencing.
The current study consisted of 615 female participants with abnormal cervical cytological pathology (ASC-US, LSIL or HSIL). In total, 35.61% (219/615) of the participants were <30 years of age, 32.36% (199/615) were 30–44 years old, 28.62% (176/615) were 45–59 years old and 3.41% (21/615) were >60 years of age. In 470 (76.42%) of these participants, HPV infection was detected, and 419 (89.15%) of them were infected with ≥1 HR-HPV type. As shown in
The present study additionally analyzed participants with ST or MT infection. The highest rate of MT infection (64.02% of all infected patients) was observed in participants <30 years old. In participants >30 years old, the percentage of patients with MT infection was lower (30–44 years, 51.61%; 45–59 years, 52.25%; ≥60 years, 46.67%). The highest prevalence of HPV type was HPV16 (HR), which was present in 32.98% of participants that tested HPV+, followed by HPV42 (LR; 18.09%), HPV31 (HR; 17.66%), HPV51 (HR; 13.83%), HPV56 (HR; 10.00%), HPV66 (HR; 8.72%) and HPV53 (HR; 8.72%). HR-HPV type infections that may be directly prevented by vaccination or via cross-protection with available vaccines are marked in green (HPV16 and 18) and yellow (HPV31) in
In a subgroup of 86 participants, cervical conization specimens were obtained, and 80 of these participants exhibited cervical intraepithelial neoplasia, which were classified as CIN1 (22/80), CIN2 (27/80) or CIN3 (31/80). The prevalence of HR-HPVs was associated with a more severe cervical intraepithelial neoplasia (HR-HPV appearances: CIN1, 77.27%; CIN2, 81.48%; and CIN3, 90.32%). The total prevalence of HR-HPV types 16, 18 and 31 was highest in participants whose neoplasms were classified as CIN3 (64.52%; 20/31), followed by 55.56% (15/27) of neoplasms classified as CIN2 and 50.00% (11/22) of neoplasms classified as CIN1. By contrast, there was no evidence that MT infections were more prevalent in neoplasms classified as CIN3 (48.39%), compared with those classified as CIN2 (50.85%). In addition, the present study demonstrated that in participants with CIN3 lesions, a cytological diagnosis of ASC-US was present in 19.35% of participants, LSIL in 32.26% participants and HSIL in 48.39% of participants. Participants that possessed CIN1 or CIN2 neoplasms were more likely to exhibit LSIL (50.00%; 66.67%), compared with HSIL (31.82%; 18.52%). There was no accumulation of specific HPV types among the non-vaccinated HR-HPV participants that could be assigned to one of the CIN classes.
In Germany, a routine HPV vaccine has been recommended for 12–17 year-old girls since 2007 (
The present study evaluated the prevalence of important HPV types in a local cohort of participants from Southern Bavaria, and analyzed the HPV type distribution in participants with abnormal cytological diagnostic findings, who were separated by various age groups. It is well known that the distribution of HPV types varies around the world (
In addition, the present study observed that the prevalence of HPV16 and 18 in participants >30 years old was 33.81%, which is close to the prevalence of 34.50% identified in a previous study (
It was previously demonstrated that MT HPV are more common in women with cervical lesions, compared with women with normal cervical cytology (
In addition to the prevalence of specific HPV types, the present study analyzed the distribution of HPV types in participants with abnormal cervical cytology. Since HPV may cause the development of precancerous lesions (
In the current study, 80 participants with cervical intraepithelial neoplasia were classified as grade CIN1 (22/80), CIN2 (27/80) or CIN3 (31/80). As previously reported, the incidences of particular HR-HPV infections were observed to increase in association with the severity of the cervical intraepithelial neoplasia in the present study (
Although the present study revealed that the highest prevalence of HPV16 was in participants <30 years of age, it is important to emphasize that a subgroup of the youngest women, namely those aged <23 years old, exhibited a significantly lower incidence, compared with women aged 23–29 years old (28.57 vs. 45.71%, respectively). The lower relative incidences of HPV16, 18 and 31 observed in the present study may be explained by a higher rate of vaccinated participants aged <23 years old. The low vaccination coverage in Germany (
Consequently, adequate prevention strategies against HPV depend on adapted HPV testing, which should cover all non LR-HPV types. Notably, certain FDA-approved used HPV tests, such as Hybrid Capture 2 (Digene Corporation, Gaithersburg, MD, USA), do not cover all HR-HPV types, which were detected with the test platform used in the current study, including Papillocheck® (Greiner Bio One Ltd., Stonehouse, UK) and Linear Array® (Roche Diagnostics GmbH). The present results indicate that ~9% of samples with HR-HPV infections may have been incorrectly reported as HPV− by the Hybrid Capture 2 test. The information that eight HPV types formerly classified as low-risk or probably high-risk are indeed HR-HPV types increases the percentage of non-vaccine HR-HPV to higher values than previously expected (
In participants from the present cohort study, high incidences of HR-HPV types (89.15%) and a high incidence of HPV16 (32.98%) were observed. In all grades of cytological diagnosis (ASC-US, LSIL and HSIL), HPV16 was the most common HPV type identified. The prevalence of specific HR-HPV infections increased with a more severe CIN grade. The number of findings of HR-HPV types, which may be suppressed directly (HPV16 and 18) or via cross-protection (HPV31) following vaccination, was considerably lower in participants aged ≤22 years old (HPV16, 28.57%; HPV18, 2.04%; HPV31, 6.12%), compared with those aged 23–29 years old (HPV16, 45.71%; HPV18, 7.86%; HPV31, 22.86%). Consequently, in HPV+ women of 16–22 years of age, who are more likely to be protected against certain HPV types by vaccination, compared with older woman, the present study observed an alteration in the incidences of HPV type. Consequently, future studies are required to investigate the risk of non-vaccine HR-HPV types for cervical cancer.
The authors of the present study would like to thank Mrs. Andrea Becher for using the data included in her dissertation thesis in the elaboration of the present manuscript. The authors acknowledge the support of all the staff members of the Part Shared Practice Molecular Pathology South Bavaria (Munich, Germany).
Prevalence of HR- and LR-HPV types among participants in the present study, represented according to their incidences as ST or MT infection. HPV types targeted directly by vaccination are labelled green. HPV types where a cross-protective effect has been described for current vaccines are labelled yellow. HR-HPV types without vaccination coverage and a high incidence are labelled red. HPV types were identified using Chipron LCD Array HPV 3.5 or DNA sequencing (in the case of HPV87). HPV, human papilloma virus; HR, high risk; LR, low risk; ST, single type; MT, multiple type; LCD, liquid-crystal display.
Prevalence of HPV types among participants in the present study with ASC-US, LSIL or HSIL. HPV types targeted directly by vaccination are labelled green. HPV types where a cross-protective effect has been described for current vaccines are labelled yellow. HR-HPV types without vaccination coverage and high incidence are labelled red. HPV types were identified using Chipron LCD Array HPV 3.5 or DNA sequencing (in the case of HPV87). HPV, human papilloma virus; ASC-US, atypical squamous cell of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; HR, high risk; LR, low risk; LCD, liquid-crystal display.
Relative distribution of HPV+ and HPV− participants according to the Bethesda cervical cytological classification (ASC-US, LSIL or HSIL). The distribution of cervical cytological classification among participants in the present study with ≥1 HR-HPV or 1 probably HR-HPV type with additional LR-HPV appearance, and the distribution of cervical cytological classification among participants with ≥1 LR-HPV type without any HR-HPV appearance, are depicted. Furthermore, the relative distribution of participants according to single or multiple infection is represented in the graph. HPV, human papilloma virus; ASC-US, atypical squamous cell of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; HR, high risk; LR, low risk.
(A) Number of findings and (B) incidences of HPV types that are impaired directly by vaccination or potentially via cross-protection by available vaccines. Participants <30 years of age are divided into two subgroups (group 1, <23 years old; group 2, 23–29 years old), where women ≤22 years old are more often protected against certain HPV types by vaccination, compared with participants aged >23 years old. HPV, human papilloma virus.
Type-specific HPV incidence in 615 women, grouped according to their age and risk of developing risk cancer.
Age groups (HPV+ participants only) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
HPV+ participants | Group I (<30 years) | Group II (30–44 years) | Group III (45–59 years) | Group IV (≥60 years) | ||||||||
Variable | Total n (%) | n (%) | ST | MT | n (%) | MT | n (%) | MT | n (%) | MT | n (%) | MT |
Abnormal cytology | 615 (NA) | 470 (76.42) | 204 | 266 | 189 (NA) | 121 | 155 (NA) | 80 | 111 (NA) | 58 | 15 (NA) | 7 |
HPV findings | 979 (NA) | 979 (NA) | 204 | 775 | 440 (NA) | 372 | 294 (NA) | 219 | 219 (NA) | 166 | 26 (NA) | 18 |
Patients with ≥1 HR-HPV type | 419 (68.13) | 419 (89.15) | – | – | 173 (91.53) | – | 143 (92.26) | – | 92 (82.88) | – | 11 (73.33) | – |
HPV type | ||||||||||||
16 | 155 (25.20) | 155 (32.98) | 51 | 104 | 78 (41.27) | 52 | 48 (30.97) | 30 | 25 (22.52) | 19 | 4 (26.67) | 3 |
18 | 30 (4.88) | 30 (6.38) | 10 | 20 | 12 (6.35) | 10 | 8 (5.16) | 4 | 9 (8.11) | 5 | 1 (6.67) | 1 |
31 | 83 (13.50) | 83 (17.66) | 21 | 62 | 35 (18.52) | 32 | 29 (18.71) | 18 | 19 (17.12) | 12 | 0 (0.00) | 0 |
33 | 18 (2.93) | 18 (3.83) | 3 | 15 | 8 (4.23) | 8 | 5 (3.23) | 3 | 4 (3.60) | 3 | 1 (6.67) | 1 |
35 | 14 (2.28) | 14 (2.98) | 1 | 13 | 8 (4.23) | 7 | 4 (2.58) | 4 | 2 (1.80) | 2 | 0 (0.00) | 0 |
39 | 29 (4.72) | 29 (6.17) | 8 | 21 | 12 (6.35) | 10 | 11 (7.10) | 6 | 5 (4.50) | 4 | 1 (6.67) | 1 |
45 | 24 (3.90) | 24 (5.11) | 4 | 20 | 8 (4.23) | 8 | 7 (4.52) | 6 | 7 (6.31) | 4 | 2 (13.33) | 2 |
51 | 65 (10.57) | 65 (13.83) | 11 | 54 | 36 (19.05) | 31 | 16 (10.32) | 13 | 12 (10.81) | 9 | 1 (6.67) | 1 |
52 | 36 (5.85) | 36 (7.66) | 6 | 30 | 15 (7.94) | 14 | 12 (7.74) | 8 | 9 (8.11) | 8 | 0 (0.00) | 0 |
53 | 41 (6.67) | 41 (8.72) | 4 | 37 | 16 (8.47) | 13 | 12 (7.74) | 12 | 11 (9.91) | 11 | 2 (13.33) | 1 |
56 | 47 (7.64) | 47 (10.00) | 14 | 33 | 18 (9.52) | 15 | 18 (11.61) | 12 | 11 (9.91) | 6 | 0 (0.00) | 0 |
58 | 15 (2.44) | 15 (3.19) | 5 | 10 | 6 (3.17) | 4 | 4 (2.58) | 3 | 4 (3.60) | 3 | 1 (6.67) | 0 |
59 | 29 (4.72) | 29 (6.17) | 2 | 27 | 16 (8.47) | 16 | 10 (6.45) | 8 | 3 (2.70) | 3 | 0 (0.00) | 0 |
66 | 41 (6.67) | 41 (8.72) | 8 | 33 | 20 (10.58) | 18 | 9 (5.81) | 8 | 8 (7.21) | 4 | 4 (26.67) | 3 |
67 | 28 (4.55) | 28 (5.96) | 1 | 27 | 16 (8.47) | 15 | 8 (5.16) | 8 | 3 (2.70) | 3 | 1 (6.67) | 1 |
68 | 6 (0.98) | 6 (1.28) | 2 | 4 | 2 (1.06) | 2 | 2 (1.29) | 1 | 2 (1.80) | 1 | 0 (0.00) | 0 |
70 | 22 (3.58) | 22 (4.68) | 6 | 16 | 8 (4.23) | 7 | 8 (5.16) | 5 | 6 (5.41) | 4 | 0 (0.00) | 0 |
73 | 13 (2.11) | 13 (2.77) | 2 | 11 | 7 (3.70) | 6 | 2 (1.29) | 1 | 4 (3.60) | 4 | 0 (0.00) | 0 |
82 | 5 (0.81) | 5 (1.06) | 0 | 5 | 2 (1.06) | 2 | 3 (1.94) | 3 | 0 (0.00) | 0 | 0 (0.00) | 0 |
83 | 9 (1.46) | 9 (1.91) | 1 | 8 | 5 (2.65) | 5 | 2 (1.29) | 1 | 2 (1.80) | 2 | 0 (0.00) | 0 |
Patients with ≥1 LR-HPV type | 212 (34.47) | 212 (45.11) | – | – | 90 (47.62) | – | 59 (38.06) | – | 55 (49.55) | – | 8 (53.33) | – |
HPV type | ||||||||||||
6 | 21 (3.41) | 21 (4.47) | 6 | 15 | 9 (4.76) | 7 | 7 (4.52) | 5 | 5 (4.50) | 3 | 0 (0.00) | 0 |
11 | 7 (1.14) | 7 (1.49) | 1 | 6 | 1 (0.53) | 1 | 3 (1.94) | 3 | 1 (0.90) | 1 | 2 (13.33) | 1 |
42 | 85 (13.82) | 85 (18.09) | 18 | 67 | 38 (20.11) | 33 | 18 (11.61) | 14 | 28 (25.23) | 20 | 1 (6.67) | 0 |
44 | 17 (2.76) | 17 (3.62) | 2 | 15 | 7 (3.70) | 6 | 6 (3.87) | 6 | 4 (3.60) | 3 | 0 (0.00) | 0 |
54 | 32 (5.20) | 32 (6.81) | 2 | 30 | 16 (8.47) | 14 | 7 (4.52) | 7 | 9 (8.11) | 9 | 0 (0.00) | 0 |
61 | 13 (2.11) | 13 (2.77) | 0 | 13 | 8 (4.23) | 8 | 2 (1.29) | 2 | 3 (2.70) | 3 | 0 (0.00) | 0 |
62 | 24 (3.90) | 24 (5.11) | 3 | 21 | 5 (2.65) | 4 | 12 (7.74) | 12 | 6 (5.41) | 4 | 1 (6.67) | 1 |
72 | 1 (0.16) | 1 (0.21) | 1 | 0 | 0 (0.00) | 0 | 1 (0.65) | 0 | 0 (0.00) | 0 | 0 (0.00) | 0 |
81 | 8 (1.30) | 8 (1.70) | 1 | 7 | 3 (1.59) | 3 | 2 (1.29) | 2 | 3 (2.70) | 2 | 0 (0.00) | 0 |
84 | 19 (3.09) | 19 (4.04) | 0 | 19 | 9 (4.76) | 9 | 6 (3.87) | 6 | 3 (2.70) | 3 | 1 (6.67) | 1 |
87 | 1 (0.16) | 1 (0.21) | 1 | 0 | 1 (0.53) | 0 | 0 (0.00) | 0 | 0 (0.00) | 0 | 0 (0.00) | 0 |
90 | 26 (4.23) | 26 (5.53) | 5 | 21 | 9 (4.76) | 8 | 7 (4.52) | 4 | 8 (7.21) | 8 | 2 (13.33) | 1 |
91 | 15 (2.44) | 15 (3.19) | 4 | 11 | 6 (3.17) | 4 | 5 (3.23) | 4 | 3 (2.70) | 3 | 1 (6.67) | 0 |
Single infection | 204 (33.17) | 204 (43.40) | – | – | 68 (35.98) | – | 75 (48.39) | – | 53 (47.75) | – | 8 (53.33) | – |
Double infection | 129 (20.98) | 129 (27.45) | – | – | 57 (30.16) | – | 41 (26.45) | – | 27 (24.32) | – | 4 (26.67) | – |
Multiple (>1) infections | 266 (43.25) | 266 (56.60) | – | – | 121 (64.02) | – | 80 (51.61) | – | 58 (52.25) | – | 7 (46.67) | – |
Abnormal cytology consists of atypical squamous cell of undetermined significance, low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion. HPV, human papilloma virus; HR, high risk; LR, low risk; ST, single type; MT, multiple type; NA, not available.